A composite gel microsphere and its preparation method

Abstract

The present invention discloses a composite gel microsphere and a preparation method thereof, comprising the following steps: preparing silica colloidal spheres by the Stober method; synthesizing magnesium silicate nanoparticles from the silica colloidal spheres by the hydrothermal method; loading soluble drugs or growth factors on the magnesium silicate nanoparticles by physical adsorption to obtain a loaded material; adding an aqueous solution of the loaded material to an aqueous solution of GelMA / sodium alginate to obtain a bioink; wherein, the mass ratio of the magnesium silicate nanoparticles to GelMA is (1:200)-(1:25); adding the bioink to a photocuring 3D printer for printing to obtain the composite gel microsphere. The composite gel microsphere of the present invention contains both inorganic nanoparticles and organic hydrogels, and loads soluble drugs or growth factors to superimpose biological effects, perfectly combining the three of biomaterials, seed cells, and soluble drugs or growth factors, aiming to play a full role in the field of bone tissue engineering and providing a good solution for bone regeneration and reconstruction.

Description

Technical Field

[0001] The present invention relates to the technical field of gel microspheres, and in particular to a composite gel microsphere and a preparation method thereof. Background Art

[0002] Nanomaterials have controllable physical and chemical properties and are easy to modify, which has always been a hot topic in the medical field. Nanostructures have been widely used in the medical field and are often used in drug delivery, molecular imaging, waste clearance, tumor killing and other fields. Among them, silicon-based nanoparticles have achieved great success in the fields of biomedicine and bioengineering, and also show great potential in the field of bone reconstruction. As early as 1968, Stober successfully synthesized homogeneous silica colloids with different particle sizes. Later, scholars improved on this basis to prepare nanoparticles with different morphologies, such as spherical, flower-shaped, and dendritic. The present invention first synthesizes silica colloids by the classical Stober method, and then uses this as a precursor material to prepare hollow porous magnesium silicate nanoparticles by hydrothermal method. Magnesium silicate nanoparticles have a large specific surface area and a rich mesoporous structure, and have broad application prospects in biomolecular delivery.

[0003] However, nanoparticles have a high surface energy and are extremely prone to aggregation, and the aggregation morphology is difficult to control, thus losing their excellent properties. It is a bioactive glass material in the form of powder, etc., which is not suitable for hard tissue repair and may face problems such as difficult fixation and difficult shaping. In addition, when nanoparticles are locally applied to bone defects, the nanoparticles are first internalized by the phagocytic system as foreign bodies, greatly reducing their bioavailability. Therefore, selecting a suitable carrier to load magnesium silicate nanoparticles can effectively solve this problem. At present, domestic and foreign research scholars mostly load nanoparticles onto materials such as hydrogels, micelles, and metal-organic frameworks. Among them, hydrogels have attracted much attention due to their properties such as porosity and adjustable structure.

[0004] Ideal bone repair materials should also possess properties such as osteoconductivity, osteoinduction, osteogenicity, biodegradability, and good biocompatibility. As a transplantation material for bone defects, hydrogels need to provide mechanical structural support on the one hand and a space for osteoblasts to grow into on the other hand. Gelatin methacrylate (GelMA) hydrogels can improve their mechanical strength through the photocrosslinking process by introducing methacrylate groups. They have good biocompatibility and show strong application advantages in bone regeneration. The broad conditions under which GelMA can polymerize at room temperature, neutral pH, and in an aqueous solution environment are very conducive to cell survival. Its photocrosslinkable property gives it flexible formability. It can not only be used as a substrate for two-dimensional cell culture to promote cell adhesion and proliferation, but also construct a three-dimensional environment or specific pattern structure, acting similar to the extracellular matrix, thereby manufacturing various biomimetic tissue structures and organoids. However, due to the low degree of methacrylation, the rapid decomposition of the structure affects the stability of the mechanical structure. Research shows that adding nanosilicates to GelMA bioinks can enhance the biological properties and physical strength of GelMA. It is worth emphasizing that magnesium silicate nanoparticles have osteogenic properties. Their silanol groups can interact with calcium ions and phosphate ions and can continuously release magnesium ions. Therefore, they are an effective material for promoting bone maturation. Combining the two has been proven to be able to improve the initial adhesion, cell proliferation, and differentiation of cells within or on the surface of the hydrogel.

[0005] The three key elements of tissue engineering are seed cells, scaffolds, and cytokines. With suitable tissue seed cells and biomaterials, advanced biomanufacturing technologies are also needed to combine them to manufacture implants that match the structure and characteristics of the target repair site. In recent years, 3D printing technology has been widely used in biomanufacturing due to its characteristics such as speed, personalization, low cost, and high manufacturing precision. Currently, the 3D printing technologies most commonly used in tissue engineering can be mainly divided into three categories: extrusion printing, inkjet printing, and photocuring printing technologies. Among them, the electrospray / photocuring printer used in this paper can print gel microspheres with a diameter of 200 - 1000 μm. The microsphere cell microcarriers can not only amplify cells in large quantities in vitro but also serve as carriers for cells and drugs. By injecting the cells, they can be transported to the defect site, which can not only provide sufficient space to meet the needs of three-dimensional cell culture in vitro but also protect the internal cells from damage caused by extrusion and friction during tissue injection. Due to the above excellent properties, microspheres have been used in many fields such as bone defect repair, cartilage regeneration, and myocardial repair. However, microspheres often lack bioactivity and cannot directly promote cell proliferation and differentiation after in vitro cell proliferation and injection into the body. Methods such as adding growth factors often also face problems such as the fragility and easy inactivation of growth factors and rapid release. Therefore, there is an urgent need for a microsphere with good bioactivity. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention provides a composite gel microsphere and a preparation method thereof. The composite gel microsphere contains both inorganic nanoparticles and organic hydrogels, and loads soluble drugs or growth factors to superimpose biological effects, perfectly combining biomaterials, seed cells, and soluble drugs or growth factors, aiming to play a full role in the field of bone tissue engineering and providing a good solution for bone regeneration and reconstruction. Further, the present invention composites magnesium silicate nanoparticles loaded with benidipine with a polymer material. Under the encapsulation of the polymer matrix, the nanoparticles can slowly release active silicon and magnesium ions, and a microcarrier with both good biological activity and osteoinductivity and a sufficient strength scaffold structure can be obtained, rapidly amplifying cells in vitro to meet the demand for cell amplification during tissue repair.

[0007] The first aspect of the present invention provides a preparation method of a composite gel microsphere, comprising the following steps:

[0008] S10. Preparation step of silica colloidal spheres: Prepare silica colloidal spheres by the stober method;

[0009] S20. Synthesis step of magnesium silicate nanoparticles: Synthesize magnesium silicate nanoparticles from silica colloidal spheres by the hydrothermal method;

[0010] S30. Loading step: Load soluble drugs or growth factors onto magnesium silicate nanoparticles by physical adsorption to obtain a loaded material;

[0011] S40. Preparation step of bioink:

[0012] Add methacrylated gelatin (GelMA) to an aqueous sodium alginate solution to form a first stock solution, add a photoinitiator, fully dissolve and mix evenly to obtain a GelMA / sodium alginate aqueous solution for standby;

[0013] Ultrasonically disperse the loaded material in deionized water to obtain a loaded material aqueous solution for standby;

[0014] Prepare an aqueous calcium chloride solution as a crosslinking agent for standby;

[0015] Add the loaded material aqueous solution and the crosslinking agent to the GelMA / sodium alginate aqueous solution to obtain bioink; wherein, the mass ratio of magnesium silicate nanoparticles to GelMA is (1:200)-(1:25);

[0016] S50. Printing step: Add the bioink to a photocuring 3D printer for printing. The printed microspheres fall into the crosslinking agent and are initially cured, and then secondarily cured by ultraviolet light to finally obtain the composite gel microspheres.

[0017] In the present invention, as a preferred embodiment, in S30, the loading step, the soluble drug is benidipine.

[0018] In the present invention, as a preferred embodiment, the specific process of step S10, the preparation step of silica colloidal spheres, is as follows: Add 2 mol / L ammonia water and 10 mol / L deionized water to 70 ml of absolute ethanol and stir for 25 min. After mixing evenly, quickly drop 0.2 mol of tetraethyl orthosilicate into the above solution. The solution gradually turns milky white in 10 min. Continue to stir at 400 rpm for 4 h. After centrifuging at 8000 rpm, wash the colloidal spheres 3 times with deionized water and absolute ethanol respectively, and dry at 60 °C for 12 h.

[0019] In the present invention, as a preferred embodiment, the specific process of step S20, the synthesis step of magnesium silicate nanoparticles, is as follows: Weigh 200 mg of silica colloidal spheres and put them into 40 ml of deionized water and disperse them evenly by ultrasonic wave; Add 1.5 mmol of magnesium chloride hexahydrate, 20 mmol of ammonium chloride and 2 ml of ammonia water to 40 ml of water and stir to mix evenly; Then mix the above two and put them into a muffle furnace at 160 °C for 12 h at high temperature; After cooling to room temperature, centrifuge to collect the precipitate, wash it 3 times with water and absolute ethanol respectively, and dry at 60 °C for 12 h; Reserve for use.

[0020] In the present invention, as a preferred embodiment, in step S20, the synthesis step of magnesium silicate nanoparticles, the magnesium silicate nanoparticles have a porous hollow structure.

[0021] In the present invention, as a preferred embodiment, the specific process of step S30, the loading step, is as follows: Weigh 6 mg of benidipine hydrochloride and dissolve it in 10 ml of absolute ethanol, and gradually drop it into 10 ml of an aqueous solution containing 10 mg of magnesium silicate nanoparticles, stir at 500 rpm for 24 h, wash it 3 times with water, centrifuge to collect the precipitate, and dry at 60 °C.

[0022] In the present invention, as a preferred embodiment, the specific process of step S40, the preparation step of the bioink, is as follows:

[0023] Take a 15 ml centrifuge tube, weigh 1 g of GelMA freeze-dried sponge and add it to an aqueous solution of sodium alginate with a concentration of 0.5% to prepare a first stock solution with a concentration of 10%. After fully dissolving at 50 °C, introduce a photoinitiator LAP with a mass-volume ratio of 0.5%, and fully dissolve and mix evenly.

[0024] Weigh 40 mg of benidipine-loaded magnesium silicate nanoparticles and disperse them ultrasonically in 4 ml of deionized water to prepare an aqueous solution of the loading material with a concentration of 10 mg / mL, reserve for use;

[0025] Prepare 100 ml of a 5% aqueous solution of calcium chloride as a crosslinking agent.

[0026] In the present invention, as a preferred embodiment, in S40, the bio-ink configuration step, the mass ratio of magnesium silicate nanoparticles to GelMA is 1:200, 1:100, 1:50 or 1:25.

[0027] In the present invention, as a preferred embodiment, in S50, the printing step, the printing parameters are set to 20KPa of air pressure and 6.66kV of voltage; the bio-ink is added to the syringe and fixed with a constant temperature heating belt, the air pressure pump is connected, the DC power supply is turned on, the printing device status is checked, the cross-linking agent calcium chloride aqueous solution is placed in a 50ml small beaker to hold the microspheres, and the ultraviolet light is irradiated on the air-liquid interface to make the gel spheres solidify quickly; the precipitate is collected by centrifugation and observed under an electron microscope, and stored at -4°C

[0028] The second aspect of the present invention further provides a composite gel microsphere, which is prepared by the preparation method of the composite gel microsphere of the first aspect.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The composite gel microspheres prepared according to the present invention contain both inorganic nanoparticles and organic hydrogels, and are loaded with soluble drugs or growth-promoting factors to superimpose biological effects, perfectly combining biomaterials, seed cells, soluble drugs or growth-promoting factors, giving full play to the field of bone tissue engineering, and providing a good solution for bone regeneration and reconstruction. Furthermore, the present invention composites magnesium silicate nanoparticles loaded with benidipine with polymer materials. Under the wrapping of the polymer matrix, the nanoparticles can slowly release active silicon and magnesium ions, so that microcarriers with good biological activity and bone inductivity and a sufficiently strong scaffold structure can be obtained, which can rapidly expand cells in vitro and meet the demand for cell expansion in the process of tissue repair.

[0031] The magnesium silicate nanoparticle composite gel microspheres loaded with benidipine prepared by the present invention have controllable particle size, rounded surface and good biocompatibility. The average particle size of the magnesium silicate nanoparticles is 380nm, and the porous hollow structure has a very high specific surface area, a drug loading rate of up to 340.1mg / g, a potential of -25mv, a benidipine release rate of less than 10% within 24 hours, and a continuous release time of silicon and magnesium elements of up to 28d. The composite gel microspheres have a diameter of 230nm, an increased mechanical strength, and better biocompatibility. Compared with simple GelMA microspheres, the biological activity of the present invention is significantly improved in rat bone marrow mesenchymal stem cell culture for 1d, 3d, and 7d, the number of cells adhering to the surface of the microspheres is increased, the number of adhesion spots is increased, and the osteogenic differentiation ability is stronger. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1-1 This is a comparison chart of the cell proliferation results after the composite gel microspheres of Examples 1-5 were co-cultured with rat bone marrow mesenchymal stem cells for 1 day;

[0033] Figure 1-2 Comparison chart of cell proliferation results after 4 days of co - culture of the composite gel microspheres of Examples 1 - 5 with rat bone marrow mesenchymal stem cells;

[0034] Figure 1-3 Comparison chart of cell proliferation results after 7 days of co - culture of the composite gel microspheres of Examples 1 - 5 with rat bone marrow mesenchymal stem cells;

[0035] Figure 2-1 Scanning electron micrograph of magnesium silicate nanoparticles when WD = 8.6;

[0036] Figure 2-2 Scanning electron micrograph of magnesium silicate nanoparticles when WD = 9.4;

[0037] Figure 3-1 Scanning electron micrograph of pure GelMA microspheres when MAG = 500X;

[0038] Figure 3-2 Scanning electron micrograph of pure GelMA microspheres when MAG = 3.00KX;

[0039] Figure 4-1 Scanning electron micrograph of the composite gel microspheres of the group with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:100 (Example 2); MAG = 2.90KX;

[0040] Figure 4-2 Scanning electron micrograph of the composite gel microspheres of the group with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:100 (Example 2); MAG = 1.99KX;

[0041] Figure 5 Scanning electron micrograph of the composite gel microspheres of the group with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:100 (Example 2); MAG = 5.0KX;

[0042] Figure 6-1 Cross - sectional scanning electron micrograph of pure GelMA gel spheres;

[0043] Figure 6-2 Cross - sectional scanning electron micrograph of the composite gel microspheres;

[0044] Figure 7-1 、 7-2 Laser confocal image of benidipine - loaded magnesium silicate nanoparticle composite gel microspheres under GFP (green fluorescent protein).

[0045] Figure 8-1 Laser confocal microscopy images of the composite gel microspheres of pure GelMA (Comparative Example 1) in co - culture with cells;

[0046] Figure 8-2 Confocal laser microscopy images of the composite gel microspheres with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:50 (Example 3) in co-culture with cells;

[0047] Figure 8-3 Confocal laser microscopy images of the composite gel microspheres with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:100 (Example 2) in co-culture with cells;

[0048] Figure 8-4 Confocal laser microscopy images of the composite gel microspheres with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:200 (Example 1) in co-culture with cells. Detailed implementation manners

[0049] Next, in combination with the accompanying drawings and specific implementation manners, the invention will be further described. It should be noted that, on the premise of no conflict, the following-described embodiments or technical features can be arbitrarily combined with each other to form new embodiments. Except as otherwise specified, the materials and equipment used in this embodiment can be purchased from the market.

[0050] The present invention provides a method for preparing composite gel microspheres, including the following steps:

[0051] S10. Preparation step of silica colloidal spheres: Prepare silica colloidal spheres by the Stober method;

[0052] S20. Synthesis step of magnesium silicate nanoparticles: Synthesize magnesium silicate nanoparticles from silica colloidal spheres by the hydrothermal method;

[0053] S30. Loading step: Load soluble drugs or growth factors on magnesium silicate nanoparticles by physical adsorption to obtain a loaded material;

[0054] S40. Bioink preparation step:

[0055] Add methacrylated gelatin (GelMA) to an aqueous sodium alginate solution to form a first stock solution, add a photoinitiator, and dissolve and mix well to obtain a GelMA / sodium alginate aqueous solution for standby;

[0056] Ultrasonically disperse the loaded material in deionized water to obtain a loaded material aqueous solution for standby;

[0057] Prepare an aqueous calcium chloride solution as a crosslinking agent for standby;

[0058] Add the loaded material aqueous solution to the GelMA / sodium alginate aqueous solution to obtain a bioink; wherein, the mass ratio of magnesium silicate nanoparticles to GelMA is (1:200)-(1:25);

[0059] S50, Printing step: Add the bioink into a stereolithography 3D printer for printing. The printed microspheres fall into the crosslinking agent and are preliminarily cured, and then are secondarily cured by ultraviolet light to finally obtain composite gel microspheres.

[0060] As a preferred embodiment, in S30, the soluble drug in the loading step is benidipine.

[0061] As a preferred embodiment, the specific process of S10, the preparation step of silica colloidal spheres is as follows: Add 2mol / L ammonia water and 10mol / L deionized water into 70ml anhydrous ethanol and stir for 25min. After mixing evenly, quickly drop 0.2mol of tetraethyl orthosilicate into the above solution. The solution gradually turns milky white in 10min, continue to stir at 400rpm for 4h, centrifuge at 8000rpm, and then wash the colloidal spheres with deionized water and anhydrous ethanol 3 times respectively, and dry at 60℃ for 12h.

[0062] In the present invention, as a preferred embodiment, the specific process of S20, the synthesis step of magnesium silicate nanoparticles is as follows: Weigh 200mg of silica colloidal spheres and put them into 40ml of deionized water and disperse them evenly by ultrasonic wave; Add 1.5mmol of magnesium chloride hexahydrate, 20mmol of ammonium chloride and 2ml of ammonia water into 40ml of water and stir and mix evenly; Then mix the above two evenly, put them into a muffle furnace and keep at a high temperature of 160℃ for 12h; After cooling to room temperature, centrifuge to collect the precipitate, wash it 3 times with water and anhydrous ethanol respectively, and dry at 60℃ for 12h; Reserve for use.

[0063] As a preferred embodiment, in S20, the synthesis step of magnesium silicate nanoparticles, the magnesium silicate nanoparticles are porous hollow structures.

[0064] As a preferred embodiment, the specific process of S30, the loading step is as follows: Weigh 6mg of benidipine hydrochloride and dissolve it in 10ml of anhydrous ethanol, and gradually drop it into 10ml of an aqueous solution containing 10mg of magnesium silicate nanoparticles, stir at 500rpm for 24h, wash with water 3 times, centrifuge to collect the precipitate, and dry at 60℃.

[0065] As a preferred embodiment, the specific process of S40, the bioink preparation step is as follows:

[0066] Take a 15ml centrifuge tube, weigh 1g of GelMA freeze-dried sponge and add it into an aqueous solution of sodium alginate with a concentration of 0.5% to prepare a first stock solution with a concentration of 10%. After fully dissolving at 50℃, introduce a photoinitiator LAP with a mass-volume ratio of 0.5%, and fully dissolve and mix evenly.

[0067] Weigh 40mg of magnesium silicate nanoparticles loaded with benidipine, disperse them by ultrasonic wave in 4ml of deionized water to prepare an aqueous solution of the loading material with a concentration of 10mg / mL, and reserve for use;

[0068] Prepare 100 ml of 5% calcium chloride aqueous solution as the crosslinking agent.

[0069] As a preferred embodiment, in step S40, the preparation step of the bioink, the mass ratio of magnesium silicate nanoparticles to GelMA is 1:200, 1:100, 1:50 or 1:25.

[0070] As a preferred embodiment, in step S50, the printing step, the printing parameters are set as air pressure 20 KPa and voltage 6.66 kv; add the bioink into a syringe, fix a constant temperature heating belt, connect an air pressure pump, turn on the DC power supply, check the status of the printing equipment, use a 50 ml small beaker to hold the microspheres with the crosslinking agent calcium chloride aqueous solution, irradiate the gas-liquid interface with an ultraviolet lamp to quickly solidify the gel beads; collect the precipitate by centrifugation, observe it under an electron microscope, and store it at -4°C.

[0071] The present invention also provides a composite gel microsphere, which is prepared by the preparation method of the composite gel microsphere.

[0072] The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and should not be construed as a limitation to the present application.

[0073] Example 1:

[0074] This embodiment provides a preparation method of a composite gel microsphere, including the following steps:

[0075] S10, the preparation step of silica colloidal spheres: Prepare silica colloidal spheres by the stober method; the specific process is as follows: Add 2 mol / L ammonia water and 10 mol / L deionized water to 70 ml of absolute ethanol and stir for 25 min. After mixing evenly, quickly drop 0.2 mol of tetraethyl orthosilicate into the above solution. The solution gradually turns milky white in 10 min. Continue to stir at 400 rpm for 4 h. After centrifugation at 8000 rpm, wash the colloidal spheres with deionized water and absolute ethanol 3 times respectively, and dry at 60°C for 12 h;

[0076] S20, the synthesis step of magnesium silicate nanoparticles: Synthesize magnesium silicate nanoparticles from silica colloidal spheres by the hydrothermal method; the specific process is as follows: Weigh 200 mg of silica colloidal spheres and put them into 40 ml of deionized water and disperse them evenly by ultrasonic waves; add 1.5 mmol of magnesium chloride hexahydrate, 20 mmol of ammonium chloride and 2 ml of ammonia water to 40 ml of water and stir evenly; then mix the above two, put them into a muffle furnace and keep at a high temperature of 160°C for 12 h; after cooling to room temperature, collect the precipitate by centrifugation, wash it 3 times with water and absolute ethanol respectively, and dry at 60°C for 12 h; reserve;

[0077] S30, loading step: loading soluble drugs or growth-promoting factors on magnesium silicate nanoparticles by physical adsorption to obtain a loaded material; the specific process is as follows: weigh 6 mg of benidipine hydrochloride and dissolve it in 10 ml of anhydrous ethanol, add it dropwise into 10 ml of an aqueous solution containing 10 mg of magnesium silicate nanoparticles, stir at 500 rpm for 24 hours, wash with water 3 times, collect the precipitate by centrifugation, and dry at 60°C;

[0078] S40, Bio-ink configuration steps:

[0079] Take 1 g of GelMA freeze-dried sponge in a 15 ml centrifuge tube and add 0.5% sodium alginate aqueous solution to make a first stock solution with a concentration of 10%. After fully dissolving at 50° C., introduce 0.5% of the mass volume ratio of the photoinitiator LAP and fully dissolve and mix.

[0080] Weigh 40 mg of magnesium silicate nanoparticles loaded with benidipine and ultrasonically disperse them in 4 ml of deionized water to prepare a 10 mg / mL aqueous solution of the loaded material for later use;

[0081] Prepare 100 ml of 5% calcium chloride aqueous solution as a cross-linking agent;

[0082] The loaded material aqueous solution is added into the GelMA / sodium alginate aqueous solution to obtain a biological ink, wherein the mass ratio of magnesium silicate nanoparticles to GelMA is 1:200;

[0083] S50, printing step: adding the bio-ink into a light-curing 3D printer for printing to obtain composite gel microspheres; the specific process is: setting the printing parameters to an air pressure of 20 KPa and a voltage of 6.66 kV; adding the bio-ink into a syringe to fix a constant temperature heating belt, connecting an air pressure pump, turning on a DC power supply, checking the status of the printing device, filling a 50 ml small beaker with a cross-linking agent calcium chloride aqueous solution to receive the microspheres, irradiating the air-liquid interface with an ultraviolet lamp to rapidly solidify the gel spheres; collecting the precipitate by centrifugation and observing it under an electron microscope, and storing it at -4°C.

[0084] Embodiment 2:

[0085] The characteristic of this embodiment is that, in S40, the bio-ink preparation step, the aqueous solution of the load material is added to the GelMA / sodium alginate aqueous solution to obtain the bio-ink; wherein the mass ratio of magnesium silicate nanoparticles to GelMA is 1:100; and the rest is the same as in Embodiment 1.

[0086] Embodiment 3:

[0087] The characteristic of this embodiment is that, in S40, the bio-ink preparation step, the aqueous solution of the load material is added to the GelMA / sodium alginate aqueous solution to obtain the bio-ink; wherein the mass ratio of magnesium silicate nanoparticles to GelMA is 1:50; and the rest is the same as in Embodiment 1.

[0088] Example 4:

[0089] The feature of this example is that in step S40, the aqueous solution of the loading material is added to the GelMA / sodium alginate aqueous solution to obtain the bioink; wherein, the mass ratio of magnesium silicate nanoparticles to GelMA is 1:25; others are the same as in Example 1.

[0090] Example 5:

[0091] The feature of this example is that in step S40, the aqueous solution of the loading material is added to the GelMA / sodium alginate aqueous solution to obtain the bioink; wherein, the mass ratio of magnesium silicate nanoparticles to GelMA is 1:400; others are the same as in Example 1.

[0092] Comparative Example 1:

[0093] The feature of this example is that in step S40, the aqueous solution of the loading material is not added, and only the GelMA / sodium alginate aqueous solution is used to obtain the bioink; others are the same as in Example 1.

[0094] Figure 1-1 It is a comparison chart of the proliferation results of the composite gel microspheres of Examples 1-5 with rat bone marrow mesenchymal stem cells after 1 day of co-culture;

[0095] Figure 1-2 It is a comparison chart of the proliferation results of the composite gel microspheres of Examples 1-5 with rat bone marrow mesenchymal stem cells after 4 days of co-culture;

[0096] Figure 1-3 It is a comparison chart of the proliferation results of the composite gel microspheres of Examples 1-5 with rat bone marrow mesenchymal stem cells after 7 days of co-culture;

[0097] From Figure 1-1 , 1-2 , and 1-3, it can be seen that after different concentrations of magnesium silicate nanoparticles are incorporated into GelMA, the cells on the surface of the microspheres are significantly amplified, and the group with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:50 (Example 3) shows the highest cell viability.

[0098] Figure 2-1 It is a scanning electron microscope image of magnesium silicate nanoparticles when WD = 8.6;

[0099] Figure 2-2 It is a scanning electron microscope image of magnesium silicate nanoparticles when WD = 9.4;

[0100] In the figure, WD is the working distance, which refers to the distance from the imaging surface of the sample to the objective lens;

[0101] from Figure 2-1 , 2-2 It can be seen that the magnesium silicate nanoparticles have a hollow mesoporous structure and a particle size of about 380 nm.

[0102] Figure 3-1 This is the scanning electron microscopy image of pure GelMA microspheres when MAG=500X;

[0103] Figure 3-2 The scanning electron micrograph of pure GelMA microspheres when MAG = 3.00KX;

[0104] from Figure 3-1 , 3-2 It can be seen that a small amount of cells adhere to the surface of the microspheres, and the surface is smooth and round.

[0105] Figure 4-1 The scanning electron microscope image of the composite gel microspheres with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:100 (Example 2); MAG = 2.90KX;

[0106] Figure 4-2 The scanning electron microscope image of the composite gel microspheres of the group with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:100 (Example 2); MAG = 1.99KX;

[0107] from Figure 4-1 , 4-2 It can be seen that in the same period of time, more cells adhere to the surface of the gel microspheres doped with nanoparticles. The locally enlarged surface is uneven, showing a porous and nanoparticle-like micro-nano surface.

[0108] Figure 5 The scanning electron microscope image of the composite gel microspheres with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:100 (Example 2); MAG = 5.0KX;

[0109] from Figure 5 It can be seen that the higher the content of nanoparticles, the relatively reduced organic polymer matrix, the reduced water content, the increased brittleness, and the fragile microspheres.

[0110] Figure 6-1 This is a cross-sectional scanning electron micrograph of a pure GelMA gel sphere;

[0111] Figure 6-2 is a cross-sectional scanning electron microscope image of the composite gel microsphere;

[0112] from Figure 6-1 It can be observed that the pure GelMA gel microspheres are loose and porous, and cells can easily enter and grow and differentiate. Figure 6-2 It can be observed that the pores of magnesium silicate nanoparticle composite gel microspheres loaded with benidipine are reduced and the degradation is slower.

[0113] Figure 7-1 and 7-2 is the laser confocal image of the benidipine-loaded magnesium silicate nanoparticle composite gel microspheres under GFP (green fluorescent protein), which verifies that the nanoparticles are evenly distributed and relatively dispersed inside the microspheres.

[0114] Figure 8-1 is the laser confocal microscopic image of the composite gel microspheres of pure GelMA (Comparative Example 1) co-cultured with cells;

[0115] Figure 8-2 is the laser confocal microscopic image of the composite gel microspheres of the group with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:50 co-cultured with cells;

[0116] Figure 8-3 is the laser confocal microscopic image of the composite gel microspheres of the group with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:100 co-cultured with cells;

[0117] Figure 8-4 is the laser confocal microscopic image of the composite gel microspheres of the group with a mass ratio of magnesium silicate nanoparticles to GelMA of 1:200 co-cultured with cells.

[0118] From Figure 8-1 and 8-2 , 8-3, 8-4, it can be seen that the green fluorescence shows the distribution of nanoparticles, the blue is the nuclear staining, the red is the phalloidin skeleton staining, and the FITC labels the focal adhesions. The results show that as the ratio of nanoparticles increases, the number of cells on the surface of the microspheres increases, spreads on the surface, and the focal adhesion staining increases, indicating better biocompatibility.

[0119] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantive changes and substitutions made by those skilled in the art based on the present invention fall within the scope of protection required by the present invention.

Claims

1. A method for preparing composite gel microspheres, characterized in that, The following steps are involved: S10, step of preparing silica colloidal spheres: preparing silica colloidal spheres by using the Stober method; S20, synthesis steps of magnesium silicate nanoparticles: weigh 200 mg of silica colloidal spheres and put them into 40 mL of deionized water for ultrasonic dispersion; add 1.5 mmol of magnesium chloride hexahydrate, 20 mmol of ammonium chloride and 2 mL of ammonia water into 40 mL of water and stir to mix; then mix the two and put them into a muffle furnace at 160° C. for 12 hours; cool to room temperature and collect the precipitate by centrifugation, wash it with water and anhydrous ethanol for 3 times respectively, and dry it at 60° C. for 12 hours; set aside; magnesium silicate nanoparticles are porous hollow structures; the average particle size of magnesium silicate nanoparticles is 380 nm; S30, loading step: weigh 6 mg of benidipine and dissolve it in 10 mL of anhydrous ethanol, add it dropwise into 10 mL of an aqueous solution containing 10 mg of magnesium silicate nanoparticles, stir at 500 rpm for 24 h, wash with water 3 times, collect the precipitate by centrifugation, and dry at 60°C; S40, biological ink preparation steps: take a 15mL centrifuge tube, weigh 1g of GelMA freeze-dried sponge, add 0.5% sodium alginate aqueous solution to make a first stock solution with a concentration of 10%, fully dissolve at 50°C, introduce 0.5% of the mass volume ratio of the photoinitiator LAP, fully dissolve and mix; weigh 40mg of magnesium silicate nanoparticles loaded with benidipine, ultrasonically disperse in 4mL of deionized water to make a 10mg / mL loaded material aqueous solution, and set aside; prepare 100mL of 5% calcium chloride aqueous solution as a cross-linking agent; add the loaded material aqueous solution to the GelMA / sodium alginate aqueous solution to obtain biological ink; wherein the mass ratio of magnesium silicate nanoparticles to GelMA is 1:200, 1:100 or 1:50; S50, printing step: adding biological ink to the DLP printer for printing to obtain composite gel microspheres; the specific process is: setting the printing parameters to 20KPa air pressure and 6.66kv voltage; adding the biological ink to the syringe to fix the constant temperature heating belt, connecting the air pressure pump, turning on the DC power supply, checking the status of the printing equipment, placing the microspheres in a 50mL beaker with calcium chloride aqueous solution, irradiating the air-liquid interface with ultraviolet light to quickly solidify the gel microspheres; collecting the precipitate by centrifugation and observing it under an electron microscope, and storing it at -4°C to finally obtain composite gel microspheres.

2. A composite gel microsphere, characterized in that, The composite gel microsphere is prepared by the preparation method of the composite gel microsphere as claimed in claim 1.

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