CN115947957B

CN115947957B - A microsphere composite hydrogel and its preparation method and application

Info

Publication number: CN115947957B
Application number: CN202211589623.8A
Authority: CN
Inventors: 吴婷婷; 许为康; 施奕万; 王兆振; 高博韬; 郭瑞
Original assignee: Institute of Biological and Medical Engineering of Guangdong Academy of Sciences
Current assignee: Institute of Biological and Medical Engineering of Guangdong Academy of Sciences
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2024-12-10
Anticipated expiration: 2042-12-12
Also published as: CN115947957A

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

Microsphere composite hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the field of medical materials, and particularly relates to microsphere composite hydrogel and a preparation method and application thereof.

Background

In recent years, the number of bone defect patients caused by wounds, tumors, infections and the like is obviously increased, and bone defects larger than a critical size are difficult to repair by themselves, and the global bone grafting operation related to the treatment of the bone defects exceeds 200 ten thousand times per year, so that the bone grafting operation is the second most common tissue grafting after blood transfusion. Autologous bone grafting is considered the most desirable option for bone defect surgery, but has limitations in terms of donor source and donor site morbidity. Accordingly, bone tissue engineering is attracting attention of researchers. The ideal bone defect repair biological material has biodegradability, biocompatibility and good mechanical property, and can mediate the reaction of bone repair cells. The natural polymer material is combined with a better drug carrier model, so that the bone repair cell can be supported, and simultaneously, the controlled release drug can continuously induce the stem cell to osteoblast differentiation, regulate and control vascular cells to form new blood vessels, thereby being beneficial to bone repair and being a research and development hot spot of the bone repair biological material.

Compared with the synthetic polymer material, the polymer material constructed by natural proteins and polysaccharides has better biocompatibility. Among them, silk fibroin (silk fibroin, SF) is a natural protein derived from silkworm cocoons, has good biocompatibility, biodegradability and easy extraction, and thus SF is considered as a good tissue regeneration biomaterial. However, weak mechanical properties, poor osteoinductive and osteoconductive properties limit the application of SF in bone tissue engineering. Therefore, SF is usually combined with other biological materials such as chitosan, inorganic particles and the like to construct a composite material, so that the mechanical property, the antibacterial property and the biological property of the composite material are enhanced. Carboxymethyl chitosan (carboxymethyl chitosan, CMCS) is a derivative of chitosan. Compared with chitosan, CMCS has higher water solubility, can chelate with more Ca ²⁺, has excellent bone mineralization activity, has certain antibacterial property, reduces the infection risk after artificial bone grafting, promotes the repair of surgical wounds in clinical practical application, but has higher degradation rate. Sodium alginate (sodium alginate, SA) is an anionic copolymer natural polysaccharide derived from brown seaweed, has good biocompatibility, can promote bone tissue to grow in along a scaffold material, has proper biodegradability and viscosity, and is widely applied to tissue engineering. The natural protein and the polysaccharide are combined, so that the mechanical property of the composite material can be improved, meanwhile, the composite material is endowed with a certain antibacterial property, and the degradation rate of the scaffold material can be regulated and controlled to be suitable for the osteogenesis rate. Although a certain electrostatic interaction exists between amino groups in proteins and carboxyl groups, hydroxyl groups and other groups in polysaccharides, different proteins and polypeptides are mixed and formed, and a cross-linking agent is still required to be added to solidify the proteins and polypeptides into a complex network structure.

Microspheres have long been widely used for drug delivery with their excellent controlled release capability. Among them, degradable polyester microsphere-based composites are receiving a great deal of attention. Among them, PLGA is a copolymer of PGA and PLA, and has a wide range of control of degradation time, and thus is widely used for drug release. However, the hydrophilic property is poor, and the degradation products are acidic, so that the biological activity is poor, and serious local inflammation is easily caused, which prevents the application of the degradable polyester biological material in bone tissue engineering to a certain extent. Mesoporous hydroxyapatite, MS, MBG and mesoporous silicate particles have good biocompatibility and bioactivity. The MS has a mesoporous structure and a high specific surface area, can be used as a carrier for loading drugs and molecules through surface modification, has a drug slow release function, and is a good drug carrier. It is still of great significance how to prepare bone repair materials with excellent comprehensive properties such as biocompatibility, bioactivity, mechanical properties, bone mineralization promotion and the like.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the microsphere composite hydrogel provided by the invention has good biocompatibility and strong mechanical property, and can promote bone mineralization and accelerate bone defect repair.

The invention also provides a preparation method of the microsphere composite hydrogel.

The invention also provides a preparation method and application of the drug-loaded microsphere composite hydrogel material containing the microsphere composite hydrogel.

In a first aspect of the invention, a microsphere composite hydrogel is provided, wherein the microsphere composite hydrogel comprises microspheres and hydrogel, the microspheres are dispersed in the hydrogel, and the microspheres comprise degradable polyester materials and mesoporous materials wrapped in the degradable polyester materials.

Compared with the prior art, the mesoporous material adopted by the invention has rich mesoporous structure and high specific surface area, can be used as a carrier of medicines and molecules, has the capability of forming mineral calcium phosphate similar to the surface of bones, can stimulate bone mesenchymal stem cells to differentiate into bone by a bone morphogenetic protein 2 pathway and an adenylate activating protein kinase pathway so as to promote the formation of new bone, and can effectively improve the biocompatibility and mechanical property of the microspheres and prolong the medicine slow release time. Meanwhile, researches show that the compressive strength of the microsphere obtained by wrapping and dispersing the mesoporous material in the degradable polyester material is obviously higher than that of the pure polyester microsphere, and the microsphere is more suitable for bone repair. Therefore, the microsphere composite hydrogel provided by the invention has good biocompatibility, biodegradability and strong mechanical properties, and simultaneously has good bone mineralization capacity, is beneficial to bone repair, and can further load medicines and molecules to realize slow release of the medicines and the molecules.

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

Preferably, the hydrogel comprises at least one of a natural protein, a natural polysaccharide.

Preferably, the mass ratio of the natural protein to the natural polysaccharide is 1:1-8, more preferably 1:1-6, and even more preferably 1:1.5-4.

Preferably, the natural protein comprises at least one of fibrin, fibrinogen, silk Fibroin (SF), collagen, elastin.

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

Preferably, the hydrogel comprises a combination of Silk Fibroin (SF), carboxymethyl chitosan (CMCS) and Sodium Alginate (SA), wherein the mass ratio of the silk fibroin to the carboxymethyl chitosan to the 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, and the like.

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

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

Preferably, the hydrogel further comprises a cross-linking agent including at least one of a protein cross-linking agent, an ionic cross-linking agent, including but not limited to genipin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, metal salts, and the like. The invention selects the protein cross-linking agent and the ion cross-linking agent with good biocompatibility, which have no cytotoxicity, do not affect the biocompatibility, have high cross-linking degree, and are beneficial to improving the mechanical strength and the stability of the hydrogel.

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% and the like.

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

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.

Preferably, the metal salt comprises at least one of CaCl ₂、SrCl₂、CuCl₂、ZnCl₂, 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, zinc ions can further promote repair and reconstruction of tissue (e.g., bone tissue).

Preferably, the mesoporous material comprises at least one of Mesoporous Silicon (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.

Preferably, the mesoporous material is a sphere or a near sphere, and the particle size of the mesoporous material is 100 nm-1 μm, more preferably 200 nm-600 nm.

Preferably, the pore diameter of the mesoporous material is 1-20 nm, and more preferably 1-10 nm.

Preferably, the mesoporous material is wrapped in the 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.

Preferably, the particle size of the microspheres is 30-500 μm, more preferably 90-150 μm.

Preferably, the degradable polyester comprises at least one of polylactic acid-glycolic acid copolymer (PLGA), polyglycolic acid, polylactic acid (PLA), polyhydroxyalkanoate (PHA), polycaprolactone (PCL), polytrimethylene carbonate, polybutylene succinate, epsilon-polylysine.

In a second aspect of the present invention, the preparation method of the microsphere composite hydrogel comprises the following steps:

Dispersing the microsphere in hydrogel to obtain the microsphere composite hydrogel.

Preferably, the preparation method of the microsphere composite hydrogel comprises the following steps of dissolving and mixing natural proteins and natural polysaccharides, adding a cross-linking agent for mixing and cross-linking, and adding microspheres to obtain the microsphere composite hydrogel.

The cross-linking agent is added into the system in a solution form, the cross-linking agent solution comprises genipin solution and metal salt solution, the mass concentration of the genipin is 0.1-10%, more preferably 0.25-0.5%, the volume dosage is determined according to the mass ratio of the genipin to the hydrogel (natural protein and natural polysaccharide) in the microsphere composite hydrogel, for example, about 1mL of genipin solution is added per 100mg of the natural protein and natural polysaccharide mixture.

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, for example, 25 mu L of the metal salt solution is added per 100mg of the natural protein and natural polysaccharide mixture.

Preferably, the microsphere shell is prepared by one of an emulsifying solvent volatilization method, a microfluidic method, a phase separation method, a salting-out method, a spray drying method, a membrane emulsification method and a nano precipitation method, and more preferably, the emulsifying solvent volatilization method.

Preferably, the microsphere is prepared by a preparation method comprising the following steps of mixing a degradable polyester material and a mesoporous material, adding a surfactant solution, stirring and separating to obtain the microsphere with the surface coated with the degradable polyester material.

Preferably, the mass ratio of the degradable polyester material to the mesoporous material is 1-10:1, more preferably 5-10:1, and even more preferably 6-7:1.

Preferably, the preparation method of the microsphere comprises the steps of dissolving a degradable polyester material and a mesoporous silicon material in dichloromethane to obtain a mixed solution, adding a surfactant, stirring for 12 hours, centrifugally separating and cleaning to obtain the microsphere. The solvent of the mixed solution comprises at least one of dichloromethane and ethanol.

Preferably, the microspheres obtained by washing can be further freeze-dried and stored at low temperature.

Preferably, the surfactant comprises at least one of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), gelatin, methylcellulose.

Preferably, the mesoporous silicon used in the invention can be selected from commercial products or self-made mesoporous silicon, more preferably the mesoporous silicon is prepared by a template method, specifically, dodecylamine and tetraethyl orthosilicate are dissolved in absolute ethanol/water solution and mixed for reaction to obtain Mesoporous Silicon (MS).

Preferably, the mass volume ratio of the dodecylamine to the tetraethyl orthosilicate is 1 g:3-5 ml, more preferably 1 g:4-4.5 ml.

Preferably, the preparation method of the mesoporous silicon comprises the steps of mixing a dodecylamine solution with a tetraethyl orthosilicate solution, stirring, ageing, extracting, drying and calcining to obtain the mesoporous silicon, wherein the stirring time is 15-20 hours, more preferably about 18 hours, the ageing time is 20-50 minutes, more preferably about 30 minutes, the solvent used for extraction is an ethanol hydrochloride solution, the extracting time is 3-6 hours, more preferably about 4 hours, the drying temperature is 70-100 ℃, more preferably about 80 ℃, the drying time is 4-16 hours, more preferably about 12 hours, the calcining temperature is 400-800 ℃, more preferably 600-700 ℃, and the calcining time is 2-4 hours, more preferably 2-3 hours.

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

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

Preferably, the silk fibroin is not limited in source, can be obtained by self-making from commercial products, and is more preferably prepared by a preparation method comprising the following steps:

Step S11, placing the silkworm cocoons from which the silkworm chrysalis are removed in a sodium carbonate solution, and performing heating degumming for two times;

and step S12, mixing the lithium bromide solution with degummed cocoons, and filtering to obtain the silk fibroin.

Preferably, in the step S11, the mass-volume ratio of the silkworm cocoons from which the silkworm chrysalis is removed to the sodium carbonate is 1 g:40-60 mL, and more preferably about 1g:50 mL.

Preferably, in step S11, the concentration of the sodium carbonate solution used for the first heat degumming is about 1.0 to 2.0wt%, more preferably about 1.0wt%, and the concentration of the sodium carbonate solution used for the second heat degumming is about 0.5 to 0.9wt%, more preferably about 0.5 wt%.

Preferably, in step S11, the step of finishing the first heat degumming further includes washing with water for 5-7 times, rubbing during the washing, and performing the second heat degumming after the washing is finished.

Preferably, in step S11, the temperature of the two heating degumming is independently selected from 80-110 ℃, more preferably 90-100 ℃, and the heating time is 20-50 min, more preferably about 30 min.

Preferably, in step S11, the cocoons obtained after the degumming are further dried at a drying temperature of 50-70 ℃, and more preferably about 60 ℃.

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

Preferably, the filtering in the step S12 is performed by using 8-10 layers of filter cloth, and the filtering is followed by centrifugation, dialysis and freeze-drying, wherein the rotation speed of centrifugation is 6000-7000 rpm, more preferably 6700rpm, the centrifugation time is 5-20 min, more preferably about 10min, the molecular weight cut-off of a dialysis bag used for dialysis is 7000-9000 Da, more preferably 8000Da, and the dialysis time is 2-4 days, more preferably about 3 days.

In a third aspect of the present invention, a drug-loaded microsphere composite hydrogel is provided, the drug-loaded microsphere composite hydrogel comprising the microsphere composite hydrogel and an active drug loaded in the microsphere.

The microsphere in the microsphere composite hydrogel comprises a degradable polyester material and a mesoporous material wrapped in the degradable polyester, wherein the mesoporous material contains a rich mesoporous structure, has a high specific surface area, can be used as a carrier of an active drug, further loads the active drug, can prolong the release of the drug, can protect the drug in the mesoporous structure from being damaged or deactivated, can improve the slow release effect, and can further prolong the release process of the drug. The drug-loaded microsphere composite hydrogel prepared by the invention has good biocompatibility, mechanical property and bone mineralization capacity, and also has good drug slow release effect.

Preferably, the drug loading rate of the drug-loaded microsphere composite hydrogel is 0.00001-10%, more preferably 0.1-5%, and even more preferably about 1.5%.

Preferably, the active drug is not limited, and different active drugs can be selected according to the required curative effect, including but not limited to bone repair promoting factors, anti-infective/antitumor drugs, etc.

In a fourth aspect of the present invention, the preparation method of the drug-loaded microsphere composite hydrogel comprises the following steps:

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

s32, mixing the drug-loaded mesoporous material with the degradable polyester material, adding a surfactant solution, stirring and separating to obtain the drug-loaded microspheres;

S33, dispersing the drug-loaded microsphere in the hydrogel to obtain the drug-loaded microsphere composite hydrogel.

Preferably, in step S31, the mass of the active agent is about 0.0001 to 50%, more preferably about 0.1 to 30%, and still more preferably about 20% of the mass of the mesoporous material.

In the invention, the preparation method of the drug-loaded microsphere composite hydrogel and the microsphere composite hydrogel are mainly different in that the mesoporous material is loaded with active drugs before being coated with the degradable polyester material, and other experimental processes are similar.

In a fifth aspect of the invention, the microsphere composite hydrogel and the drug-loaded microsphere composite hydrogel are applied to the preparation of bone implantation or bone repair materials and anti-infection and anti-tumor drugs.

Compared with the prior art, the invention has at least the following beneficial effects:

The microsphere composite hydrogel disclosed by the invention has good biocompatibility, strong mechanical property, certain bone mineralization and biological activity, and the microsphere comprises a degradable polyester material and a mesoporous material wrapped in the degradable polyester material, wherein the mesoporous material contains rich mesoporous structures, can load active drugs (such as bone repair promoting factors, anti-infection/anti-tumor drugs and the like) to realize effective slow release of the drugs at defect parts, and can have the effects of accelerating bone healing/resisting infection/anti-tumor, so that the microsphere composite hydrogel is a hydrogel system suitable for bone defect repair caused by different causes.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

characterization of MS/PLGA microspheres of comparative example 1 FIG. 1 (a) SEM image, (b) particle size distribution, and (c) energy spectrum scan.

FIG. 2 is a representation of comparative example 2 and examples 1-3, showing (a) FITR spectra, (b) XRD spectra, and (c) SEM images.

Fig. 3 mechanical properties of comparative example 2 and examples 1 to 3, and drug release conditions of comparative example 3 and example 4:

A stress-strain curve, (b) a stress at 60% strain, and (c) a compressive elastic modulus between 5% and 15% strain;

(d) Rhodamine accumulates released amounts.

FIG. 4 shows the properties of comparative example 2 and examples 1 to 3 in buffers (a) swelling ratio, (b) saturated swelling ratio, (c) porosity, (d) degradation under enzyme-free conditions and (e) degradation under enzyme conditions.

FIG. 5 shows the evaluation of cell compatibility of comparative example 2 and examples 1 to 3, (a) live-dead staining test, and (b) cell proliferation test.

FIG. 6 shows the osteogenic properties of comparative example 2 and examples 1 to 3, (a) ALP staining and ARS staining, (b) expression of the osteogenic related gene on day 7, and (c) expression of the osteogenic related gene on day 14.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

The starting materials used in the examples below were all commercially available, unless otherwise specified, the processes employed were all conventional in the art, and the operating temperatures employed were all room temperature (20.+ -. 5 ℃ C.) unless otherwise specified.

Example 1

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

1. Synthesis of MS/PLGA microspheres

MS was prepared by dissolving 10g of dodecylamine (DDA) in 70mL of absolute ethanol and 80mL of distilled water at room temperature, sonicating for several minutes and magnetically stirring for 1h to give solution A. Solution B was prepared by mixing 44.6mL of hot tetraethyl orthosilicate (TEOS) and 40mL of absolute ethanol at room temperature for about 30min under magnetic stirring. The two solutions were then stirred and mixed at room temperature for about 18h. The mixture was then aged for 30min, washed with 800mL distilled water, extracted with 600mL HCl-ethanol for 4h, then dried at 80 ℃ for 12h, and finally calcined at 600 ℃ for 3h to give MS powder.

MS/PLGA microspheres were prepared using a modified single emulsion solvent evaporation method, 1g PLGA and 150mg MS powder were dissolved in 8mL dichloromethane and sonicated for several minutes before adding 1.0% PVA in water and mechanically stirring for 12h. And (3) washing the obtained MS/PLGA microspheres with deionized water for 5 times, and finally freeze-drying and preserving at low temperature for later use.

2. Extraction of SF

A certain amount of silkworm cocoons from which silkworm chrysalis are removed are placed in a 1.0wt% anhydrous sodium carbonate solution (wherein 1g of silkworm cocoons are required to be 50mL of anhydrous sodium carbonate solution), boiled at 95 ℃ for 30min, washed with deionized water 7 times after boiling, kneaded during washing, and boiled again under the same conditions with a 0.5wt% anhydrous sodium carbonate solution, and the above operations are repeated. After degumming, the cocoons are put into a 60 ℃ oven for drying, and are stored in a sealed manner.

40G of lithium bromide was prepared as a 50mL solution, and 5g of degummed cocoons were added thereto and placed in a shaker until completely dissolved. The solution was filtered using 8 layers of gauze and centrifuged at 6700rpm for 10min, after centrifugation transferred to dialysis bags (8000 Da) for 3 days, and finally lyophilized for use.

3. Preparation of SF-CMCS-SA hydrogel carrying 0.5% MS/PLGA microspheres

120Mg SF is taken and placed in 2.925mL of deionized water to be stirred and dissolved, and 120mg CMCS is added to be stirred and dissolved after the dissolution is completed. After dissolution, 60mg of SA was added thereto and the mixture was stirred for dissolution. After dissolution, 3mL of 1% genipin solution and 75 mu L of 10% CaCl ₂ solution are added, after stirring and mixing, a magnetic stirrer is taken out, then MS/PLGA microspheres are added into the prepared solution according to the proportion of 0.5%, finally, the solution is placed into a mould, and the mould is placed at 37 ℃ for 30min for solidification, frozen in a refrigerator after solidification, and stored after freeze-drying. Final SF, CMCS, SA, microsphere, genipin and CaCl ₂ were at final concentrations of 2%,2%,1%,0.5%,0.5% and 0.125%, respectively.

Example 2

The embodiment is a preparation method of SF/CMCS/SA composite gel containing 1% MS/PLGA microspheres, which comprises the following steps:

After MS/PLGA microspheres were synthesized and SF was extracted according to the method of example 1, SF, SA, CMCS, microspheres were mixed at a final concentration ratio of 2%,1%, stirred well, added with genipin solution and CaCl ₂ solution, then placed in a mold, cured at 37℃for 30min, frozen in a refrigerator after curing, and stored after lyophilization. The final concentrations of genipin solution and CaCl ₂ solution were 0.25% and 0.2%.

Example 3

The embodiment is a preparation method of SF/CMCS/SA composite gel containing 2% MS/PLGA microspheres, which comprises the following steps:

After MS/PLGA microspheres were synthesized and SF was extracted according to the method of example 1, SF, SA, CMCS, microspheres were mixed at a final concentration ratio of 2%, 1%, 2%, stirred well, added with genipin solution and CaCl ₂ solution, then placed in a mold, cured at 37℃for 30min, frozen in a refrigerator after curing, and stored after lyophilization. The final concentrations of genipin solution and CaCl ₂ solution were 0.25% and 0.2%.

Example 4

In the embodiment, rhodamine B is taken as a micromolecular drug model, and 1% microsphere composite hydrogel loaded rhodamine B in the embodiment 2 is taken as an example for illustration, and the specific process is as follows:

1. Drug-loaded MS/PLGA microspheres 1g of MS (prepared in example 1) was placed in 200mg of rhodamine solution, the supernatant was removed by centrifugation, and the drug-loaded MS powder was obtained after drying, PLGA and drug-loaded MS powder were dissolved in methylene chloride to obtain solution A, PVA solution was prepared to obtain solution B, and then the solution A and solution B were mixed and mechanically stirred for 12 hours. And then washing by deionized water, and finally freeze-drying and preserving at low temperature for standby to obtain the microsphere with the drug loading rate of 1.5%.

2.1% Drug-loaded MS/PLGA composite hydrogel, extracting SF according to the method of example 1, mixing SF, SA, CMCS solutions according to the final concentration ratio of 2%, 1% and 2%, adding drug-loaded MS/PLGA microspheres into the prepared solution after uniform mixing, adding genipin solution and CaCl ₂ solution after uniform stirring, placing in a mould, curing for 30min at 37 ℃, freezing in a refrigerator after curing, and preserving after freeze-drying. The final concentrations of genipin solution and CaCl ₂ solution were 0.25% and 0.2%.

Example 5

The SF/CMCS/SA composite hydrogel containing 1% mesoporous bioglass/PLGA microspheres is prepared in the embodiment, and the specific process is as follows:

Mixing SF, SA, CMCS solutions according to the final concentration ratio of 2%, 1% and 2%, adding 1% mesoporous bioglass/polylactic acid microspheres into the prepared solution after uniformly mixing, adding genipin solution and SrCl ₂ solution after uniformly stirring, placing the mixture into a mould, curing the mixture at 45 ℃ for 10min, freezing the mixture in a refrigerator after curing, and preserving the mixture after freeze-drying. The final concentrations of genipin solution and SrCl ₂ solution were 0.25% and 0.2%.

Example 6

The fibrin/chitosan composite hydrogel containing 1% mesoporous calcium silicate/PLGA microspheres is prepared by the following specific processes:

Mixing fibrin and chitosan according to a final concentration ratio of 1% and 4%, adding 1% mesoporous calcium silicate/PLGA microspheres into the prepared solution after uniformly mixing, adding genipin and ZnCl ₂ solution after uniformly stirring, placing into a mould, curing for 10min at 45 ℃, freezing in a refrigerator after curing, and preserving after freeze-drying. The final concentrations of genipin solution and ZnCl ₂ solution were 1% and 0.1%.

Example 7

The collagen/SA composite hydrogel containing 1% mesoporous calcium silicate/PLGA microspheres is prepared in the embodiment, and the specific process is as follows:

Mixing collagen and SA according to the final concentration ratio of 1% and 4%, adding 1% mesoporous calcium silicate/PLGA microspheres into the prepared solution after uniformly mixing, adding genipin solution and CuCl ₂ solution after uniformly stirring, placing into a mould, curing for 10min at 45 ℃, freezing in a refrigerator after curing, and preserving after freeze-drying. The final concentrations of genipin solution and CuCl ₂ solution were 0.6% and 0.05%.

Comparative example 1

MS/PLGA microspheres were synthesized as described in example 1.

Comparative example 2

The embodiment is a preparation method of SF/CMCS/SA composite gel without microspheres, which comprises the following steps:

After SF is extracted according to the method of example 1, SF, SA, CMCS solutions are mixed according to the final concentration ratio of 2%, 1% and 2%, genipin solution and CaCl ₂ solution are added after uniform mixing, the mixture is placed in a mould after uniform stirring, and the mould is cured for 30min at 37 ℃, frozen in a refrigerator after curing, and stored after freeze-drying. The final concentrations of genipin solution and CaCl ₂ solution were 00.5% and 0.125%.

Comparative example 3

The comparative example is a preparation method of a purely drug-loaded microsphere, and MS/PLGA is synthesized by taking rhodamine as a model drug according to the method of the example 4.

Test examples

The performance of the microsphere composite hydrogels prepared in examples and comparative examples and drug-loaded composite hydrogels was tested in this test example. Wherein:

(1) Morphology characterization of microspheres and microsphere composite hydrogels

The microsphere morphology of comparative example 1 and the composite gel of examples 1-3 were characterized in that microsphere (non-drug-loaded microsphere, drug-loaded microsphere) powder and 4 microsphere gels of different solid contents were stuck to a sample stage with a conductive adhesive, subjected to metal spraying treatment, and observed for microsphere and gel morphology and energy spectrum scanning analysis using a bench scanning electron microscope.

As shown in FIG. 1, the microspheres prepared in comparative example 1 have good spheronization effect, rough surface and MS particle coverage, and are caused by the fact that the particles are dispersed on the surface of the polyester in the emulsification process, and the microspheres have good diameter uniformity and the size of 113.79 +/-23.87 mu m. EDS scanning results show that the microspheres contain C, O and Si elements, and are consistent with the raw material elements used for preparing the microspheres.

The microspheres with different contents are loaded into an SF/CMCS/SA gel system, and the morphology of the composite gel is observed, so that the microspheres are more uniformly distributed in a gel network and are tightly combined with the gel from the figure 2 b. Although the pores of the composite gel show a tendency to shrink as the content of the microspheres increases, the macroporous structure of the gel remains. EDS energy spectrum scanning analysis shows that C, N, O, na, cl and Ca exist in the composite gel, and the energy spectrum scanning result shows that Si element also exists in the composite gel due to the fact that 0.5%, 1% and 2% groups contain microspheres with different solid contents.

(2) Infrared scanning of microsphere composite hydrogels

The infrared scanning of comparative example 2 and examples 1-3 comprises loading microspheres of comparative example 1 with different contents into SF, CMCS, SA composite gel, freeze-drying, testing by using an ATR-FTIR mode of a Fourier infrared spectrometer, setting specific parameters to be tested within the range of 600-4000cm ^-1, setting resolution to be 4cm ^-1, and performing cumulative scanning for 32 times to finally obtain an infrared absorption spectrum.

As a result, as shown in FIG. 2a, the infrared spectrum showed a stretching vibration peak of-OH at 3283cm ^-1, and characteristic absorption bands of amide I band, amide II band and amide III band on SF were observed at 1644, 1529 and 1231cm ^-1, respectively, which are related to beta sheet conformation of SF. Wherein the amide I band is mainly c=o stretching vibration and C-N stretching vibration, the amide II band is mainly N-H in-plane bending vibration and C-N stretching vibration, and the amide III band is mainly C-N stretching vibration and N-H in-plane bending vibration. 1024. 1308, 1419 and 1616cm ^-1 are respectively the stretching vibration, C-N stretching vibration, -COO symmetrical stretching vibration and-COO asymmetrical stretching vibration of the primary alcohol C-O bond of CMCS, and 1419 and 1616cm ^-1 are respectively the SA symmetrical stretching vibration and asymmetrical stretching vibration peaks. Infrared energy of the microgels of different solids content corresponds to characteristic absorption peaks at SF, CMCS and SA.

(3) X-ray diffraction (XRD) characterization of microsphere composite hydrogels

X-ray diffraction (XRD) characterization of comparative example 2 and examples 1-3, prepared gel was freeze-dried and tested with X-ray diffractometer, cuK alpha-ray, X-ray wavelength: The super-energy detection counter records a diffraction intensity curve between 2 theta = 10-60 degrees, and the scanning speed is 2 degrees/min. Finally obtaining the XRD pattern of the sample. As a result, as shown in FIG. 2c, the XRD characteristic diffraction peaks of gels containing different amounts of MS/PLGA microspheres were almost the same, and all were around 20℃and were consistent with the XRD characteristic peaks of CMCS and SF. As a result of the crosslinking curing by adding CaCl ₂ solution, its characteristic main peak appears in the 0% group (comparative example 2). This shows that SF/CMCS/SA gels of different solid content MS/PLGA microspheres did not change the respective secondary structure.

(4) Mechanical property analysis of microsphere composite hydrogel

Mechanical property analysis of comparative example 2 and examples 1 to 3 the gel was immersed in PBS buffer solution, and after reaching the swelling equilibrium, compression test was performed on a mechanical tester, and the mechanical properties of the four sets of scaffold materials were tested. The diameter of the bracket is about 6-7 mm, the height is 4-6 mm. The test was set at 2mm/min. The stress (sigma) at 5% -15% strain is calculated according to formula 1:

Sigma=f/s×100 equation 1

F and S represent the load area and the compression area, respectively.

The results are shown in fig. 3 a-c, which respectively show stress-strain curves, average compressive strength at 60% strain and average compressive modulus of the microsphere composite gels with different solid contents. As the solids content of the microspheres increases, the mechanical properties of the composite gel also gradually increase and then decrease, which may be related to microsphere sedimentation and maldistribution, with 1% of the microsphere composite hydrogels (example 2) exhibiting the strongest mechanical properties compared to the other groups. The average compressive strength and elastic modulus at 60% strain of example 2 were 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.+ -. 0.61 kPa).

(5) Drug release properties of microsphere composite hydrogels

Example 4 and comparative example 3 drug release in vitro drug release performance tests were performed on drug-loaded microspheres (comparative example 3) and 1% drug-loaded microsphere composite gel (example 4) using rhodamine B as a small molecule drug model. The drug-loaded microspheres and 1% drug-loaded microsphere composite gel were placed in 10mL centrifuge tubes, respectively, and subjected to in vitro release testing with PBS buffer to 5mL, and placed in a 37 ℃ shaker with gentle shaking. 1.5mL of the supernatant was taken at predetermined times, filtered and the rhodamine B concentration was detected by ultraviolet-visible spectrophotometry. Immediately after each sampling 1.5mL of fresh PBS solution was replenished and the cumulative release rate was calculated.

The results are shown in FIG. 3d, and the comparative example 3 and the example 4 show that no drug burst occurs in the early stage, thus the microspheres have better drug slow release effect. As the microspheres degrade, the drug of comparative example 3 began to release slowly at 7 days, whereas the microsphere/gel composite of example 4 released a greater amount of drug than comparative example 3 before 7 days, which was the time the microsphere composite gel was prepared, the microspheres released a portion of the drug. After 14 days, the drug released by the microspheres is greatly increased, and the release of the microsphere composite gel is slower, so that the swelling property and the degradation property of the gel are not separated, the drug of the microspheres in the gel shallow network is released first, and the gel can form a coating layer on the surface of the microspheres, thereby being beneficial to improving the controlled release property of the microspheres. The drug release time of each group of materials reaches more than 2 months, which indicates that the constructed microsphere and gel have good controlled release and sustained release performances.

(6) Swelling property, porosity and degradation property of microsphere composite hydrogel

Swelling, porosity and degradation Properties of comparative example 2 and examples 1 to 3A lyophilized material (weight: W ₁) was soaked in a 10ml EP tube, 5ml PBS solution (pH=7.4) was added, and placed in a shaker at 90rpm at 37℃until gel swelling reached equilibrium. During this period, excess water on the surface was adsorbed with filter paper every 2 hours, the gel weight was recorded as W ₂, and the weight of the fully swollen gel was recorded until the weight of the swollen gel was no longer changed. The swelling ratio SI is then calculated according to the formula.

Si= (W ₂ - W₁) / W₁ x 100% equation 2)

The lyophilized microsphere gels with different solid contents are immersed in absolute ethanol until saturated, and no bubbles come out. The gel was weighed before (W ₃) and after (W ₄) immersion in absolute ethanol. The porosity P was calculated using the following formula:

P= (W ₄-W₃)/ρ_EthanolV_Gel x 100% equation 3)

Wherein, W ₃ and W ₄ represent the weights of the samples before and after immersing in alcohol, V _Gel is the volume of the gel, and ρ _Ethanol is a constant (density of absolute ethanol at normal temperature), respectively.

The degradation behavior of the scaffold material was tested in PBS (volume ratio 1:100) with or without XIV type protease (2U/mL) in a shaker at 37℃at 90 rpm. The initial weight of the dried sample was determined (W ₅). Samples were washed with water over a defined time interval, lyophilized and weighed (W ₆). And calculating the degradation rate according to a formula.

Degradation rate (%) = (W ₅–W₆)/W₅ x 100% equation 4

The results are shown in FIGS. 4 a-b, showing the swelling properties of the composite gel. With time, all four groups of composite gels showed the fastest water absorption within 0.h and the water absorption was still increasing after 0.5h, but the water absorption was very small, slowly tending to stabilize, reaching an equilibrium swelling level after 24 h. As the solids content of the microspheres increased, the equilibrium swelling of the microsphere composite gel tended to decrease, with water absorption, and 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. Fig. 4c shows the porosity of each set of composite gels, with the porosity of all microsphere gels exceeding 80%, indicating that the composite gel has good porosity. As the solid content of the microspheres increases, the porosity decreases, which indicates that the more the solid content of the microspheres is, the larger the solid content of the microspheres is, and the more compact the three-dimensional network structure is formed. Fig. 4d and 4e present the degradation properties of the respective sets of composite gels with and without enzymes. With the increase of the solid content of the microspheres, the degradation rate of each group of composite gel is in a decreasing trend, but the degradation rate is approximately the same, and the composite gel is in a rapid and then stable state. The degradation rate of the composite gel under the condition of enzyme is obviously faster than that under the condition of no enzyme, however, after 28d, each group of composite gel still remains about half, which indicates that the composite gel has good degradation performance.

(7) Cytotoxicity experiment and live-dead staining of microsphere composite hydrogel

Cytotoxicity test, firstly preparing leaching solution of microsphere composite gel, preparing leaching solution and complete culture medium 1:1, mixing the leaching solution and cells to form cell suspension, placing the cell suspension into holes of 48-hole culture plate, every hole is 2 ten thousand cells, and every 2d is the culture medium replaced. Cell proliferation was detected by CCK-8 method at 1,3 and 7d of culture, respectively.

Cell live and dead staining experiments, namely cell culture is carried out for 1d and 7d in an orifice plate respectively, and then activity of mesenchymal stem cells in microsphere gels with different solid contents is detected by using a live/dead cell staining kit

The results are shown in FIG. 5, and CCK-8 shows that each set of microsphere/gel materials has good cell compatibility, and that example 3 has slightly lower cell activity than comparative example 2. The results of staining the living and dead cells showed that the cells of comparative example 2 and examples 1-2 were very active and proliferated well, while the number of cells of example 3 was less than that of comparative example 2, which was consistent with the CCK-8 results.

(8) Evaluation of osteogenic Property of microsphere composite hydrogel

The osteogenic differentiation performance of comparative example 2 and examples 1-3 was characterized by first preparing a leaching solution of microsphere composite gel, preparing the leaching solution with complete medium 1:1, mixing the leaching solution with cells to form a cell suspension, and placing the cell suspension in 48-well culture for osteogenic performance evaluation.

Alkaline phosphatase (ALP) staining experiments microsphere gels of different solids content were placed in wells of 24 well plates, 2.5X10 ⁴ rat BMSCs per well. After 24h of culture, the cell culture medium was replaced with osteoinductive differentiation medium, and cultured for 7d and 14d, respectively, with medium replaced every 2 d. After termination of the incubation, the gel samples were washed three times with pre-chilled PBS and fixed with 4% paraformaldehyde fixing solution, then stained and photographed using an ALP staining kit according to instructions.

Alizarin Red (ARS) staining experiments microsphere gels of different solids content were placed in wells of 24 well plates, 2.5×10 ⁴ rat BMSCs per well. After 24h of culture, the cell culture medium was replaced with osteogenic differentiation medium, and the osteogenic differentiation medium was replaced every 2 d. After 7d and 21d of culture, the original culture medium is discarded, the cells of each group are washed by PBS buffer solution for 2 times, the cells of each group are fixed by alizarin red dye fixing solution, each hole is added with alizarin red dye solution to cover the cells after PBS washing, the cells are incubated and dyed for 30min at room temperature, and after deionized water is used for washing the dye solution, the cells are photographed under an inverted microscope to observe the formation amount of calcium nodules.

Quantitative real-time polymerase chain reaction microsphere gels of different solid contents were placed in wells of 24-well plates, 2.5X10 ⁴ rat BMSCs were grown per well. After 24h of culture, the cell culture medium was changed to osteoinductive differentiation medium, and the medium was changed every 2 d. Collecting cells in the pore plate at 7d and 14d, extracting total RNA of each group of cells, using cDNA of a reverse transcription reaction product as a template, and detecting the osteogenic related genes OPN and RunX2 by adopting a real-time quantitative RT-PCR method.

Fig. 6a shows that examples 1-3 all have good osteogenic properties. From ALP staining, it was found that each of the composite gels exhibited certain osteogenic properties, with 1% (example 2) and 2% (example 3) being significantly stronger than 0% (comparative example 2) and 0.5% (example 1). As can be seen from ARS staining results, as the solid content of the microspheres increased, more red-stained calcium nodules were observed under the microscope, indicating that the osteogenic differentiation performance was also significantly enhanced.

FIG. 6b and FIG. 6c show the expression of OPN and RunX2 genes at 7day and 14day, respectively, OPN and RunX-2 being osteogenic genes. From the figure, it was found that OPN and RunX2 gene expression in culture of 14day was significantly higher than that in culture of 7day, and that the osteogenic gene expression in 1% and 2% groups was significantly higher than in 0% and 0.5% groups. In summary, the osteogenic properties of the composite gel of 1% MS/PLGA microspheres in example 2 are best.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather to enable any modification, equivalent replacement, improvement or the like to be made within the spirit and principles of the invention.

Claims

1. The microsphere composite hydrogel is characterized by comprising microspheres and hydrogel, wherein the microspheres are dispersed in the hydrogel, the microspheres comprise degradable polyester materials and mesoporous materials wrapped in the degradable polyester materials, the hydrogel comprises a combination of silk fibroin, carboxymethyl chitosan and sodium alginate, the hydrogel further comprises a cross-linking agent, the cross-linking agent comprises at least one of genipin and metal salt, the mesoporous materials comprise at least one of mesoporous silicon, mesoporous bioglass, mesoporous calcium silicate, mesoporous zinc silicate, mesoporous strontium silicate and mesoporous magnesium silicate, and the degradable polyester materials comprise polylactic acid-glycolic acid copolymer.

2. The microsphere composite hydrogel according to claim 1, wherein the mass ratio of 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, comprising the steps of:

4. A drug-loaded microsphere composite hydrogel, comprising the microsphere composite hydrogel of any one of claims 1-2 and an active drug loaded in the microsphere.

5. The method for preparing the drug-loaded microsphere composite hydrogel according to claim 4, which is characterized by comprising the following steps:

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