A microsphere composite hydrogel and its preparation method and application

Abstract

The present invention belongs to the field of medical materials, and specifically relates to a microsphere composite hydrogel and a preparation method and application thereof. The microsphere composite hydrogel of the present invention comprises microspheres and hydrogels, wherein the microspheres are dispersed in the hydrogels; the microspheres comprise a degradable polyester material and a mesoporous material wrapped in the degradable polyester material. The microsphere composite hydrogel disclosed in the present invention has good biocompatibility, biological activity, strong mechanical properties and bone mineralization ability; and the microspheres comprise a degradable polyester material and a mesoporous material, and the mesoporous material contains a rich mesoporous structure, which can load active drugs (bone repair promoting factors, anti-infection / anti-tumor drugs, etc.), and realize effective slow and controlled release of drugs in the defect site, so that it can accelerate bone healing / anti-infection / anti-tumor effects, and is a hydrogel system suitable for bone defect repair caused by different causes.

Description

Technical Field

[0001] The invention belongs to the field of medical materials, and specifically relates to a microsphere composite hydrogel and a preparation method and application thereof. Background Art

[0002] In recent years, the number of patients with bone defects caused by trauma, tumors, and infections has increased significantly. Bone defects larger than a critical size are difficult to self-repair. Every year, more than 2 million bone transplants are performed worldwide to treat bone defects, making it the second most common tissue transplant after blood transfusion. Autologous bone transplantation is considered the most ideal choice for bone defect surgery, but there are limitations in terms of donor sources and donor site morbidity. Therefore, bone tissue engineering has attracted much attention from researchers. Ideal bone defect repair biomaterials should be biodegradable, biocompatible, and have good mechanical properties, and be able to mediate the response of bone repair cells. The selection of natural polymer materials combined with a better drug carrier model can provide support for bone repair cells while controlling the release of drugs to continuously induce osteogenic differentiation of stem cells and regulate the formation of new blood vessels by vascular cells, which is beneficial to bone repair and is a hot spot in the research and development of bone repair biomaterials.

[0003] Compared with artificial synthetic polymer materials, polymer materials constructed from natural proteins and polysaccharides show better biocompatibility. Among them, silk fibroin (SF) is a natural protein derived from silkworm cocoons. It has good biocompatibility, biodegradability and easy extraction. Therefore, SF is considered to be a good tissue regeneration biomaterial. However, weak mechanical properties, poor osteoinductivity and osteoconductivity limit the application of SF in bone tissue engineering. Therefore, SF is usually combined with other biomaterials such as chitosan and inorganic particles to construct composite materials to enhance their mechanical properties, antibacterial properties and biological properties. Carboxymethyl chitosan (CMCS) is a derivative of chitosan. Compared with chitosan, CMCS has higher water solubility and can react with more Ca 2+Chelation has superior bone mineralization activity and certain antibacterial properties, which reduces the risk of infection after artificial bone transplantation and promotes surgical wound repair in clinical practice, but its degradation rate is relatively fast. Sodium alginate (SA) is an anionic copolymer natural polysaccharide derived from brown seaweed. It has good biocompatibility, can promote the growth of bone tissue along the scaffold material, and has suitable biodegradability and viscosity. It is widely used in tissue engineering. Combining natural proteins and polysaccharides can not only improve the mechanical properties of composite materials, but also give the composite materials certain antibacterial properties, and can regulate the degradation rate of scaffold materials to adapt to the rate of bone formation. Although there is a certain electrostatic interaction between the amino groups in the protein and the carboxyl, hydroxyl and other groups in the polysaccharide, it is still necessary to add a cross-linking agent to solidify different proteins and polypeptides into a complex network structure.

[0004] For a long time, microspheres have been widely used in drug delivery for their excellent controlled release ability. Among them, degradable polyester microsphere-based composites have received widespread attention. Among them, PLGA is a copolymer of PGA and PLA, and the degradation time has a wide range of regulation, so it is widely used for drug release. However, its hydrophilicity is poor and the degradation products are acidic, so the biological activity is poor and it is easy to cause severe local inflammation, which to a certain extent hinders the application of degradable polyester biomaterials in bone tissue engineering. Mesoporous hydroxyapatite, MS, MBG, and mesoporous silicate particles have good biocompatibility and bioactivity. Among them, MS has a mesoporous structure and a high specific surface area. It can be made into a carrier for loading drugs and molecules through surface modification, and has the function of sustained drug release, which is a good drug carrier. How to prepare bone repair materials with excellent comprehensive properties such as biocompatibility, bioactivity, mechanical properties, and bone mineralization promotion is still of great significance. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a microsphere composite hydrogel, which has good biocompatibility, strong mechanical properties, and can promote bone mineralization and accelerate bone defect repair.

[0006] The invention also provides a method for preparing the microsphere composite hydrogel.

[0007] The present invention also provides a preparation method and application of a drug-loaded microsphere composite hydrogel material comprising the microsphere composite hydrogel.

[0008] In a first aspect of the present invention, a microsphere composite hydrogel is provided, wherein the microsphere composite hydrogel comprises microspheres and hydrogel, wherein the microspheres are dispersed in the hydrogel; and the microspheres comprise a degradable polyester material and a mesoporous material encapsulated in the degradable polyester material.

[0009] Compared with the prior art, the mesoporous material used in the present invention has a rich mesoporous structure and a high specific surface area, and can be used as a carrier of drugs and molecules. Moreover, the mesoporous material also has the ability to form calcium phosphate similar to the mineral on the surface of bones, and can stimulate bone marrow mesenchymal stem cells to differentiate into osteoblasts through the bone morphogenetic protein 2 pathway and the adenylate activated protein kinase pathway, thereby promoting new bone formation; the mesoporous material is mixed with a degradable polyester material to form microspheres, which can effectively improve the biocompatibility and mechanical properties of the microspheres, and at the same time prolong the drug release time. At the same time, studies have shown that the compressive strength of the microspheres obtained by encapsulating the mesoporous material and dispersing it in the degradable polyester material is significantly higher than that of the pure polyester microspheres, which is more suitable for bone repair. Therefore, the microsphere composite hydrogel of the present invention has good biocompatibility, biodegradability and strong mechanical properties, and at the same time has good bone mineralization ability, which is beneficial to bone repair, and can further load drugs and molecules to achieve drug and molecule sustained release.

[0010] Preferably, the mass of the microspheres is 0.1-100% of the mass of the hydrogel, more preferably 0.5-50%, including but not limited to 2%, 6%, 10%, 20%, 30%, 40%, etc.

[0011] Preferably, the hydrogel comprises at least one of natural protein and natural polysaccharide.

[0012] Preferably, the mass ratio of the natural protein to the natural polysaccharide is 1:1-8, more preferably 1:1-6, further preferably 1:1.5-4.

[0013] Preferably, the natural protein includes at least one of fibrin, fibrinogen, silk fibroin (SF), collagen and elastin.

[0014] Preferably, the natural polysaccharide comprises at least one of carboxymethyl chitosan (CMCS), starch, hyaluronic acid, sodium alginate (SA), cellulose and chitosan.

[0015] Preferably, the hydrogel comprises a combination of silk fibroin (SF), carboxymethyl chitosan (CMCS) and sodium alginate (SA), and the mass ratio of the silk fibroin, carboxymethyl chitosan and sodium alginate is 1:0.1-5:0.1-5; more preferably 1:1:0.5, including but not limited to 1:1:0.5, 1:1:1, 1:1:1.5, etc.

[0016] Preferably, when the hydrogel comprises fibrin and chitosan, the mass ratio of fibrin to chitosan is 1:0.1-5, more preferably 1:3-5, including but not limited to 1:3, 1:4, 1:5, etc.

[0017] Preferably, when the hydrogel comprises collagen and sodium alginate, the mass ratio of collagen to sodium alginate is 1:0.1-5, more preferably 1:3-5, including but not limited to 1:3, 1:4, 1:5 and the like.

[0018] Preferably, the hydrogel further comprises a crosslinking agent, which comprises at least one of a protein crosslinking agent and an ionic crosslinking agent, including but not limited to genipin, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, metal salts, etc. The present invention selects protein crosslinking agents and ionic crosslinking agents with good biocompatibility, which are non-cytotoxic, do not affect biocompatibility, and have a high degree of crosslinking, which is conducive to improving the mechanical strength and stability of the hydrogel.

[0019] Preferably, the mass of the cross-linking agent accounts for 5-30% of the mass of the hydrogel, more preferably 9-22%, including but not limited to 9%, 12.5%, 13%, 22%, etc.

[0020] Preferably, the cross-linking agent includes at least one of genipin and a metal salt; the mass ratio of genipin to the metal salt is 1 to 20:1, more preferably 1 to 15:1, including but not limited to 1.25:1, 4:1, 10:1, 12:1, 15:1, etc.

[0021] Preferably, the ratio of genipin to the total mass of the hydrogel (natural protein and natural polysaccharide) is 1:5-30, more preferably 1:5-20, including but not limited to 1:5, 1:8, 1:9, 1:10, 1:11, 1:12, 1:15, 1:20, etc.

[0022] Preferably, the metal salt includes at least one of CaCl2, SrCl2, CuCl2, and ZnCl2, and the ratio of the mass of the metal salt to the mass of the hydrogel is 1:10-150, more preferably 1:25-100, including but not limited to 1:35, 1:37, 1:40, 1:42, 1:45, 1:50, 1:100, etc. Calcium, strontium, copper, and zinc ions can further promote the repair and reconstruction of tissues (such as bone tissue).

[0023] Preferably, the mesoporous material includes at least one of mesoporous silica (MS), mesoporous bioglass, mesoporous calcium silicate, mesoporous zinc silicate, mesoporous strontium silicate and mesoporous magnesium silicate. The silicon, calcium, zinc, strontium and magnesium ions in the mesoporous material can further promote the repair and reconstruction of tissues.

[0024] Preferably, the mesoporous material is a sphere or a nearly sphere, and the particle size of the mesoporous material is 100 nm to 1 μm, more preferably 200 nm to 600 nm.

[0025] Preferably, the pore size of the mesoporous material is 1 to 20 nm, more preferably 1 to 10 nm.

[0026] Preferably, the mesoporous material is wrapped in a degradable polyester material, and the mass ratio of the mesoporous material to the degradable polyester is 1:0.2-20, more preferably 1:0.5-10, including but not limited to 1:0.5, 1:3, 1:6, 1:10, etc.

[0027] Preferably, the particle size of the microspheres is 30 to 500 μm, more preferably 90 to 150 μm.

[0028] Preferably, the degradable polyester includes at least one of polylactic acid-co-glycolic acid (PLGA), polyglycolic acid, polylactic acid (PLA), polyhydroxyalkanoate (PHA), polycaprolactone (PCL), polytrimethylene carbonate, polybutylene succinate, and ε-polylysine.

[0029] In a second aspect of the present invention, the method for preparing the microsphere composite hydrogel comprises the following steps:

[0030] The microspheres are dispersed in the hydrogel to obtain the microsphere composite hydrogel.

[0031] Preferably, the preparation method of the microsphere composite hydrogel comprises the following steps: dissolving and mixing natural protein and natural polysaccharide, adding a cross-linking agent for mixing and cross-linking, and adding microspheres to obtain the microsphere composite hydrogel.

[0032] The cross-linking agent is added to the system in the form of a solution, and the cross-linking agent solution includes a genipin solution and a metal salt solution. The mass concentration of the genipin is 0.1-10%, and more preferably 0.25-0.5%. The volume dosage is determined according to the mass ratio of genipin to the hydrogel (natural protein and natural polysaccharide) in the microsphere composite hydrogel, such as adding about 1 mL of genipin solution to every 100 mg of the natural protein and natural polysaccharide mixture.

[0033] Preferably, the mass concentration of the metal salt solution is 10-20%, and the volume dosage is determined according to the mass ratio of the metal salt to the hydrogel (natural protein and natural polysaccharide) in the microsphere composite hydrogel, such as adding 25 μL of metal salt solution to every 100 mg of natural protein and natural polysaccharide mixture.

[0034] Preferably, the microsphere shell is prepared by one of the following methods: emulsification solvent evaporation method, microfluidics method, phase separation method, salting-out method, spray drying method, membrane emulsification method, and nanoprecipitation method, and the emulsification solvent evaporation method is more preferred.

[0035] Preferably, the microspheres are prepared by a preparation method comprising the following steps: mixing a degradable polyester material and a mesoporous material, adding a surfactant solution, stirring, and separating to obtain the microspheres with the surface coated with the degradable polyester material.

[0036] Preferably, the mass ratio of the degradable polyester material to the mesoporous material is 1 to 10:1, more preferably 5 to 10:1, and further preferably 6 to 7:1.

[0037] Preferably, the preparation method of the microspheres is specifically as follows: dissolving the degradable polyester material and the mesoporous silicon material in dichloromethane to obtain a mixed solution, adding a surfactant, stirring for 12 hours, centrifuging and washing to obtain the microspheres. The solvent of the mixed solution includes at least one of dichloromethane and ethanol.

[0038] Preferably, the washed microspheres can be further freeze-dried and stored at low temperature.

[0039] Preferably, the surfactant includes at least one of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), gelatin, and methyl cellulose.

[0040] Preferably, the mesoporous silicon used in the present invention can be a commercially available product or homemade, and more preferably the mesoporous silicon is prepared by a template method; specifically, dodecylamine and tetraethyl orthosilicate are dissolved in anhydrous ethanol / water solution, mixed and reacted to obtain mesoporous silicon (MS).

[0041] Preferably, the mass volume ratio of dodecylamine to tetraethyl orthosilicate is 1 g: 3 to 5 mL, more preferably 1 g: 4 to 4.5 mL.

[0042] Preferably, the preparation method of the mesoporous silicon is specifically, mixing a dodecylamine solution with a tetraethyl orthosilicate solution, stirring, aging, extracting, drying and calcining to obtain the mesoporous silicon; the stirring time is 15 to 20 hours, more preferably about 18 hours; the aging time is 20 to 50 minutes, more preferably about 30 minutes; the solvent used for the extraction is a hydrochloric acid ethanol solution, and the extraction time is 3 to 6 hours, more preferably about 4 hours; the drying temperature is 70 to 100°C, more preferably about 80°C, and the drying time is 4 to 16 hours, more preferably about 12 hours; the calcination temperature is 400 to 800°C, more preferably 600 to 700°C, and the calcination time is 2 to 4 hours, more preferably 2 to 3 hours.

[0043] Preferably, the mass volume ratio of dodecylamine to solvent in the dodecylamine solution is 1g:1-2mL, more preferably 1g:1-1.5mL; the solvent of the dodecylamine solution includes ethanol and water, and the mass ratio of ethanol to water is 1:1-1.3, more preferably 1:1-1.15.

[0044] Preferably, the volume ratio of tetraethyl orthosilicate to solvent in the tetraethyl orthosilicate solution is 1 to 1.5: 1, more preferably 1 to 1.2: 1. The solvent of the tetraethyl orthosilicate solution includes at least one of ethanol and water.

[0045] Preferably, the source of the silk fibroin is not limited, and a commercial product can be selected or it can be obtained by self-production; more preferably, the silk fibroin is prepared by a preparation method comprising the following steps:

[0046] Step S11, placing the silkworm cocoons from which the pupae have been removed in a sodium carbonate solution, and performing heating degumming twice;

[0047] Step S12, mixing the lithium bromide solution with the degummed silkworm cocoons, and filtering to obtain the silk fibroin.

[0048] Preferably, in step S11, the mass volume ratio of the silk cocoons from which the silkworm pupae are removed to the sodium carbonate is 1 g:40-60 mL; more preferably, it is about 1 g:50 mL.

[0049] Preferably, in step S11, the concentration of the sodium carbonate solution used in the first heating degumming is 1.0-2.0wt%, more preferably about 1.0wt%; the concentration of the sodium carbonate solution used in the second heating degumming is 0.5-0.9wt%, more preferably about 0.5wt%.

[0050] Preferably, in step S11, the first heating degumming step further includes washing, washing with water for 5 to 7 times, kneading during the washing process, and then performing a second heating degumming after the washing is completed.

[0051] Preferably, in step S11, the temperatures of the two heating degumming processes are independently selected from 80 to 110° C., more preferably 90 to 100° C.; the heating time is 20 to 50 min, more preferably about 30 min.

[0052] Preferably, step S11 further involves drying the cocoons obtained after the two degumming processes, with the drying temperature being 50-70°C, more preferably about 60°C.

[0053] Preferably, in step S12, the mass ratio of lithium bromide to degummed silkworm cocoons is 5 to 10:1, more preferably 8 to 10:1.

[0054] Preferably, the filtration in step S12 uses 8 to 10 layers of filter cloth, and after filtration, it also includes centrifugation, dialysis, and freeze-drying treatment. The centrifugal speed is 6000 to 7000 rpm, more preferably 6700 rpm; the centrifugal time is 5 to 20 minutes, more preferably about 10 minutes; the dialysis bag used for dialysis has a molecular weight cutoff of 7000 to 9000 Da, more preferably 8000 Da, and the dialysis time is 2 to 4 days, more preferably about 3 days.

[0055] The third aspect of the present invention provides a drug-loaded microsphere composite hydrogel, which comprises the microsphere composite hydrogel and an active drug loaded in the microsphere.

[0056] In the present invention, the microspheres in the microsphere composite hydrogel include a degradable polyester material and a mesoporous material wrapped in the degradable polyester. The mesoporous material contains a rich mesoporous structure and a high specific surface area, and can be used as a carrier of active drugs, further loading active drugs, and can prolong the release of drugs; the degradable polyester material can protect the drugs in the mesoporous structure from being destroyed or inactivated, and can improve the sustained release effect; the hydrogel can further prolong the release process of the drug. The drug-loaded microsphere composite hydrogel prepared by the present invention has good biocompatibility, mechanical properties and bone mineralization ability, and also has a good drug sustained release effect.

[0057] Preferably, the drug loading amount of the drug-loaded microsphere composite hydrogel is 0.00001-10%, more preferably 0.1-5%, and further preferably about 1.5%.

[0058] Preferably, there is no limitation on the active drug, and different active drugs can be selected according to the desired therapeutic effect, including but not limited to bone repair promoting factors, anti-infection / anti-tumor drugs, etc.

[0059] In a fourth aspect of the present invention, the method for preparing the drug-loaded microsphere composite hydrogel comprises the following steps:

[0060] S31, mixing the mesoporous material with the active drug to obtain a drug-loaded mesoporous material;

[0061] S32, mixing the drug-loaded mesoporous material and the degradable polyester material, adding a surfactant solution, stirring, and separating to obtain the drug-loaded microspheres;

[0062] S33, dispersing the drug-loaded microspheres in the hydrogel to obtain the drug-loaded microsphere composite hydrogel.

[0063] Preferably, in step S31, the mass of the active drug is 0.0001 to 50% of the mass of the mesoporous material, more preferably 0.1 to 30%, and further preferably about 20%.

[0064] In the present invention, the main difference between the preparation method of the drug-loaded microsphere composite hydrogel and the microsphere composite hydrogel is that the mesoporous material is first loaded with active drugs before coating the degradable polyester material, and the other experimental processes are similar.

[0065] A fifth aspect of the present invention is the use of the microsphere composite hydrogel and the drug-loaded microsphere composite hydrogel in the preparation of bone implant or bone repair materials, anti-infection and anti-tumor drugs.

[0066] Compared with the prior art, the present invention has at least the following beneficial effects:

[0067] The microsphere composite hydrogel disclosed in the present invention has good biocompatibility, strong mechanical properties, certain bone mineralization and biological activity, and the microspheres include a degradable polyester material and a mesoporous material wrapped in the degradable polyester material. The mesoporous material contains a rich mesoporous structure and can be loaded with active drugs (such as bone repair promoting factors, anti-infection / anti-tumor drugs, etc.), so as to achieve effective sustained release of drugs at the defect site, thereby having the effects of accelerating bone healing / anti-infection / anti-tumor, and is a hydrogel system suitable for repairing bone defects caused by different causes. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0069] Figure 1 Characterization of the MS / PLGA microspheres of Comparative Example 1: (a) SEM image; (b) particle size distribution; (c) energy spectrum scanning.

[0070] Figure 2 Characterization of Comparative Example 2 and Examples 1 to 3: (a) FITR spectrum; (b) XRD pattern; (c) SEM image.

[0071] Figure 3 Mechanical properties of Comparative Example 2 and Examples 1 to 3, and drug release of Comparative Example 3 and Example 4:

[0072] (a) Stress-strain curve; (b) stress at 60% strain; (c) compressive elastic modulus between 5% and 15% strain;

[0073] (d) Cumulative release of rhodamine.

[0074] Figure 4 Performance of Comparative Example 2 and Examples 1 to 3 in buffer: (a) swelling ratio, (b) saturated swelling ratio, (c) porosity, (d) degradation ratio in the absence of enzymes, and (e) degradation ratio in the presence of enzymes.

[0075] Figure 5 Evaluation of cell compatibility of Comparative Example 2 and Examples 1 to 3: (a) live-dead staining assay; (b) cell proliferation assay.

[0076] Figure 6 Osteogenesis performance of Comparative Example 2 and Examples 1 to 3: (a) ALP staining and ARS staining; (b) expression of osteogenesis-related genes on day 7; (c) expression of osteogenesis-related genes on day 14. DETAILED DESCRIPTION

[0077] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0078] The raw materials used in the following examples, unless otherwise specified, can be obtained from conventional commercial sources; the processes used, unless otherwise specified, are conventional processes in the art; the operating temperatures used, unless otherwise specified, are room temperature (20±5° C.).

[0079] Example 1

[0080] This example prepared a SF / CMCS / SA composite hydrogel containing 0.5% MS / PLGA microspheres.

[0081] 1. Synthesis of MS / PLGA microspheres

[0082] Preparation of MS: Solution A was obtained by dissolving 10 g of dodecylamine (DDA) in 70 mL of anhydrous ethanol and 80 mL of distilled water at room temperature, ultrasonically treating for a few minutes and then magnetically stirring for 1 h. Solution B was prepared by mixing 44.6 mL of hot tetraethyl orthosilicate (TEOS) and 40 ml of anhydrous ethanol under magnetic stirring at room temperature for about 30 min. The two solutions were then stirred and mixed at room temperature for about 18 h. The mixture was then aged for 30 min, washed with 800 mL of distilled water, extracted with 600 mL of HCl-ethanol for 4 h, then dried at 80 ° C for 12 h, and finally calcined at 600 ° C for 3 h to obtain MS powder.

[0083] MS / PLGA microspheres were prepared by an improved single emulsion solvent evaporation method. 1 g PLGA and 150 mg MS powder were dissolved in 8 mL dichloromethane, and then ultrasonicated for several minutes before adding 1.0% PVA aqueous solution and mechanically stirred for 12 h. The obtained MS / PLGA microspheres were washed 5 times with deionized water and finally freeze-dried and stored at low temperature for later use.

[0084] 2. Extraction of SF

[0085] A certain amount of silk cocoons from which the pupae have been removed are placed in a 1.0wt% anhydrous sodium carbonate solution (50mL of anhydrous sodium carbonate solution is required for 1g of silk cocoons), boiled at 95°C for 30min, washed with deionized water 7 times after boiling, rubbed during the washing process, replaced with a 0.5wt% anhydrous sodium carbonate solution and boiled again under the same conditions, and the above operation is repeated. After degumming is completed, the silk cocoons are dried in a 60°C oven and sealed for storage.

[0086] Take 40g of lithium bromide to make a 50mL solution, add 5g of degummed silkworm cocoons, and place in a shaker until completely dissolved. Filter the solution through 8 layers of gauze and centrifuge at 6700rpm for 10min. After centrifugation, transfer to a dialysis bag (8000Da) and dialyze for 3 days, and finally freeze-dry for use.

[0087] 3. Preparation of SF-CMCS-SA hydrogel loaded with 0.5% MS / PLGA microspheres

[0088] Take 120mg SF and place it in 2.925mL deionized water and stir to dissolve. After complete dissolution, add 120mg CMCS and stir to dissolve. After dissolution, add 60mg SA and stir to dissolve. After dissolution, add 3mL 1% genipin solution and 75μL 10% CaCl2 solution, stir and mix, then remove the magnetic stirrer, and then add MS / PLGA microspheres to the prepared solution at a ratio of 0.5%, and finally place it in a mold and solidify it at 37℃ for 30min. After solidification, freeze it in a refrigerator and store it after freeze-drying. The final concentrations of SF, CMCS, SA, microspheres, genipin and CaCl2 are 2%, 2%, 1%, 0.5%, 0.5% and 0.125% respectively.

[0089] Example 2

[0090] This embodiment is a method for preparing SF / CMCS / SA composite gel containing 1% MS / PLGA microspheres, comprising the following steps:

[0091] After synthesizing MS / PLGA microspheres and extracting SF according to the method of Example 1, SF, SA, CMCS, and microspheres were mixed at a final concentration ratio of 2%, 1%, 2%, and 1%, and then genipin solution and CaCl2 solution were added after stirring evenly, and then placed in a mold, cured at 37° C. for 30 minutes, frozen in a refrigerator after curing, and stored after freeze-drying. The final concentrations of genipin solution and CaCl2 solution were 0.25% and 0.2%.

[0092] Example 3

[0093] This embodiment is a method for preparing SF / CMCS / SA composite gel containing 2% MS / PLGA microspheres, comprising the following steps:

[0094] After synthesizing MS / PLGA microspheres and extracting SF according to the method of Example 1, SF, SA, CMCS, and microspheres were mixed at a final concentration ratio of 2%, 1%, 2%, and 2%, and then genipin solution and CaCl2 solution were added after stirring evenly, and then placed in a mold, cured at 37° C. for 30 minutes, and frozen in a refrigerator after curing, and stored after freeze-drying. The final concentrations of genipin solution and CaCl2 solution were 0.25% and 0.2%.

[0095] Example 4

[0096] This example uses rhodamine B as a small molecule drug model and uses 1% microsphere composite hydrogel loaded with rhodamine B in Example 2 as an example for illustration. The specific process is as follows:

[0097] 1. Drug-loaded MS / PLGA microspheres: 1 g of MS (prepared in Example 1) was placed in a 200 mg rhodamine solution, centrifuged to remove the supernatant, and dried to obtain drug-loaded MS powder; PLGA and drug-loaded MS powder were dissolved in dichloromethane to obtain solution A, and PVA solution was prepared to obtain solution B, and then A and B were mixed and mechanically stirred for 12 hours. Then, the microspheres were washed with deionized water, and finally freeze-dried and stored at low temperature for future use, to obtain microspheres with a drug loading of 1.5%.

[0098] 2.1% drug-loaded MS / PLGA composite hydrogel: SF was extracted according to the method of Example 1, and SF, SA, and CMCS solutions were mixed at a final concentration ratio of 2%, 1%, and 2%. After mixing evenly, the drug-loaded MS / PLGA microspheres were added to the prepared solution, and after stirring evenly, genipin solution and CaCl2 solution were added, and then placed in a mold, and solidified at 37°C for 30 minutes. After solidification, it was frozen in a refrigerator and stored after freeze-drying. The final concentrations of genipin solution and CaCl2 solution were 0.25% and 0.2%.

[0099] Example 5

[0100] This example prepares a SF / CMCS / SA composite hydrogel containing 1% mesoporous bioglass / PLGA microspheres, and the specific process is as follows:

[0101] SF, SA, CMCS solutions were mixed at a final concentration ratio of 2%, 1%, and 2%, and after mixing evenly, 1% of mesoporous bioglass / polylactic acid microspheres were added to the prepared solution, and after stirring evenly, genipin solution and SrCl2 solution were added, and then placed in a mold, cured at 45°C for 10 minutes, and frozen in a refrigerator after curing, and stored after freeze-drying. The final concentrations of genipin solution and SrCl2 solution were 0.25% and 0.2%.

[0102] Example 6

[0103] This example prepares a fibrin / chitosan composite hydrogel containing 1% mesoporous calcium silicate / PLGA microspheres, and the specific process is as follows:

[0104] Fibrin and chitosan were mixed at a final concentration ratio of 1% and 4%, and after mixing evenly, 1% mesoporous calcium silicate / PLGA microspheres were added to the prepared solution, and after stirring evenly, genipin and ZnCl2 solutions were added, and then placed in a mold, and solidified at 45°C for 10 minutes, and then frozen in a refrigerator after solidification, and stored after freeze-drying. The final concentrations of genipin solution and ZnCl2 solution were 1% and 0.1%.

[0105] Example 7

[0106] In this example, a collagen / SA composite hydrogel containing 1% mesoporous calcium silicate / PLGA microspheres was prepared. The specific process was as follows:

[0107] Collagen and SA were mixed at a final concentration ratio of 1% and 4%, and after mixing evenly, 1% mesoporous calcium silicate / PLGA microspheres were added to the prepared solution, and after stirring evenly, genipin solution and CuCl2 solution were added, and then placed in a mold, and solidified at 45°C for 10 minutes, and then frozen in a refrigerator after solidification, and stored after freeze-drying. The final concentrations of genipin solution and CuCl2 solution were 0.6% and 0.05%.

[0108] Comparative Example 1

[0109] MS / PLGA microspheres were synthesized according to the method of Example 1.

[0110] Comparative Example 2

[0111] This embodiment is a method for preparing a SF / CMCS / SA composite gel without microspheres, comprising the following steps:

[0112] After SF was extracted according to the method of Example 1, SF, SA, and CMCS solutions were mixed at a final concentration ratio of 2%, 1%, and 2%, and after mixing evenly, genipin solution and CaCl2 solution were added, stirred and mixed, and placed in a mold, and solidified at 37° C. for 30 minutes. After solidification, it was frozen in a refrigerator and stored after freeze-drying. The final concentrations of genipin solution and CaCl2 solution were 00.5% and 0.125%.

[0113] Comparative Example 3

[0114] This comparative example is a method for preparing simple drug-loaded microspheres, using rhodamine as a model drug and synthesizing MS / PLGA according to the method of Example 4.

[0115] Test example

[0116] This test example tests the performance of the microsphere composite hydrogel prepared in the embodiment and the comparative example, as well as the drug-loaded composite hydrogel.

[0117] (1) Morphological characterization of microspheres and microsphere composite hydrogels

[0118] The microsphere morphology of comparative example 1 and the composite gel of implementations 1 to 3 were characterized by pasting microsphere (microspheres without drug loading, microspheres loaded) powder and 4 microsphere gels with different solid contents onto the sample stage with conductive glue, spraying them with gold, and observing the microsphere and gel morphology and energy spectrum scanning analysis using a desktop scanning electron microscope.

[0119] The results are as follows Figure 1 As shown, the microspheres prepared in Comparative Example 1 have a good balling effect, and the surface is rough and covered with MS particles. This is caused by the dispersion of particles on the polyester surface during the emulsification process. The uniformity of the microsphere diameter is good, and the size is 113.79±23.87μm. The EDS scanning results show that it contains C, O and Si elements, which are consistent with the raw material elements used in the preparation of the microspheres.

[0120] The microspheres with different contents were loaded into the SF / CMCS / SA gel system, and the morphology of the composite gel was observed. Figure 2 b It was found that the microspheres were evenly distributed in the gel network, and the microspheres were tightly bound to the gel. Although the pores of the composite gel tended to shrink with the increase of the microsphere content, the macroporous structure of the gel was still retained. Its EDS energy spectrum scanning analysis showed that C, N, O, Na, Cl and Ca were all present in the composite gel; since the 0.5%, 1% and 2% groups contained microspheres with different solid contents, the energy spectrum surface scanning results showed that Si element also appeared in the composite gel.

[0121] (2) Infrared scanning of microsphere composite hydrogel

[0122] Infrared scanning of Comparative Example 2 and Examples 1 to 3: The microspheres of Comparative Example 1 with different contents were loaded into SF, CMCS, and SA composite gels, and then freeze-dried and tested using the ATR-FTIR mode of the Fourier transform infrared spectrometer. The specific parameters were set to 600-4000cm -1 Range test, resolution is 4cm -1 , and scanned 32 times cumulatively to finally obtain the infrared absorption spectrum.

[0123] The results are as follows Figure 2 As shown in a, the infrared spectrum shows that 3283cm -1 The stretching vibration peaks of -OH are at 1644, 1529 and 1231 cm -1The characteristic absorption bands of amide I band, amide II band and amide III band on SF can be observed at 1024, 1308, 1419 and 1616 cm-1, which are related to the β-sheet conformation of SF. Among them, amide I band is mainly C=O stretching vibration and CN stretching vibration, amide II band is mainly NH in-plane bending vibration and CN stretching vibration, and amide III band is mainly C-N stretching vibration and N-H in-plane bending vibration. -1 The stretching vibrations of the primary alcohol CO bond, CN stretching vibration, symmetric stretching vibration of -COO and asymmetric stretching vibration of -COO are at 1419 and 1616 cm -1 The symmetric stretching vibration and asymmetric stretching vibration peaks of -COO of SA are also at the positions. The infrared energy of microsphere gels with different solid contents corresponds to the characteristic absorption peaks of SF, CMCS and SA.

[0124] (3) X-ray diffraction (XRD) characterization of microsphere composite hydrogel

[0125] X-ray diffraction (XRD) characterization of Comparative Example 2 and Examples 1 to 3: After freeze-drying, the prepared gel was tested with an X-ray diffractometer, CuKα radiation, X-ray wavelength: The ultra-powerful detection counter records the diffraction intensity curve between 2θ=10° and 60°, with a scanning speed of 2° / min. Finally, the XRD spectrum of the sample is obtained. The results are as follows Figure 2 As shown in Figure c, the XRD characteristic diffraction peaks of the gels containing different amounts of MS / PLGA microspheres are roughly the same, all around 20°, which is consistent with the XRD characteristic peaks of CMCS and SF. Since CaCl2 solution is added for cross-linking and curing, its characteristic main peak appears in the 0% group (Comparative Example 2). This indicates that the SF / CMCS / SA gels with different solid contents of MS / PLGA microspheres have not changed their respective secondary structures.

[0126] (4) Mechanical properties analysis of microsphere composite hydrogel

[0127] Mechanical properties analysis of Comparative Example 2 and Examples 1 to 3: The gel was immersed in PBS buffer solution, and after reaching swelling equilibrium, a compression test was performed on a mechanical testing machine to test the mechanical properties of four groups of scaffold materials. The scaffold diameter is about 6 to 7 mm and the height is 4 to 6 mm. The test setting is 2 mm / min. The stress (σ) at 5% to 15% strain is calculated according to Formula 1:

[0128] σ=F / S×100 Formula 1

[0129] F and S represent the loading area and compression area, respectively.

[0130] The results are as follows Figure 3As shown in a to c, the stress-strain curves of microsphere composite gels with different solid contents, the average compressive strength at 60% strain, and the average compression modulus are respectively shown. With the increase of the solid content of the microspheres, the mechanical properties of the composite gel gradually increase and then weaken, which may be related to the sedimentation and uneven distribution of the microspheres. Compared with other groups, the 1% microsphere composite hydrogel (Example 2) exhibits the strongest mechanical properties. The average compressive strength and elastic modulus of Example 2 at 60% strain are 415.24±3.72kPa and 13.13±1.38kPa, respectively, which are much higher than those of Comparative Example 1 (179.36±13.47kPa, 1.48±0.61kPa).

[0131] (5) Drug release properties of microsphere composite hydrogel

[0132] Drug release of Example 4 and Comparative Example 3: Using rhodamine B as a small molecule drug model, the drug-loaded microspheres (Comparative Example 3) and 1% drug-loaded microsphere composite gel (Example 4) were tested for in vitro drug release performance. The drug-loaded microspheres and 1% drug-loaded microsphere composite gel were placed in 10mL centrifuge tubes, fixed to 5mL with PBS buffer, and placed in a 37°C shaker with slight shaking for in vitro release testing. 1.5mL of supernatant was taken at a predetermined time, filtered, and the rhodamine B concentration was detected by UV-visible spectrophotometry. 1.5mL of fresh PBS solution was immediately added after each sampling, and then the cumulative release rate was calculated.

[0133] The results are as follows Figure 3 As shown in Figure d, no sudden drug release occurred in the early stage of Comparative Example 3 and Example 4, indicating that the microspheres have a good drug sustained release effect. As the microspheres degrade, the drug in Comparative Example 3 begins to be slowly released at 7 days, while the release amount of the microsphere / gel composite material of Example 4 before 7 days is higher than that of Comparative Example 3. This is because the microspheres released part of the drug during the preparation of the microsphere composite gel. After 14 days, the amount of drug released by the microspheres increased greatly, while the release of the microsphere composite gel was relatively slow, which is inseparable from the swelling and degradation properties of the gel itself. The drug in the microspheres located in the shallow network of the gel is released first, and the gel can form a coating layer on the surface of the microspheres, which is conducive to improving its controlled release performance. The drug release time of each group of materials reached more than 2 months, indicating that the constructed microspheres and gels have good controlled release and sustained release properties.

[0134] (6) Swelling properties, porosity and degradation properties of microsphere composite hydrogels

[0135] Swelling performance, porosity and degradation performance of Comparative Example 2 and Examples 1 to 3: Soak the freeze-dried material (weighed and recorded as W1) in a 10ml EP tube, add 5ml PBS solution (pH=7.4), and place it in a shaker at 37°C and 90rpm until the gel swelling reaches equilibrium. During this period, use filter paper to absorb excess water on the surface every 2 hours, weigh the gel and record it as W2, measure until the weight of the swollen gel no longer changes, and record the weight of the completely swollen gel. Then calculate the swelling rate SI according to the formula.

[0136] SI= (W2 - W1) / W1*100% Formula 2

[0137] The freeze-dried microsphere gels with different solid contents were immersed in anhydrous ethanol until saturated and no bubbles came out. The gels were weighed before (W3) and after (W4) immersion in anhydrous ethanol. The porosity P was calculated using the following formula:

[0138] P=(W4-W3) / ρ 乙醇 V 凝胶 *100% Formula 3

[0139] Where W3 and W4 represent the weight of the sample before and after immersion in alcohol, respectively, and V 凝胶 is the volume of the gel, ρ 乙醇 is a constant (the density of anhydrous ethanol at room temperature).

[0140] The degradation behavior of the scaffold material was tested in PBS (volume ratio 1:100) with or without type XIV protease (2U / mL) in a shaker at 37°C and 90rpm. The initial weight of the dried sample was measured (W5). The sample was washed with water, freeze-dried and weighed at specified time intervals (W6). The degradation rate was calculated according to the formula.

[0141] Degradation rate (%) = (W5–W6) / W5*100% Formula 4

[0142] The results are as follows Figure 4 As shown in a-b, the swelling properties of the composite gel. With the increase of time, the four groups of composite gels all showed the fastest water absorption within 0.h, and the water absorption was still increasing after 0.5h, but the water absorption rate was very small, slowly tending to stabilize, and reaching the equilibrium swelling level after 24 hours. With the increase of the solid content of the microspheres, the equilibrium swelling of the microsphere composite gel showed a downward trend, and the water absorption decreased. The equilibrium swelling rates of the 0% (Comparative Example 1), 0.5% (Example 1), 1% (Example 2) and 2% (Example 3) groups were 1573.56±2.85%, 1272.99±4.05%, 1116.02±27.25 and 952.58±42.32%, respectively. Figure 4c shows the porosity of each group of composite gels. The porosity of all microsphere gels exceeds 80%, which indicates that the composite gel has good porosity. As the solid content of microspheres increases, the porosity decreases, indicating that the more solid content of microspheres, the tighter the three-dimensional network structure formed. Figure 4 d and Figure 4 e shows the degradation performance of each group of composite gels under enzyme and no enzyme conditions. With the increase of microsphere solid content, the degradation rate of each group of composite gels showed a downward trend, but the degradation rate was roughly the same, first fast and then stable. The degradation rate of composite gel under enzyme conditions was significantly faster than that under no enzyme conditions. However, after 28 days, about half of each group of composite gels remained, indicating that the composite gel has good degradability.

[0143] (7) Cytotoxicity test and live-dead staining of microsphere composite hydrogel

[0144] Cytotoxicity experiment: First, prepare the extract of microsphere composite gel, mix the extract with complete culture medium in a ratio of 1:1, mix with cells to form a cell suspension, and place it in the wells of a 48-well culture plate, with 20,000 cells per well, and replace the culture medium every 2 days. CCK-8 method was used to detect cell proliferation on the 1st, 3rd, and 7th day of culture.

[0145] Cell live / dead staining experiment: The cells were cultured in the well plate for 1 day and 7 days respectively, and then the live / dead cell staining kit was used to detect the activity of bone marrow mesenchymal stem cells in microsphere gels with different solid contents

[0146] The results are as follows Figure 5 As shown, the CCK-8 results show that each group of microsphere / gel materials has good cell compatibility, and the cell activity of Example 3 is slightly lower than that of Comparative Example 2. The live and dead cell staining results show that the cells of Comparative Example 2 and Examples 1 to 2 have good activity and can proliferate well, while the number of cells in Example 3 is less than that in Comparative Example 2, which is consistent with the CCK-8 results of the cells.

[0147] (8) Evaluation of the osteogenic properties of microsphere composite hydrogel

[0148] The osteogenic differentiation performance of Comparative Example 2 and Examples 1 to 3 was characterized: first, an extract of the microsphere composite gel was prepared, the extract was mixed with complete culture medium in a 1:1 ratio, mixed with cells to form a cell suspension, and placed in a 48-well culture for osteogenic performance evaluation.

[0149] Alkaline phosphatase (ALP) staining experiment: Microsphere gels with different solid contents were placed in the wells of a 24-well culture plate, with 2.5×10 4Rat BMSCs. After culturing for 24 hours, the cell culture medium was replaced with osteogenic differentiation medium, and cultured for 7 days and 14 days, respectively, with the medium replaced every 2 days. After terminating the culture, the gel samples were washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde fixative, and then stained with the ALP staining kit according to the instructions and photographed for observation.

[0150] Alizarin red (ARS) staining experiment: Microsphere gels with different solid contents were placed in the wells of a 24-well culture plate, with 2.5×10 4 Rat BMSCs. After 24 hours of culture, the cell culture medium was replaced with osteogenic differentiation medium, and the osteogenic differentiation medium was replaced every 2 days. After 7 days and 21 days of culture, the original culture medium was discarded, and the cells were rinsed twice with PBS buffer. The cells in each group were fixed with Alizarin Red staining fixative. After washing with PBS, Alizarin Red staining solution was added to each well to cover the cells, and the cells were incubated and stained for 30 minutes at room temperature. After the staining solution was washed with deionized water, the cells were photographed under an inverted microscope to observe the amount of calcium nodules formed.

[0151] Quantitative real-time polymerase chain reaction: Microsphere gels with different solid contents were placed in the wells of a 24-well culture plate, with 2.5×10 4 Rat BMSCs. After culturing for 24 hours, the cell culture medium was replaced with osteogenic differentiation medium, and the medium was replaced every 2 days. The cells in the well plate were collected on the 7th and 14th days, and the total RNA of each group of cells was extracted. The cDNA of the reverse transcription reaction product was used as a template, and the osteogenesis-related genes OPN and RunX2 were detected by real-time quantitative RT-PCR.

[0152] Figure 6 a shows that Examples 1 to 3 all have good osteogenic properties. From ALP staining, it can be found that each group of composite gels exhibits certain osteogenic properties, among which the 1% (Example 2) and 2% groups (Example 3) are significantly stronger than the 0% (Comparative Example 2) and 0.5% groups (Example 1). From the ARS staining results, it can be seen that with the increase of the solid content of the microspheres, more red-stained calcium nodules can be observed under the microscope, which indicates that the osteogenic differentiation performance is also significantly enhanced.

[0153] Figure 6 b and Figure 6 c shows the expression of OPN and RunX2 genes at 7 days and 14 days, respectively. OPN and RunX-2 are osteogenesis-related genes. It can be seen from the figure that the expression of OPN and RunX2 genes at 14 days of culture is significantly higher than that at 7 days of culture, and the expression of osteogenic genes in the 1% and 2% groups is significantly higher than that in the 0% and 0.5% groups. In summary, the composite gel of 1% MS / PLGA microspheres in Example 2 has the best osteogenesis performance.

[0154] 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A microsphere composite hydrogel, characterized in that: The microsphere composite hydrogel includes microspheres and hydrogel, wherein the microspheres are dispersed in the hydrogel; the microspheres include a degradable polyester material and a mesoporous material wrapped in the degradable polyester material; the hydrogel includes a combination of silk fibroin, carboxymethyl chitosan, and sodium alginate; the hydrogel also includes a cross-linking agent; the cross-linking agent includes at least one of genipin and a metal salt; the mesoporous material includes at least one of mesoporous silicon, mesoporous bioglass, mesoporous calcium silicate, mesoporous zinc silicate, mesoporous strontium silicate, and mesoporous magnesium silicate; the degradable polyester material includes a polylactic acid-glycolic acid copolymer.

2. The microsphere composite hydrogel according to claim 1, characterized in that: The mass ratio of the silk fibroin, carboxymethyl chitosan and sodium alginate is 1:0.1-5:0.1-5.

3. The method for preparing the microsphere composite hydrogel according to any one of claims 1 to 2, characterized in that: The steps include: The microspheres are dispersed in the hydrogel to obtain the microsphere composite hydrogel.

4. A drug-loaded microsphere composite hydrogel, characterized in that: The drug-loaded microsphere composite hydrogel comprises the microsphere composite hydrogel according to any one of claims 1 to 2 and an active drug loaded in the microsphere.

5. The method for preparing the drug-loaded microsphere composite hydrogel according to claim 4, characterized in that: The steps include: S31, mixing the mesoporous material with the active drug to obtain a drug-loaded mesoporous material; S32, mixing the drug-loaded mesoporous material and the degradable polyester material, adding a surfactant solution, stirring, and separating to obtain the drug-loaded microspheres; S33, dispersing the drug-loaded microspheres in the hydrogel to obtain the drug-loaded microsphere composite hydrogel.

6. Use of the microsphere composite hydrogel according to any one of claims 1 to 2 and the drug-loaded microsphere composite hydrogel according to claim 4 in the preparation of bone implant or bone repair materials, anti-infection and anti-tumor drugs.

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