CN114377212A

CN114377212A - BMP-2 synergistic induction system for bone regeneration and construction method thereof

Info

Publication number: CN114377212A
Application number: CN202210064791.9A
Authority: CN
Inventors: 蒋欣泉; 林思涵; 周名亮; 杨光正; 殷实
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-04-22
Anticipated expiration: 2042-01-20
Also published as: CN114377212B

Abstract

The invention discloses a BMP-2 synergistic induction system for bone regeneration and a construction method thereof, wherein the synergistic induction system comprises a carrier and a synergistic system component loaded on the carrier, and the synergistic system component comprises bone morphogenetic protein BMP-2 and bioactive ion Mg²⁺(ii) a Said Mg²⁺The concentration is 2.5-20 mmol/L; the carrier comprises any one or the combination of more than two of natural polymer materials such as gelatin, methacrylic acid modified gelatin, polycaprolactone, calcium phosphate, titanium alloy and the like and derivatives thereof, synthetic polymer materials and derivatives thereof, inorganic materials, metal materials and the like; the carrier form of the synergistic induction system can be any one of microspheres, a scaffold and a microsphere composite scaffold. The BMP-2 synergistic induction system provided by the invention is assisted by Mg²⁺Assisting BMP-2 in osteogenesis to reduce the dosage of BMP-2 and increase the dosageThe BMP-2 is used for treating the bone regeneration, so that the effects of high efficiency, safety and accurate bone regeneration are achieved.

Description

BMP-2 synergistic induction system for bone regeneration and construction method thereof

Technical Field

The invention relates to the field of bone defect repair materials, in particular to a BMP-2 synergistic induction system for bone regeneration and a construction method thereof.

Background

In recent years, bone defect diseases caused by inflammation, trauma, tumor, deformity and the like become common diseases and frequently encountered diseases which are second to circulatory diseases, respiratory diseases and digestive system diseases and threaten the health of residents in China. With the accelerating aging of population and the change of aging, such as osteoporosis, the pressure of clinical bone defect treatment is further increased, and how to quickly and efficiently complete bone defect repair is of great significance.

The treatment method based on bone morphogenetic protein-2 (BMP-2), also called bone morphogenetic protein, is a commonly used treatment means for bone defect in clinic at present, and the effect similar to or even exceeding the gold standard of bone defect treatment is desirable. However, the dosage of BMP-2 used in clinical application is "super-physiological", and the use of BMP-2 with high dosage often causes complications such as ectopic osteogenesis, inflammation, tumor formation, osteolysis and the like; the existing clinically approved product carrying the BMP-2 uses collagen sponge, has weak slow release effect on the BMP-2, is easy to form factor burst release, and causes the phenomena of uncontrollable osteogenesis range and irregular osteogenesis of new bone structure, thereby obviously reducing the osteogenesis efficiency and doubling the work and half the work; the heavy use of BMP-2 also places a heavy economic burden on the patient.

In conclusion, a method for effectively reducing the dosage of BMP-2, reducing the cost, reducing the risk of side effects, ensuring the osteogenesis efficiency and achieving the effect of achieving the maximum result with little effort is needed.

Disclosure of Invention

The invention aims to solve the problem that the safety and the efficiency of the bone morphogenetic protein BMP-2 used in the bone defect regeneration treatment are difficult to be considered at the same time.

To achieve the above objectThe invention provides a BMP-2 synergistic induction system for bone regeneration, which comprises a carrier and a synergistic system component loaded on the carrier, wherein the synergistic system component comprises bone morphogenetic protein BMP-2 and bioactive ion Mg²⁺(ii) a Said Mg²⁺The concentration of (A) is 2.5-20 mmol/L; the carrier can be prepared from natural polymer materials and derivatives thereof, synthetic polymer materials and derivatives thereof, inorganic materials, metal materials and other materials, and comprises but is not limited to any one or the combination of more than any two of gelatin, sodium alginate, hyaluronic acid, methacrylic acid modified gelatin, methacrylic acid modified sodium alginate, methacrylic acid modified hyaluronic acid, polycaprolactone, calcium phosphate, calcium silicate, titanium alloy and the like; the carrier form of the synergistic induction system can be any one of microspheres, a scaffold and a microsphere composite scaffold.

Preferably, said Mg²⁺The concentration of (b) is 5 to 10 mmol/L.

The invention also provides a construction method of the BMP-2 synergistic induction system for bone regeneration, the carrier form of the synergistic induction system is microsphere, and the preparation method of the microsphere comprises the following steps:

s1, screening Mg²⁺Optimum concentration, in conditioned Medium, Mg²⁺The final concentration is 0.8-20 mmoL/L, and Mg capable of improving osteogenic effect of bone morphogenetic protein BMP-2 is screened²⁺Concentration;

s2, preparing a methacrylic acid modified sodium alginate solution, and mixing bone morphogenetic proteins BMP-2 and Mg²⁺Dissolving the BMP in the mixed solution to obtain BMP-2/Mg by utilizing a microfluidic technology²⁺And (3) microspheres.

Preferably, in S1, the Mg is²⁺The optimum concentration is 5-10 mmoL/L.

Preferably, in S2, the concentration of the methacrylic acid modified sodium alginate solution is 4% to 7%.

Preferably, in S2, the microfluidic technology uses the mixed solution as an aqueous phase and any one of corn oil, paraffin oil, dichloromethane and perfluorinated oil as an oil phase.

The invention also provides a construction method of the BMP-2 synergistic induction system for bone regeneration, the carrier form of the synergistic induction system is a microsphere composite scaffold, and the preparation method of the microsphere composite scaffold comprises the following steps:

s2, preparing a methacrylic acid modified sodium alginate solution, and mixing bone morphogenetic proteins BMP-2 and Mg²⁺Dissolving the BMP in the mixed solution to obtain BMP-2/Mg by utilizing a microfluidic technology²⁺Microspheres;

s3, treating the BMP-2/Mg²⁺The microspheres are embedded into methacrylic acid modified gelatin solution to be used as printing material, and BMP-2/Mg is prepared by utilizing 3D printing technology²⁺A microsphere composite scaffold.

Preferably, in S3, the concentration of the methacrylic acid modified gelatin solution is 4% to 7%.

Preferably, in S3, the BMP-2/Mg²⁺Mg in microspheres²⁺The concentration of (2) was 5 mmol/L.

The invention has the beneficial effects that:

(1) the BMP-2 synergistic induction system constructed by the invention uses Mg²⁺The BMP-2 is assisted to form bone to reduce the dosage of the BMP-2 and ensure the realization of high-efficiency bone formation, namely, the BMP-2 is suitable for promoting the bone formation effect of low-dosage BMP-2, and the safety and economic problems possibly brought by clinical use of the super-physiological dosage of the BMP-2 are solved;

(2)Mg²⁺the concentration is in the range of 2.5-20mmol/L, the osteogenesis effect of BMP-2 can be improved, and the optimal concentration is preferably in the range of 5-10 mmol/L;

(3) the preparation method comprises the following steps of selecting a natural polymer material and derivatives thereof, a synthetic polymer material and derivatives thereof, an inorganic material, a metal material and the like, wherein the natural polymer material and derivatives thereof, the synthetic polymer material and derivatives thereof, the inorganic material, the metal material and the like comprise but are not limited to any one or combination of more than any two of gelatin, sodium alginate, hyaluronic acid, methacrylic acid modified gelatin, methacrylic acid modified sodium alginate, methacrylic acid modified hyaluronic acid, polycaprolactone, calcium phosphate, calcium silicate, titanium alloy and the like as factors and an ion carrier, the carrier can be a bracket, the early-stage load of a bone defect area can be realized through the bracket, the carrier can also be microspheres, the injection type noninvasive delivery of the carrier can be realized in the form of the microspheres, and the carrier can also be a combination of two forms, so that the application range of a system is expanded;

(4) the invention does not need special and complex equipment, has wide raw material source and simple operation flow, and is beneficial to popularization and use.

Drawings

FIG. 1 shows the cells in BMP-2 and Mg at various concentrations²⁺Synergistic alkaline phosphatase staining results.

FIG. 2 shows the cells in BMP-2 and Mg at various concentrations²⁺Analysis of expression of osteogenesis-related genes under synergistic stimulation (#, ###) indicates differences p from Con group and BMP-2 group, respectively<0.01)。

FIG. 3 shows cells in Mg²⁺And the immunofluorescence staining pattern of the expression condition of osteocalcin (Ocn) which is an osteogenic differentiation marker under the synergistic stimulation of different concentrations of BMP-2.

FIG. 4 shows the BMP-2 co-induction system obtained by the preparation.

Fig. 5 is a quantitative analysis of the in vivo osteogenesis effect and related parameters of the BMP-2 co-induction system by X-ray and CT reconstruction analysis (X and # # indicate p <0.01 difference from 20 μ g/mL and 50 μ g/mL groups, respectively).

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.

Magnesium (Mg) is the fourth most abundant metal element in the human body and plays an important role in bone development and reconstruction. Research shows that magnesium deficiency may cause osteogenesis inhibition, osteoporosis and other problems, magnesium transport receptor high expression phenomenon exists in the chondrogenesis area in the embryonic development, and Mg is locally enriched²⁺Can simulate bone development microenvironment to induce vascularization bone regeneration and display Mg²⁺Good bone regeneration induction ability, in particular, BMP-2 induced bone regeneration process is similar to the process of bone formation in embryonic cartilage. Therefore, different concentrations of Mg were explored²⁺Has osteogenic effect on BMP-2The method is of great significance.

Mg²⁺Screening for optimum concentration

1 test materials and test cells

1.1 test cells: rat Bone Marrow Stem Cells (BMSCs) isolated from 3-week-old Sprague Dawley rats.

1.2 Experimental materials: the recombinant BMP-2 is purchased from Shanghai Ribang biomaterial, Inc., an alkaline phosphatase staining solution kit, absolute ethyl alcohol, an RNA extraction kit, a reverse transcription kit and the like.

1.3 experimental equipment: a fluorescent quantitative PCR instrument, a constant temperature box, a microscope, a centrifuge, a liquid-transferring gun, a spectrophotometer and the like.

2 course of experiment

2.1Mg²⁺Preference for increasing the osteogenic Effect concentration of the bone morphogenetic protein BMP-2

Inoculating BMSCs in good growth state into a culture plate, adding a conditioned medium, wherein the final concentration of BMP-2 in the conditioned medium is 100ng/mL, and Mg is added²⁺The final concentrations are respectively 0.8 (namely BMP group), 1.6, 2.5, 5, 10 and 20 mmol/L; in a common complete medium, i.e., without BMP-2 (factor), Mg²⁺The final concentration of (ions) was 0.8mmol/L as control (extracellular Mg under physiological conditions)²⁺Concentration, i.e., Con group) as shown in table 1. After incubation for 7 days, ALP staining is carried out on the cells treated differently, cell RNA is collected, reverse transcription fluorescence quantitative PCR analysis is carried out, and the osteogenic differentiation degree of BMSCs under different stimuli is detected, so that the osteogenic induction effect of the culture solution of each group is judged.

Table 1: mg (magnesium)²⁺Concentration setting for experiments to enhance osteogenic effect of bone morphogenetic protein BMP-2

2.2 Mg²⁺Evaluation of promoting osteogenic Effect of Low dose BMP-2

Inoculating BMSCs with good growth state into the culture plate, adding a conditioned medium, wherein Mg in the conditioned medium²⁺The final concentration is 5mmol/L, and the final concentration of BMP-2 is 0 (namely Mg group)20, 50, 100 ng/mL; in a common complete medium, i.e. without BMP-2, Mg²⁺A final concentration of 0.8mmol/L is used as a control (i.e., Con group) and is shown in Table 2. After 7 days of culture, the cells were harvested, fixed and then subjected to immunofluorescence staining to analyze the expression of osteocalcin (Ocn), an osteogenic marker, to thereby evaluate Mg²⁺Whether the osteogenic effect of low dose of BMP-2 can be improved.

Table 2: mg (magnesium)²⁺Concentration setting for improving osteogenic effect experiment of low-dose BMP-2 with different concentrations

3 results of the experiment

After performing the experiments according to the grouping of Table 1, the results are shown in FIG. 1, BMSCs at BMP-2 and different concentrations of Mg²⁺Under the synergistic stimulation, different effects of alkaline phosphatase staining appear. When Mg²⁺Tissue staining became darker and darker with increasing concentration, especially Mg²⁺The tissue staining color is deepest when the concentration reaches 5mmol/L, and the tissue staining color is deepest along with Mg²⁺At concentrations above 10mmol/L, the tissue staining color began to fade gradually to BMP-2 group levels.

After experiments according to the groupings in Table 1, the results are shown in FIG. 2 when Mg²⁺After the concentration exceeds 2.5mmol/L, the expression level of osteogenic related genes Sp7, Alp, Opn and Runx-2 is obviously increased, which indicates that Mg²⁺Begin to contribute to the osteogenic effect of BMP-2; when Mg²⁺When the concentration is 5-10mmol/L, particularly 5mmol/L, the expression levels of the osteogenesis related genes Sp7, Alp, Opn and Runx-2 reach peak, which indicates that Mg²⁺The promotion effect on the osteogenesis effect of BMP-2 is strongest; when Mg²⁺After the concentration exceeds 10mmol/L, the promotion effect begins to fall back; when Mg²⁺Mg at a concentration of 20mmol/L²⁺Has little effect of promoting the osteogenic induction of BMP-2.

After the experiment was performed according to the grouping of table 2, the results are shown in fig. 3, and there were 8 groups of sample data. When Mg²⁺At a concentration of 0.8mmol/L, Ocn expression decreased gradually with decreasing BMP-2 dose, of which 20 groups (20ng/mL BMP-2+0.8mmol/L Mg²⁺) Ocn expression level and Mg group (0ng/mL BMP-2+5mmol/L Mg)²⁺) Near, and added Mg²⁺After the concentration is 5mmol/L, the expression level of Ocn induced by low-dose BMP-2 is obviously improved, and the expression levels of Ocn in the 20+ Mg groups are obviously higher than those in the 20 and 50 groups and are close to the 100 group.

In conclusion, Mg²⁺The concentration is in the range of 2.5-20mmol/L, the osteogenesis effect of BMP-2 can be improved, and the optimal concentration is preferably in the range of 5-10mmol/L, and the osteogenesis effect of low-concentration BMP-2 can be obviously improved. The BMP-2 synergistic induction system is guided to be constructed according to the concentration.

Example construction of BMP-2 synergistic Induction System (microsphere composite scaffold)

The BMP-2 synergistic induction system is constructed by using any one or a combination of any two or more of natural polymer materials and derivatives thereof, synthetic polymer materials and derivatives thereof, inorganic materials, metal materials and the like as factors and an ionic carrier, wherein the carrier is not limited to any one or a combination of any two or more of Gelatin (Gelatin), Sodium Alginate (Sodium Alginate), Hyaluronic Acid (Hyaluronic Acid), methacrylic Acid modified Gelatin (GelMA), methacrylic Acid modified Sodium Alginate (AlMA), methacrylic Acid modified Hyaluronic Acid, polycaprolactone, calcium phosphate, calcium silicate, titanium alloy and the like. And the cooperative induction system with different forms can be prepared by combining a microfluidic technology and a 3D printing technology, and the form of the cooperative induction system can be any one of microspheres, a stent and a microsphere composite stent. In this example, the preparation process of the microsphere composite scaffold is further described in detail.

1 test materials and test cells

1.1 Experimental animals: sprague Dawley rats at 8 weeks of age.

1.2 Experimental materials: BMP-2, Mg²⁺The solvent comprises a solvent, a methacrylic acid modified sodium alginate solution, Span80, methacrylic acid modified gelatin, a formalin solution, a 3% pentobarbital solution, a LAP photoinitiator and the like.

1.3 experimental equipment: the device comprises a micro-fluidic chip, a micro-injection pump, an ultraviolet lamp irradiation instrument, a particle size analyzer, a 3D printer, a fluorescence microscope, a scalpel, a micro-CT imager, an X-ray detector, a baking sheet machine, a slicing machine and the like.

2 course of experiment

2.1 preparation of BMP-2/Mg²⁺Microspheres

Preparing 6% AlMA solution containing 0.25% LAP photoinitiator, and mixing BMP-2 and Mg²⁺Dissolving in water, using corn oil containing 1% Span80 as oil phase, pumping water phase and oil phase at two inlets of the microfluidic chip respectively by using micro-sampling pump, controlling oil phase flow rate at 10mL/h, and forming uniform BMP-2/Mg in the oil phase as shown in A of figure 4²⁺And (3) microspheres. BMP-2/Mg was collected at the chip outlet²⁺Irradiating microsphere with ultraviolet light of 395nm wavelength for 20s to make BMP-2/Mg²⁺The microspheres are photo-cured to obtain BMP-2/Mg²⁺And (3) microspheres. The microspheres can also be used alone as a carrier for a synergistic induction system.

2.2 BMP-2/Mg²⁺Preparation of microsphere composite scaffold

BMP-2/Mg obtained in the above experiment step 2.1²⁺The mass ratio of the microspheres is 1: 10 is embedded in 6 percent GelMA containing 0.25 percent LAP photoinitiator, the system is placed in 4 ℃ to be cooled for 10 minutes, then is balanced for 30 minutes at 22 ℃, printing parameters are adjusted, and 3D printing is carried out by a 3D printer. For the observation of BMP-2/Mg²⁺Distribution of microspheres in GelMA scaffold, preparation of BMP-2/Mg by using red fluorescence-labeled AlMA²⁺Preparing the composite scaffold by microspheres according to the conditions to obtain BMP-2/Mg²⁺A microsphere composite scaffold.

2.3 rat skull defect model preparation

SD rats of 8 weeks old are weighed and anesthetized by injecting 3% pentobarbital solution at a dose of 100-200 muL/100 g body weight. After leg clamping reflection and corneal reflection of a rat disappear, shaving hairs, disinfecting a skull top operation area by using 75% alcohol, making a sagittal incision along a skull median suture to periosteum, separating the periosteum, preparing circular bone defects with the diameter of 5mm at symmetrical positions on two sides of the skull by using a bone taking drill of a planting machine, and flushing a drill bit by PBS (phosphate buffered saline) for cooling.

2.4 BMP-2/Mg²⁺Microsphere composite stent implantation

In vivo experiments were performed by implanting several sets of scaffolds matched in concentration as shown in table 3 at the defect: GelMA/AlMA microsphere composite scaffold group (Con group); GelMA/(20 mu g/mLBMP-2) AlMA microsphere composite scaffold group (20 group); GelMA/(20 mu g/mL BMP-2+5mmol/LMg2+) AlMA microsphere composite bracket group (20+ Mg group); GelMA/(50 mu g/mL BMP-2) AlMA microsphere composite scaffold group (50 groups); GelMA/(100 mu g/mL BMP-2) AlMA microsphere composite scaffold group (100 group); blank defects were used as control groups (Blank groups), and the amount of each sample n was 5. Sequentially suturing periosteum and skin, sterilizing the operation area again, observing the breath and the state of the rat, confirming the stable state of the rat, marking, and returning to the mouse cage.

Table 3: setting of AlMA microsphere concentration in different GelMA/AlMA microsphere composite scaffold groups

2.5 evaluation of in vivo efficacy of BMP-2 Co-Induction System

Four weeks after surgery, rats were euthanized and specimens were fixed with 10% formalin for 12-24 hours; the samples are washed by clean water for 30 minutes, and the condition of each component bone is qualitatively and quantitatively detected by utilizing X-ray and micro-CT imaging.

3 results of the experiment

FIG. 4 shows a BMP-2 co-induction system prepared by microfluidic technology, wherein A in FIG. 4 shows BMP-2/Mg obtained by "water-in-oil" method²⁺Microspheres, B of FIG. 4 shows BMP-2/Mg prepared under these conditions²⁺The microsphere size is uniformly distributed about 100 μm, and C in FIG. 4 shows BMP-2/Mg²⁺The microspheres are well supported in the scaffold.

FIG. 5A is a schematic diagram of the in vivo osteogenesis effect of BMP-2 co-induction system analyzed by X-ray and CT reconstruction. The Blank group and the Con group have no new bone formation basically, 20 groups have a certain new bone formation, the bone repair effect is gradually improved along with the increase of the dosage of BMP-2, and the formation amount of the new bone is the largest in 100 groups. Different from the 20 groups, the 20+ Mg group has obvious bone regeneration, the effect is better than the 50 groups, and the physiological structure of the skull is basically recovered by being close to the 100 groups. Quantitative analysis is carried out on relevant parameters of new bone formation in the defect area, as shown in B of figure 5, and in terms of bone volume/total volume BV/TV (%) numerical values, Blank group is 5.674 +/-0.960, Con group is 11.940 +/-1.136, 20 group is 20.860 +/-1.521, 20+ Mg group is 33.170 +/-1.730, 50 group is 27.370 +/-1.986, 100 group is 43.970 +/-1.479; in terms of BMD (mgHA/cc) value measured by bone density, Blank set is 38.550 ± 7.575, Con set is 81.340 ± 6.004, 20 set is 123.300 ± 7.349, 20+ Mg set is 206.900 ± 8.599, 50 set is 175.900 ± 8.507, and 100 set is 266.800 ± 12.780.

In conclusion, the low dose of BMP-2 in Mg²⁺Has the function of high-efficiency and rapid bone regeneration under the assistance of (2).

The method of the invention can also directly prepare the bracket by adding BMP-2 and Mg²⁺The scaffold is obtained by directly dissolving in GelMA, and can also be used as a carrier of a synergistic induction system. In conclusion, the invention provides a BMP-2 synergistic induction system for rapid and efficient regeneration of bone defects, namely screening proper Mg²⁺Concentration, co-loading with BMP-2 in a carrier material to form BMP-2 and Mg²⁺With the aid of Mg²⁺The BMP-2 is assisted to form bone so as to reduce the dosage of the BMP-2 and simultaneously expand the treatment effect of the low-dosage BMP-2, thereby achieving the effects of high efficiency, safety and accurate bone regeneration.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Claims

1. BMP-2 synergistic induction system for bone regeneration, which is characterized by comprising a carrier and a synergistic system component loaded on the carrier, wherein the synergistic system component comprisesContains bone morphogenetic protein BMP-2 and bioactive ion Mg²⁺；

Said Mg²⁺The concentration of (A) is 2.5-20 mmol/L; the carrier comprises any one or the combination of more than two of gelatin, sodium alginate, hyaluronic acid, methacrylic acid modified gelatin, methacrylic acid modified sodium alginate, methacrylic acid modified hyaluronic acid, polycaprolactone, calcium phosphate, calcium silicate and titanium alloy;

the carrier form of the synergistic induction system can be any one of microspheres, a scaffold and a microsphere composite scaffold.

2. BMP-2 co-induction system for bone regeneration according to claim 1, wherein Mg is present²⁺The concentration of (b) is 5 to 10 mmol/L.

3. A method for constructing BMP-2 synergistic induction system for bone regeneration in accordance with claim 1, wherein the carrier form of synergistic induction system is microsphere, and said microsphere preparation method comprises the following steps:

s1, screening Mg²⁺Optimum concentration: in conditioned media, Mg²⁺The final concentration is 0.8-20 mmoL/L, and Mg capable of improving osteogenic effect of bone morphogenetic protein BMP-2 is screened²⁺Concentration;

s2, preparing a methacrylic acid modified sodium alginate solution, and mixing bone morphogenetic proteins BMP-2 and Mg²⁺Dissolved therein as a mixed solution so that Mg is present²⁺At the concentration of said Mg²⁺BMP-2/Mg is prepared and obtained by utilizing the microfluidic technology within the optimal concentration range²⁺And (3) microspheres.

4. The method of claim 3, wherein the Mg 1 is Mg²⁺The optimum concentration is 5-10 mmoL/L.

5. The method for constructing the BMP-2 cooperative induction system for bone regeneration according to claim 3, wherein in S2, the concentration of the methacrylic acid modified sodium alginate solution is 4% to 7%.

6. The method of claim 3, wherein in step S2, the mixed solution is used as a water phase, and any one of corn oil, paraffin oil, dichloromethane and perfluorinated oil is used as an oil phase in the microfluidic technology.

7. A method for constructing a BMP-2 cooperative induction system for bone regeneration in accordance with claim 1, wherein the carrier of the cooperative induction system is a microsphere composite scaffold, and the method for preparing the microsphere composite scaffold comprises the following steps:

s2, preparing a methacrylic acid modified sodium alginate solution, and mixing bone morphogenetic proteins BMP-2 and Mg²⁺Dissolving in the solution, and preparing BMP-2/Mg by using a microfluidic technology²⁺Microspheres;

8. The method of claim 7, wherein the concentration of the methacrylic acid modified gelatin solution in S3 is 4-7%.

9. The method for constructing BMP-2 together with inducible system for bone regeneration of claim 7, wherein in S3, said BMP-2/Mg²⁺In the microspheres, Mg²⁺The concentration of (2) was 5 mmol/L.