Ready-to-use mesoporous mineralized silk fibroin microspheres and applications thereof

Abstract

The application provides a ready-to-use mesoporous mineralized silk fibroin microsphere and application thereof, and the mesoporous mineralized silk fibroin microsphere (referred to as mSilMA) is prepared by the following steps: preparing a SilMA hydrogel microsphere through a photo-crosslinking oil / water emulsion method, and then combining a biomimetic mineralization method and a critical point drying method. The silk fibroin material which is not easy to degrade and has good biocompatibility is selected as a carrier, so that the problems of easy loss and long-term retention of BMP-2 are solved; the biomimetic mineralization forms a calcium phosphate coating to realize efficient loading and controlled release of BMP-2; the critical point drying forms a mesoporous structure, which is beneficial to drug loading and release. The mSilMA is applied to loading and controlled release of BMP-2, and the protein loading efficiency is high and can be long-acting and low-dose released. The application has simple components, mild preparation conditions and easy acquisition, provides a new way for bone defect repair, and has a wide clinical application prospect.

Description

[0001] The present application is a divisional application of the invention patent application No. 202411111857.0, entitled "Ready-to-use mesoporous mineralized silk fibroin microspheres, preparation method and application thereof". TECHNICAL FIELD

[0002] The present application belongs to the field of biomedical materials, and specifically relates to a ready-to-use mesoporous mineralized silk fibroin microsphere and application thereof. BACKGROUND

[0003] BMP-2 (bone morphogenetic protein-2) plays a key role in bone defect repair due to its strong osteogenic ability. Since the regeneration of damaged bone tissue is a dynamic process involving coordinated responses of cells and biomolecules over several weeks, the ability to maintain BMP-2 with biological activity for a longer period of time in bone defects is expected to improve the bone healing response. However, the short in vivo retention time, high dose serious side effects, and early burst release that promote inflammation are common problems with current BMP-2 clinical products. Therefore, developing a ready-to-use delivery carrier that can long-acting low-dose release BMP-2, promote bone regeneration, and is simple and safe is an important issue in the field of bone repair.

[0004] The main defect of sponge collagen in delivering BMP-2 is the rapid release of BMP-2, which has been widely confirmed in vivo / in vitro and clinically. 40%-90% of BMP-2 is released in an initial burst mode. Only 10% or less of BMP-2 can be retained at the implant site after 1-2 weeks, which leads to the use of higher doses, thereby exacerbating local and systemic adverse reactions (inflammation, ectopic ossification, and cysts, etc.).

[0005] Calcium phosphate materials have significant advantages in bone induction and bone conduction, but they degrade quickly in the body and cannot sustain the release of BMP-2.

[0006] In addition to the currently clinically transformed collagen-based and calcium-phosphorus-based BMP-2 bone implants, numerous new BMP-2 carriers for bone regeneration have been reported in the literature, but they have complex components, multiple preparation processes, are not easy to obtain immediately, and have slow clinical transformation. For example, Liu et al. prepared a PLGA scaffold, treated it with plasma technology to increase its surface hydrophilicity, then immersed it in a mixture of zwitterionic monomers, cross-linking agents, and photoinitiators, and formed a zwitterionic hydrogel coating on the surface of the PLGA scaffold through UV curing technology. Further, the PLGA / zwitterionic hydrogel was immersed in a calcium-phosphorus mineralization solution to form calcium phosphate mineralization on the surface of the scaffold. The mineralized PLGA / zwitterionic hydrogel was freeze-dried to form a scaffold that can release BMP-2 at a low amount. This design achieves controlled release of BMP-2 through the charge interaction between zwitterionic hydrogel and BMP-2 and the strong affinity of calcium phosphate mineralization for BMP-2 (Literature source: Liu P, Bao T, Sun L, et al. In situ mineralized PLGA / zwitterionic hydrogel composite scaffold enables high-efficiency rhBMP-2 release for critical-sized bone healing [J]. Biomaterials Science, 2022); However, the PLGA scaffold has the following disadvantages: First, the PLGA scaffold after multiple steps of processing has a complex and time-consuming preparation process, high cost, and is not conducive to large-scale production and rapid application; Second, the structure and performance of the scaffold may be affected by multiple processing steps, and there are problems in stability and repeatability; In addition, due to the involvement of multiple components and complex processes in the preparation process, the biocompatibility and biodegradability of the scaffold may also be affected to some extent. Therefore, the PLGA scaffold cannot achieve rapid preparation and efficient mass production, and it is difficult to quickly meet the immediate needs of bone defect repair in actual clinical applications, and its role in rapid transformation and application in clinical treatment is limited.

[0007] Therefore, there is an urgent need to find a biomaterial carrier that can solve the problems of rapid release, short retention time, complex carrier components, etc. in existing BMP-2 controlled release carriers, has the characteristics of simple preparation process, low cost, good biocompatibility, can achieve long-acting and low-dose release of BMP-2, avoid early burst release, maintain its biological activity, promote bone regeneration, has good clinical transformation potential and application prospects, and provides a more effective solution for bone defect treatment. SUMMARY

[0008] To solve the problems in the prior art, the application provides a ready-to-use mesoporous mineralized silk fibroin microsphere and a preparation method and application thereof, the ready-to-use dry mesoporous mineralized silk fibroin microsphere (mSilMA) is successfully prepared by using silk fibroin methacrylamide as raw material and comprehensively using a biomimetic mineralization method and a critical point drying method; the silk fibroin is a regular directional fiber structure formed by layered assembly of nanofibrils, and contains a large number of polar amino acids; the hydrophilic and polar groups can be combined with positive and negative ions closely, and can induce nucleation and growth of inorganic substances on the silk fibroin template. The effective loading rate of each milligram of the mSilMA microsphere prepared in the application to BMP-2 (500 ng) is more than 85%, and in vitro slow release detection shows that the mSilMA microsphere can continuously release BMP-2 at a low amount, and the cumulative release of BMP-2 is about 2% in vitro for 4 weeks, so that the long-acting and low-dose release of BMP-2 in vitro is realized, and no early burst release occurs.

[0009] To achieve the above object, the application adopts the following scheme:

[0010] In one aspect, the application provides a ready-to-use mesoporous mineralized silk fibroin microsphere, the main component of the ready-to-use mesoporous mineralized silk fibroin microsphere has a fiber structure, the microsphere has a mesoporous pore structure, and the surface of the microsphere comprises a mineralized coating.

[0011] Further, the main component comprises silk fibroin; and the silk fibroin is silk fibroin methacrylamide.

[0012] Further, the mineralized coating is needle-shaped calcium phosphate; and the pore size of the mesoporous pore is 5-50 nm.

[0013] To solve the problems of rapid release, short retention time and complex carrier components of the BMP-2 controlled release carrier in the current bone repair field, based on the characteristic that the silk fibroin has a natural fiber structure, the application provides a ready-to-use mesoporous mineralized silk fibroin microsphere capable of long-acting and low-dose release of bone morphogenetic protein, the microsphere is formed into a mesoporous pore structure by using silk fibroin methacrylamide as a main component and using the fiber structure characteristic thereof through a specific preparation method, and a needle-shaped calcium phosphate mineralized coating is formed on the surface of the microsphere.

[0014] The loading efficiency of the microsphere prepared in the application to BMP-2 can be more than 85%, and the long-acting and low-dose release of BMP-2 can be realized, and the cumulative release of BMP-2 is only about 2% in vitro for 4 weeks. In addition, the microsphere has good biocompatibility and stability, can exist stably in a bone defect implantation site for a long time, continuously plays a role in promoting bone repair and regeneration, and provides a high-efficiency and application-prospective solution for bone defect treatment.

[0015] Further, the ready-to-use mesoporous mineralized silk fibroin microspheres are loaded with active proteins, which include any one or more of BMP-2, VEGF, FGF, and TGF-β.

[0016] Further, the active protein is BMP-2.

[0017] The ready-to-use mesoporous mineralized silk fibroin microspheres are loaded with active proteins, which include bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and the like.

[0018] In the present application, the microspheres are particularly selected to be loaded with BMP-2 because existing BMP-2 controlled release carriers have problems such as rapid release, short retention time, and complex carrier components. However, when the microspheres of the present application are used to load BMP-2, the loading efficiency of BMP-2 can be significantly improved, and long-term low-dose release can be achieved. Specifically, the mesoporous structure of the microspheres increases the specific surface area, providing more binding sites for BMP-2 and thus increasing the loading capacity. At the same time, the mineralized coating on the surface of the microspheres effectively controls the release rate of BMP-2 through interaction with BMP-2, avoids early burst release, and enables BMP-2 to continuously exert its effect for a longer period of time, thereby promoting the regeneration and repair of bone tissue. In addition, the good biocompatibility and stability of silk fibroin ensure the safety and effectiveness of the microspheres in the body, providing an efficient and reliable solution for bone defect treatment.

[0019] In some embodiments, the BMP-2 protein loading rate and long-term low-dose release capacity of the microspheres are compared and screened for different main components, different drying methods, different mineralization methods, different mineralization concentrations, microspheres without drying, and microspheres without mineralization, respectively. The experimental results show that only when silk methacrylamide is used as the main component, critical drying is used for drying, and the Ca / P method is used with a mineralization liquid concentration of 1 times the Ca / P mineralization liquid, can the mSilMA microspheres with surface-through mesoporous channels and complete structure be successfully prepared, and the BMP-2 protein loading rate can reach more than 85%, while continuously releasing BMP-2 at a low dose, with a cumulative release of about 2% of BMP-2 in vitro for 4 weeks. When any of the conditions is replaced, the effect of the present application cannot be achieved.

[0020] In the field of bone repair, achieving long-term low-dose release of bone morphogenetic protein (BMP-2) has been a key problem that needs to be solved. The present application aims to break through this difficult problem and successfully prepares ready-to-use mesoporous mineralized silk fibroin microspheres (mSilMA) using silk fibroin as raw material and combining biomimetic mineralization method and critical point drying method.

[0021] Silk fibroin as raw material is the basis of the present application, which has unique fiber structure and excellent biocompatibility. If this raw material is absent or replaced by other materials, it cannot provide a suitable carrier for subsequent mineralization and drying steps, and cannot achieve the loading and controlled release of BMP-2.

[0022] The biomimetic mineralization method (calcium chloride / phosphoric acid-ammonia reaction method) is indispensable in the preparation process. Through this method, a calcium phosphate mineralized coating is formed on the surface of the silk fibroin microspheres, which on the one hand realizes efficient loading by using the strong affinity of calcium phosphate for BMP-2, and on the other hand greatly reduces the release rate of BMP-2. If the biomimetic mineralization step is absent or other mineralization methods and concentrations are used, the mineralized coating cannot be formed, and even if the silk fibroin raw material and the critical point drying method are used, due to the lack of strong affinity and controlled release of BMP-2, effective loading and long-term stable controlled release of BMP-2 cannot be achieved, and the effect of long-acting low-dose sustained release cannot be achieved.

[0023] The critical point drying method is the key step to form mesoporous structure. The mesoporous pore structure can provide more binding sites for BMP-2, which is conducive to the residence and sustained release of cytokines and enhances the interaction between the microspheres and the body. If only the silk fibroin raw material and the biomimetic mineralization method are used, without the formation of mesoporous pore structure by critical point drying, the cytokine binding sites are limited, and efficient loading and slow and sustained release of BMP-2 cannot be achieved; and when other drying methods are used, mesoporous pores cannot be formed on the basis of the same technical scheme of the present application.

[0024] In summary, only when silk fibroin is used as raw material, and biomimetic mineralization method (calcium chloride / phosphoric acid-ammonia reaction method) and critical point drying method are used in combination, all of which are indispensable, can the mSilMA with the best effect be successfully prepared, the long-acting low-dose sustained release, high loading efficiency and good biocompatibility of BMP-2 can be achieved, and an ideal material for bone repair can be provided.

[0025] On the other hand, the present application provides a preparation method of a ready-to-use mesoporous mineralized silk fibroin microsphere, comprising the following steps:

[0026] (1) Preparation of SilMA hydrogel microspheres;

[0027] (2) Mineralization of SilMA hydrogel microspheres;

[0028] (3) Drying of the mineralized SilMA hydrogel microspheres to obtain dry mesoporous mineralized SilMA microspheres;

[0029] In the step (1), the SilMA hydrogel microspheres are prepared by using ultraviolet light crosslinking oil / water emulsion method and mechanical stirring; in the step (2), the mineralization is performed by using Ca / P method; and in the step (3), the drying is performed by using critical point drying method. The gelatin methacrylamide hydrogel microspheres (GelMA for short) are subjected to the same ethanol gradient dehydration and critical point drying, and no surface-through mesoporous structure is formed, but a non-porous structure is presented; and the SilMA microspheres form a surface interpenetrating mesoporous channel structure. Therefore, it can be initially known that not all materials can form mesopores by critical point drying.

[0030] In some embodiments, when the SilMA is prepared, different preparation methods are screened, and the experimental results show that: the SilMA prepared by the ultraviolet light crosslinking oil / water emulsion method (mechanical stirring) of the application has obvious improvement in microsphere yield and sphericity, and has significant advantages in yield and sphericity compared with the microspheres prepared by the light crosslinking 3D printing technology; and the microspheres prepared by the light crosslinking 3D printing technology are easy to adhere and cannot be separated; the sphericity is poor, and generally presents a cylindrical body. In addition, the mechanical stirring method used in the application can change the oil / water ratio, stirring speed and other parameters, so as to successfully prepare microspheres with a particle size range of 1 μm-1000 μm, but the light crosslinking 3D printing technology cannot print microspheres below 100 μm, and can only print microspheres of specific sizes, such as 200 μm, 400 μm, etc.; in addition, the yield of the microspheres is limited by the volume of the printing ink and the size of the printable stage.

[0031] Therefore, it is preferred to prepare the SilMA by using the ultraviolet light crosslinking oil / water emulsion method (mechanical stirring), which has the best yield, sphericity, controllable particle size range and other performances, and can better meet the needs of practical applications, and lays a solid foundation for subsequent preparation of mSilMA with excellent performance.

[0032] In some embodiments, to obtain the microspheres with long-acting low-dose sustained-release BMP-2 with the best performance in the application, the main components of the microspheres are first screened. The main components of the microspheres are silk fibroin methacrylamide and gelatin methacrylamide, respectively, and GelMA microspheres and SilMA microspheres are prepared, respectively. The experimental results under scanning electron microscopy show that: after the same ethanol gradient dehydration and critical point drying, the GelMA hydrogel microspheres do not form a surface-penetrating mesoporous structure, but present a non-porous structure; while the SilMA microspheres form a surface interconnected mesoporous channel structure. Therefore, it can be initially known that not all materials can form mesopores by critical point drying. According to the above results, the inventors speculate that because silk fibroin has a original fiber structure, through critical point drying, its own fiber network can be reproduced; while gelatin itself does not have a fiber structure, and in the process of ethanol gradient dehydration, as the water is lost, the gelatin itself gradually densifies the hydrogel network structure, and then through critical point drying, the fiber network structure cannot appear, so the surface is dense and non-porous; therefore, it is speculated that if the material itself has a fiber structure, critical point drying can produce a porous structure; if the material itself does not have a fiber structure, then critical point drying cannot produce a porous structure.

[0033] In the application, the mesoporous channel structure is extremely important for preparing the hydrogel microspheres with long-acting low-dose sustained-release BMP-2. The mesopores can significantly improve the drug loading capacity, the interconnected mesoporous channels are conducive to the loading and slow and continuous release of cytokines, avoid burst release, prolong the action time of BMP-2, improve the utilization efficiency and treatment effect of BMP-2, and provide stable support for bone repair; at the same time, it is helpful for the material exchange and information transmission between the microspheres and the surrounding environment, and conducive to the growth and proliferation of cells in the interstitial space of the microspheres, and improves the adaptability and biocompatibility of the microspheres in the body.

[0034] Therefore, SilMA which can form mesopores is preferably used as the main component of the microspheres to achieve more ideal drug loading and controlled release effect, and provide strong technical support and innovative ideas for the development of the field of bone tissue engineering and the like.

[0035] In some embodiments, the drying method of the SilMA hydrogel microspheres is screened during the preparation of the SilMA microspheres. The experimental results show that, by using the critical point drying method, the dry SilMA microspheres prepared do not have the phenomenon of adhesion, and the surface of the microspheres has uniformly distributed mesopores. The uniform mesoporous structure is conducive to the loading and slow release of drugs, can provide a more stable and controllable release environment for BMP-2 and other drugs, thereby better exerting the effect of BMP-2 and improving the therapeutic effect. By using the freeze-drying method, the surface of the dry SilMA microspheres cannot form mesopores, which limits the loading and slow release of BMP-2, and the microspheres are adhered to each other and have a rough surface. Such a rough surface and adhesion phenomenon can affect the dispersibility and stability of the microspheres, and thus affect the effect in actual application. Therefore, the critical point drying method is preferably used to prepare dry SilMA microspheres.

[0036] In some embodiments, different mineralization methods are screened during the preparation of the mSilMA microspheres. The experimental results show that the Ca / P method can form a more uniform and higher density calcium phosphate mineralized coating on the surface of the SilMA microspheres. The more uniform and higher density calcium phosphate mineralized coating can enhance the stability and mechanical strength of the microspheres, improve the compatibility and binding capacity of the microspheres with bone tissue, and help better promote the bone regeneration and repair process. In addition, the uniform and high-density coating can more effectively control the release rate of BMP-2, provide a more stable loading and release environment for BMP-2, and thus improve the therapeutic effect of BMP-2. Therefore, the Ca / P method is preferably used as the mineralization method of the SilMA microspheres.

[0037] Further, the mineralization concentration is 1 times the Ca / P mineralization solution, and the molar ratio of calcium chloride to phosphoric acid in the 1 times Ca / P mineralization solution is 1.5-2.0.

[0038] Meanwhile, in some embodiments, the concentration of different Ca / P mineralization solutions is screened during the preparation of the mineralized SilMA hydrogel microspheres by using the Ca / P method, and the effect of different Ca / P mineralization solution concentrations on the SilMA microspheres and the surface calcium phosphate mineralized layer is studied. The experimental results show that, under the condition that other conditions remain unchanged, increasing the concentration of the Ca / P mineralization solution will lead to the destruction of the microsphere structure. The calcium phosphate coating still maintains a needle-like structure, and with the increase of the Ca / P mineralization solution concentration, the coating density increases. However, 5 times and 10 times the Ca / P mineralization solution significantly destroys the microsphere structure. Therefore, 1 times the Ca / P mineralization solution is preferably used, under which the complete structure of the microspheres is retained, and there are relatively uniformly distributed needle-like calcium phosphate coatings on the surfaces of 20% of the SilMA microspheres. Meanwhile, the mesoporous channels of the microspheres are not completely blocked by the needle-like calcium phosphate coatings.

[0039] and under the concentration of 1 times Ca / P mineralization solution, the pore size of the dry mSilMA surface after mineralization and critical drying will be further reduced. This has the following advantages: (1) better controlled release performance: smaller pore size can more effectively load and control the release rate and release amount of drugs or cytokines, so that it can be continuously and stably released for a longer period of time, improving the treatment effect; (2) enhanced stability: smaller pore size can make the microsphere structure more stable, reduce the influence of the external environment on the internal loaded substances, and improve the stability of the microspheres during storage and use; (3) improved selectivity: it can more selectively allow specific size molecules to pass through or be adsorbed, thereby achieving more precise drug loading or dissociation; (4) reducing the burst release phenomenon: reducing the sudden massive release of drugs in the initial stage, making the drug release more uniform and controllable; therefore, in the present application, mineralization treatment is essential, only by mineralizing the SilMA hydrogel microspheres and using the Ca / P mineralization method with low calcium and phosphorus concentration, the best mSilMA can be prepared, achieving the best effect of optimizing the physical properties of the microspheres and the drug controlled release performance, and providing a more ideal material selection for the application in the field of bone repair and the like.

[0040] In some embodiments, to verify the necessity of the mineralization step for the preparation of mSilMA of the present application, the differences in the in vitro controlled release ability of BMP-2 of 20% mSilMA microspheres and 20% SilMA microspheres were compared and tested in this embodiment. The experimental results show that the 20% mSilMA microspheres can continuously and slowly release BMP-2, and the cumulative release of BMP-2 is about 2% in vitro for 4 weeks, while the 20% SilMA microspheres cannot release BMP-2 for a long time, and reach the release plateau in about 3 weeks, which may be due to the adsorption / dissolution of mesopores to drugs reaching a certain degree of balance and leading to incomplete release.

[0041] Preferably, in the 1 times Ca / P mineralization solution, the molar ratio of calcium chloride to phosphoric acid is 1.5-1.7.

[0042] Further, the mineralization solution immersion time is 12-48 hours; in the Ca / P method, the volume fraction of ammonia solution is 10%-20%, and the ammonia water action time is 30 min-2 hours.

[0043] Further, in the step (1), the concentration of the SilMA solution is 10%-30%; the dissolving temperature of the SilMA is 25℃-45℃; the LAP solution is added into the SilMA solution as the aqueous phase, and the aqueous phase is mixed with the oil phase by stirring, the final concentration of the LAP aqueous solution is 0.1%-0.5%; the volume ratio of the oil phase to the aqueous phase is 1:1-10:1; in the oil phase, the mass ratio of Span 80 to liquid paraffin is 1:10-1:30; the stirring temperature is 25℃-45℃; the stirring time and the ultraviolet irradiation time are 5-15 min.

[0044] The LAP is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0045] In some embodiments, SilMA microspheres are prepared by photo-crosslinking oil / water emulsion-critical drying method using different concentrations of SilMA solution, and the swelling and surface pore size of the SilMA microspheres are statistically analyzed. The experimental results show that: on the one hand, the water absorption and swelling capacity of 10% SilMA microspheres is the strongest, which is easy to soak and swell and break within 1 week, losing structural integrity, which indicates that the stability is insufficient and is not suitable for subsequent processing; the water absorption and swelling capacity of 20% SilMA microspheres and 30% SilMA microspheres has no obvious difference; on the other hand, according to the scanning electron microscope results, the surface pore size of 10% SilMA microspheres is the largest, with an average pore size of about 100 nm, the surface pore size of 20% SilMA microspheres is about 20 nm, and the average surface pore size of 30% SilMA microspheres is about 5 nm, while the molecular diameter of BMP-2 is about 10 nm. Combined with the partial influence of subsequent mineralization processing on the surface pore size of SilMA microspheres, it is speculated that the pore structure of 30% SilMA microspheres after mineralization is not conducive to the loading of BMP-2. While 20% SilMA microspheres can not only physically adsorb BMP-2, but also limit its rapid release to a certain extent, which is helpful to achieve better controlled release effect. Therefore, by comprehensively considering the swelling performance and surface pore size distribution of SilMA microspheres with different concentrations, 20% SilMA microspheres show a more balanced and ideal performance in stability, BMP-2 loading and controlled release, and therefore 20% SilMA microspheres are preferably selected for the next step of biomimetic mineralization.

[0046] Preferably, the dissolving temperature of the SilMA is 25℃-30℃; the stirring temperature is 25℃-30℃.

[0047] Further, in the step (3), gradient ethanol aqueous solution is used for dehydration, the microspheres are soaked in the gradient ethanol aqueous solution for 15-30 min, and the critical point drying time is 3 hours-5 hours.

[0048] Preferably, the critical point drying time is 3-5 hours.

[0049] In another aspect, the present application provides a use of the instant mesoporous mineralized silk fibroin microspheres for preparing a preparation for improving bone repair ability or promoting bone regeneration, wherein the instant mesoporous mineralized silk fibroin microspheres are prepared by the method according to any one of the above technical solutions.

[0050] In another aspect, the present application provides a use of the instant mesoporous mineralized silk fibroin microspheres for preparing a preparation for improving long-acting low-dose sustained release ability of BMP-2 or reducing burst release ability of BMP-2, wherein the instant mesoporous mineralized silk fibroin microspheres are prepared by the method according to any one of the above technical solutions.

[0051] In another aspect, the present application provides a use of the instant mesoporous mineralized silk fibroin microspheres for preparing a preparation for improving protein loading rate of BMP-2, wherein the instant mesoporous mineralized silk fibroin microspheres are prepared by the method according to any one of the above technical solutions.

[0052] In another aspect, the present application provides a use of the instant mesoporous mineralized silk fibroin microspheres for preparing a preparation for improving long-acting low-dose sustained release ability of BMP-2 or reducing burst release ability of BMP-2, wherein the instant mesoporous mineralized silk fibroin microspheres are prepared by the method according to any one of the above technical solutions.

[0053] In another aspect, the present application provides a use of the instant mesoporous mineralized silk fibroin microspheres for preparing a preparation for improving long-acting low-dose sustained release ability of BMP-2 or reducing burst release ability of BMP-2, wherein the instant mesoporous mineralized silk fibroin microspheres are prepared by the method according to any one of the above technical solutions.

[0054] The present application has the following advantages:

[0055] 1、The instant mesoporous mineralized silk fibroin microspheres, the preparation method and the application thereof provided by the present application, the SilMA hydrogel microspheres are prepared by the photo-crosslinking oil / water emulsion method, the method is simple and easy to control, and the stable microspheres can be prepared in a large amount; the SilMA hydrogel microspheres are converted into mSilMA by combining the biomimetic mineralization method and the critical point drying method, the preparation process is mild, and the structure and performance of the microspheres are not damaged, which is beneficial to large-scale production and application; and the instant dry mesoporous mineralized silk fibroin microspheres prepared by the present application have relatively simple components, and only contain the silk fibroin-based microspheres and the mineralized coating on the surface of the microspheres.

[0056] 2、The application adopts silk fibroin material which is not easy to degrade and has good biocompatibility as a delivery carrier of BMP-2, the prepared SilMA microspheres are not easy to degrade, can stably exist in the bone defect implant site, continuously release BMP-2, avoid the problems of easy degradation material leading to rapid loss of BMP-2, early large release of BMP-2 causing inflammation and inability to achieve long-term treatment effect, provide long-term stable support for bone repair; at the same time, the calcium phosphate mineralized coating is formed on the surface of the SilMA microspheres through biomimetic mineralization, on the one hand, the strong affinity of calcium phosphate material to BMP-2 is utilized to realize efficient loading, on the other hand, the release rate of BMP-2 is greatly reduced, the problems of easy degradation of pure calcium phosphate material and inability to control release of BMP-2 for a long time are solved, so that BMP-2 can play a long-acting and stable role, and promote the regeneration and repair of bone tissue; and the SilMA microspheres are formed into through mesoporous channel structures by using the critical point drying method, which provides an ideal structural basis for the loading and controlled release of drugs and proteins, and the drugs or proteins can be captured in the mesopores by physical adsorption and realize diffusion release, further enhancing the controlled release performance of the microspheres.

[0057] 3、In the application, the prepared mSilMA is applied to the loading and controlled release of BMP-2, the protein loading efficiency can reach more than 85%, and the 4-week cumulative release is only about 2%, realizing the long-acting and low-dose slow release of BMP-2; the controlled release performance can effectively maintain the effective concentration of BMP-2 in the local area, reduce the use dose and frequency of drugs, reduce the treatment cost and potential side effects, provide an efficient, safe and economical solution for the treatment of bone defects, also provide an innovative, efficient and broad application prospect technical means for the field of bone defect repair, and have important significance for improving the treatment effect of bone defects and improving the life quality of patients. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 It is the preparation process of the dry-state mesoporous mineralized silk fibroin methacrylamide microspheres (mSilMA) of the application.

[0059] Figure 2 It is the surface morphology of the GelMA microspheres and the SilMA microspheres after critical point drying under a scanning electron microscope.

[0060] Figure 3 It is the surface morphology of the dry-state SilMA microspheres prepared by freeze-drying and critical point drying methods.

[0061] Figure 4 It is a structure and content result graph of calcium phosphate substances formed on the surface of SilMA microspheres by different mineralization methods.

[0062] Figure 5The influence of three concentrations of Ca / P mineralization solution on the structure and surface mineralization coating of 20% SilMA microspheres.

[0063] Figure 6 A in FIG. 1 is the nitrogen physical adsorption test of dry-state mesoporous 20% SilMA microspheres (20% SilMA for short).

[0064] Figure 6 B in FIG. 2 is the nitrogen physical adsorption test of dry-state mesoporous 20% SilMA microspheres (20% mSilMA for short).

[0065] Figure 6 C in FIG. 3 is the pore size distribution result of 20% mSilMA microspheres and 20% SilMA microspheres.

[0066] Figure 7 A in FIG. 4 is the morphology difference of 20% mSilMA microspheres and 20% SilMA microspheres.

[0067] Figure 7 B in FIG. 5 is the difference in in-vitro controlled release capability of 20% mSilMA microspheres and 20% SilMA microspheres for BMP-2.

[0068] Figure 8 A in FIG. 6 is the swelling performance result of 10%, 20%, 30% dry-state mesoporous SilMA microspheres.

[0069] Figure 8 B in FIG. 7 is the surface pore size distribution (obtained by critical drying) of 10%, 20%, 30% SilMA microspheres obtained by statistical analysis of scanning electron microscope results.

[0070] Figure 9 is the surface morphology of 10%, 20%, 30% dry-state SilMA microspheres (obtained by critical drying).

[0071] Figure 10 A in FIG. 8 is the light microscope image of 20% SilMA hydrogel microspheres prepared by photo-crosslinking oil / water emulsion method (mechanical stirring).

[0072] Figure 10 B in FIG. 9 is the light microscope image of 20% SilMA hydrogel microspheres prepared by photo-crosslinking 3D printing technology. DETAILED DESCRIPTION

[0073] The application will be described in detail below with reference to the embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the application and do not limit the application in any way.

[0074] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0075] The materials, reagents and the like used in the following examples can be obtained commercially unless otherwise specified.

[0076] Noun explanation of related terms:

[0077] 1. Biomimetic mineralization: In the field of artificial material synthesis, biomimetic mineralization controls the nucleation and growth of minerals such as calcium carbonate and calcium phosphate to obtain inorganic or inorganic / organic composite materials with complex and advanced structures and special biological functions.

[0078] 2. Critical point drying method: To prevent the sample from being deformed due to the rapid movement of the water interface during drying, the liquid in the sample is removed in the critical state without gas and liquid interface. In the traditional drying method, the liquid is directly converted into gas under high temperature conditions, which will produce a certain surface tension change, which may damage the fine structure of the sample. For special samples such as sol-gel, biological samples, and structurally unstable porous materials, using traditional drying methods to pretreat the sample will result in the sample structure being damaged. Using the critical point drying method, the medium uses the phase change method of liquid carbon dioxide-supercritical carbon dioxide-gaseous carbon dioxide to reduce the surface stress of the interface to zero, which can save the fine structure of the sample.

[0079] 3. Main process of critical point drying sample: After the sample is dehydrated through 30%-50%-70%-90%-100% ethanol aqueous solution gradient, it is placed in a critical point drying device (high-pressure sealed container), liquid carbon dioxide is injected, and dried at a critical state of 31℃ and 72.8 atmospheres.

[0080] Example 1 Best ready-to-use dry mesoporous mineralized silk fibroin microspheres of the application and a preparation method thereof

[0081] First, the research ideas and processes of the ready-to-use dry mesoporous mineralized silk fibroin microspheres of the application

[0082] In the current field of bone repair, BMP-2, as a key bone morphogenetic protein, plays an important role in promoting bone regeneration. However, the existing BMP-2 controlled release carrier has problems such as rapid release, short retention time, complex carrier components, and the like, which seriously limits its application effect in the clinic.

[0083] In view of this, the application is dedicated to finding a new solution that can effectively solve these problems. After in-depth research and a large number of experiments, combined with a full understanding of silk fibroin, calcium phosphate materials and mesoporous structures, an innovative idea of preparing ready-to-use mesoporous mineralized silk fibroin microspheres is proposed in the application. Through the proposed preparation method of ready-to-use mesoporous mineralized silk fibroin microspheres for long-acting and low-dose release of bone morphogenetic protein, a ready-to-use mesoporous mineralized silk fibroin microsphere is finally prepared.

[0084] Silk fibroin is a natural polymer protein extracted from silk, which has adjustable mechanical properties, controllable degradation, low immunogenicity, and low cost. Various silk-based biomaterials have been approved for clinical use in China, such as silk-based ligament grafts and long-term bioabsorbable surgical meshes, indicating that silk-based derivative scaffolds have great potential for clinical transformation. Kaplan et al. prepared hydroxyapatite particles coated with silk fibroin. By combining BMP-2 with a silk fibroin coating, they were unable to achieve long-term low-dose release of BMP-2, which failed to meet the requirements for long-term stable release, with a short release time and difficulty in achieving the desired therapeutic effect. (Ding ZZ, Fan Z H, Huang X W, et al. Bioactive natural protein–hydroxyapatite nanocarriers for optimizing osteogenic differentiation of mesenchymal stem cells [J]. Journal of Materials Chemistry B, 2016.)

[0085] Calcium phosphate materials are an attractive BMP-2 controlled-release matrix that can reversibly bind proteins and interact with the characteristic conformation and amino groups of proteins through physical and electrostatic interactions. Studies have found that mineralized collagen scaffolds release about 80% of BMP-2 in vitro for 14 days. Due to the easy degradation of collagen scaffolds, it is difficult to prolong the retention time of BMP-2 and achieve long-term stable control of BMP-2 release, which cannot meet the application requirements of some applications that require long-term sustained release of BMP-2. (Yang HS, La W G, Bhang S H, et al. Apatite-coated collagen scaffold for bone morphogenetic protein-2 delivery [J]. Tissue Engineering Part A, 2011)

[0086] Mesoporous materials have attracted much attention in the field of drug delivery due to their regular pore structure, relatively narrow pore size distribution, and large specific surface area. Drugs can be captured in mesopores through the process of impregnation adsorption and diffuse release. Liu et al. used mesoporous bioactive glass scaffolds to adsorb BMP-2, but the initial release reached 27% after 1 day; therefore, the mesoporous structure is not effective in reducing early burst. Moreover, the adsorption / dissolution of drugs in mesoporous structures is easy to reach equilibrium at a certain time period, resulting in incomplete release of BMP-2, which cannot fully exert the effect of the drug and is difficult to achieve stable, sustained, and complete release of BMP-2. (Liu Y, Yang Z, Wang L, et al. Spatiotemporal Immunomodulation Using Biomimetic Scaffold Promotes Endochondral Ossification-mediated Bone Healing [J]. Advanced Science, 2021 / / Le T T, et al. Delivery of poorly soluble drugs via mesoporous silica: Impact of drug overloading on release and thermal profiles [J]. Pharmaceutics, 2019 / / He S, Pan H, Zhang J. Advances of typical mesoporous materials and the application in drug delivery [J]. Materials Research Express, 2023.)

[0087] Therefore, in the present application, silk fibroin methacrylamide (abbreviated as SilMA) is used as raw material, and biomimetic mineralization method and critical point drying method are comprehensively utilized to prepare ready-to-use dry mesoporous mineralized silk fibroin microspheres (abbreviated as mSilMA). Silk fibroin is a regular directional fiber structure formed by layering nanofibrils, containing a large number of polar amino acids. These hydrophilic and polar groups can tightly bind to positive and negative ions, and can induce nucleation and growth of inorganic substances on the silk fibroin template. The effective loading rate of each milligram of mSilMA for BMP-2 (500 ng) prepared by the present application is more than 85%. In vitro release detection found that mSilMA microspheres can continuously release BMP-2 at a low amount, and about 2% of BMP-2 is accumulated and released in vitro for 4 weeks, which can achieve long-acting and low-dose release of BMP-2.

[0088] II. Preparation method of ready-to-use mesoporous mineralized silk fibroin microspheres

[0089] The application provides a best preparation method of a ready-to-use mesoporous mineralized silk fibroin microsphere capable of long-acting low-dose slow release of BMP-2, and specifically comprises the following steps:

[0090] 1. Preparation of SilMA hydrogel microspheres by photo-crosslinking oil / water emulsion method

[0091] Dissolve SilMA in double-distilled water at room temperature to obtain a 20% (W / V) SilMA solution, and add a certain volume of LAP solution (ultraviolet initiator: lithium phenyl (2,4,6-trimethylbenzoyl) phosphate) as an aqueous phase, with a final concentration of 0.25% (W / V). The oil phase is composed of liquid paraffin and Span 80, and the mixing ratio is 1:20; the aqueous phase is added dropwise into the oil phase, and the oil / water ratio is 5:1; stirring is performed at room temperature at 400 rpm, and ultraviolet irradiation is performed during the stirring. After 10 min, the stirring and irradiation are stopped, and the microspheres are precipitated at the bottom of the beaker. The supernatant (oil phase) is discarded, and the microspheres are washed with 50% isopropanol aqueous solution for 3 times, 75% isopropanol aqueous solution for 2 times, and 80% isopropanol aqueous solution for 1 time; the microspheres are washed with a large amount of water for 3 times, and then precipitated at the bottom of the beaker. The supernatant (water) is discarded, and the microspheres are harvested by filtration.

[0092] In the application, the concentration of the SilMA solution is 10%-30%, the dissolution temperature of the SilMA is 25-45°C, preferably 25-30°C, the final concentration of the LAP aqueous solution is 0.1%-0.5% (W / V%), the mass ratio of Span 80 to liquid paraffin in the oil phase is 1:10-1:30, the volume ratio of the oil phase to the aqueous phase is 1:1-10:1, the stirring temperature is 25-45°C, preferably 25-30°C, the stirring speed is 100-500 rpm, preferably 400-450 rpm, the stirring time and ultraviolet irradiation time are 5-15 min, and the mixing and emulsifying device of the oil phase and the aqueous phase can be a mechanical stirring device, a magnetic stirring device or a homogenizing stirring device, preferably a homogenizing stirring device.

[0093] 2. Mineralization of SilMA hydrogel microspheres

[0094] The harvested SilMA hydrogel microspheres are soaked in a calcium chloride / phosphoric acid mixed solution, the molar ratio of calcium chloride to phosphoric acid is 1.67, and the soaking is performed for 24 hours, followed by filtration and discarding of the upper mineralization liquid; then, 15% (W / V) ammonia water solution is added, the reaction is performed for 3 hours, the supernatant solution is discarded by filtration, and then the prepared mineralized SilMA hydrogel microspheres are washed with water for multiple times.

[0095] The molar ratio of the calcium chloride / phosphoric acid mixed solution is 1.5-2.0, and in the embodiment, preferably 1.5-1.7; the mineralization solution immersion time is 12 hours-48 hours, and in the embodiment, preferably 24 hours; the volume fraction of the ammonia water solution is 10%-20%, and in the embodiment, preferably 15%; and the ammonia water action time is 30 min-2 hours.

[0096] 3. Critical point drying of the mineralized SilMA hydrogel microspheres

[0097] The mineralized SilMA hydrogel microspheres are dehydrated using gradient ethanol aqueous solutions, the concentrations of the ethanol aqueous solutions being 30%-50%-70%-90%-100% in turn; the immersion time for each ethanol concentration is 15 min; and then the critical point drying instrument is used for critical point drying, and the drying time is 3 hours, to obtain dry mesoporous mineralized silk fibroin methacrylamide microspheres (mSilMA for short).

[0098] The immersion time of the microspheres in the ethanol aqueous solution is 15-30 min; and the critical point drying time is 1-5 hours, and in the embodiment, preferably 3-5 hours.

[0099] 4. Calculation of the BMP-2 content combined by the mSilMA microspheres

[0100] 10 mg of the mSilMA prepared in step 3 is weighed, 500 ng of bone morphogenetic protein (BMP-2) solution is added, and after 4°C incubation for 24 hours, 5 mL of normal saline (containing 0.1% bovine serum albumin solution) is added for cleaning three times, so as to remove the BMP-2 not combined by the microspheres, the cleaning liquid is collected three times, and the BMP-2 content in the cleaning liquid is detected by using an enzyme-linked immunosorbent assay. The BMP-2 content combined by per milligram of the microspheres is calculated as follows: (the original BMP-2 addition amount minus the BMP-2 content obtained in the cleaning liquid) / the total mass of the microspheres.

[0101] 5. In vitro BMP-2 sustained release detection

[0102] 10 mg of 20% mSilMA microspheres and 20% SilMA microspheres are weighed respectively, 500 μl of 500 ng BMP-2 solution (solvent: normal saline containing 0.1% bovine serum albumin) is added respectively, 4°C incubation is performed for 24 h, then 5 mL of normal saline containing 0.1% bovine serum albumin is added respectively, and room temperature cleaning is performed three times, so that 15 mL of cleaning liquid can be collected for the 20% mSilMA microspheres and the 20% SilMA microspheres respectively. The BMP-2 enzyme-linked immunodetection kit is used to detect the BMP-2 not combined by the microspheres in the cleaning liquid; and it can be known that the combined BMP-2 is equal to the original addition amount (500 ng) minus the BMP-2 content in the cleaning liquid.

[0103] 20% mSilMA microspheres and 20% SilMA microspheres, respectively bound to BMP-2, were immersed in 5 mL of physiological saline containing 0.1% bovine serum albumin and incubated at 37°C. Supernatants were collected at 1, 4, 7, 124, 21, 28, and 35 days. The BMP-2 content in the supernatant was detected using a BMP-2 enzyme-linked immunosorbent assay (ELISA) kit. The percentage of BMP-2 released at a specific time point was defined as: the cumulative amount of BMP-2 released from the supernatant during the specified time period / the total amount of BMP-2 bound to the microspheres; the cumulative percentage of BMP-2 released was defined as: the sum of all BMP-2 release percentages within the specified time period.

[0104] In summary, the specific preparation process of the dry mesoporous mineralized silk fibroin methacrylamide microspheres (mSilMA) is as follows: Figure 1 As shown. The prepared mSilMA has a particle size of 100-500 μm, preferably 250-350 μm in this embodiment; the surface of the mSilMA has an interconnected fiber network structure and needle-like calcium phosphate material, and the pore size of the fiber network is 5-100 nm, preferably 10-15 nm in this embodiment.

[0105] Therefore, by using the optimal preparation method of this invention, based on biocompatible silk fibroin material, coupled with biomimetic mineralization method and critical point drying method, long-term low-volume release of BMP-2 in vitro was achieved without early burst release.

[0106] Example 2: Screening of the main components of microspheres and the necessity of the "mesoporous channel" structure in microspheres.

[0107] To obtain the microspheres with optimal long-lasting, low-volume sustained release of bone morphogenetic protein as shown in Example 1, the main components of the microspheres were first screened in this example. The specific screening process is as follows (all other unmentioned operations and experimental conditions are the optimal conditions in Example 1):

[0108] I. Comparison of results under scanning electron microscopy

[0109] The main components of the microspheres were silk fibroin methacrylamide and gelatin methacrylamide, respectively, to prepare gelatin methacrylamide hydrogel microspheres (GelMA) and silk fibroin methacrylamide microspheres (SilMA). The specific preparation process is as follows:

[0110] GelMA and SilMA were prepared by UV crosslinking oil / water emulsion method. The two kinds of hydrogel microspheres were immersed in 30%-50%-70%-90%-100% ethanol solution for 15 min, respectively. Then, the critical point drying instrument was used to dry the microspheres for 3 hours.

[0111] The specific results are shown in Figure 2 Figure 2 The surface morphology of GelMA and SilMA microspheres after critical point drying was observed under scanning electron microscope. It was found that the GelMA hydrogel microspheres did not form a surface penetrating mesoporous structure after the same ethanol gradient dehydration and critical point drying, but presented a non-porous structure; while the SilMA microspheres formed a surface interconnected pore structure. Therefore, it can be initially known that not all materials can form pores by critical point drying.

[0112] In the critical point drying method, the medium adopts the phase change method of liquid carbon dioxide-supercritical carbon dioxide-gaseous carbon dioxide, which reduces the surface stress of the interface to zero and can preserve the fine structure of the sample. Silk fibroin is a natural high molecular fibrous protein extracted from silk, which has a fibrous structure. Gelatin is a kind of protein obtained by partial hydrolysis of collagen fibers. Collagen has a rod-like triple helix structure, and when it is partially hydrolyzed to prepare gelatin, the triple helix structure of collagen separates and breaks, so that gelatin has lost the fibrous structure. It is speculated that because silk fibroin has the original fibrous structure, through critical point drying, its own fibrous network can be reproduced; while gelatin itself does not have a relatively complete fibrous structure, and in the process of ethanol gradient dehydration, with the loss of water, the network structure of the hydrogel gradually becomes dense, and through critical point drying, the fibrous network structure cannot appear, so the surface is dense and non-porous. Therefore, it is speculated that if the material itself has a fibrous structure, it can produce a porous structure by critical point drying; if the material itself does not have a fibrous structure, it cannot produce a porous structure by critical point drying.

[0113] ​In the present application, the mesoporous pore structure is essential to prepare the hydrogel microspheres with long-acting low-dose sustained release of BMP-2. First, the presence of mesopores can greatly increase the drug loading capacity; second, the interconnected mesoporous channels have high superiority, which is conducive to the diffusion and release of cell active factors inside the microspheres, can realize the slow and continuous release of cell active factors, avoid the burst release phenomenon of cell active factors, thereby prolong the action time of cell active factors in vivo, improve the utilization efficiency and treatment effect of cell active factors, and provide persistent and stable support for bone repair and other processes; on the other hand, the mesoporous structure plays a key role in the exchange of substances and information between the microspheres and the surrounding environment, enhances the interaction between the microspheres and the body, and improves the adaptability and biocompatibility of the microspheres in vivo.

[0114] Therefore, in the present embodiment, silk fibroin methacrylamide which can form a mesoporous pore structure is preferably used as the main component of the microspheres to achieve more ideal drug controlled release effect and biological application performance, and to provide strong technical support and innovative ideas for the development of bone tissue engineering and other fields.

[0115] II. Comparison of BMP-2 protein loading rate and long-acting low-dose sustained release ability

[0116] 1. Comparison of BMP-2 protein loading rate

[0117] Meanwhile, in order to further verify the importance of using silk fibroin material to successfully prepare mSilMA with high protein loading rate and long-acting low-dose sustained release of bone morphogenetic protein, in the present embodiment, gelatin methacrylamide hydrogel microspheres (abbreviated as GelMA) and silk fibroin methacrylamide microspheres (SilMA) in step 1 are used respectively, and GelMA microspheres and mSilMA microspheres are prepared according to the best preparation method in embodiment 1. Each is weighed 10 mg, then 500 μl of 500 ng BMP-2 solution (solvent: physiological saline containing 0.1% bovine serum albumin) is added, 4°C incubation for 24 h, then 5 mL of physiological saline containing 0.1% bovine serum albumin is added, and the room temperature is washed for 3 times, then 15 mL of washing liquid can be collected for GelMA microspheres and mSilMA microspheres respectively. The BMP-2 enzyme-linked immunoassay kit (purchased from Shenzhen Xingboshen Biological Technology Co., Ltd., model: EHC172.96) is used to detect the BMP-2 in the washing liquid which is not combined with the microspheres. The BMP-2 combined with the microspheres per milligram of microspheres is calculated as (original added amount (500 ng) minus the content of BMP-2 in the washing liquid) / total mass of the microspheres. The content of BMP-2 combined with different microspheres is shown in Table 1.

[0118] 2. Comparison of long-acting low-dose sustained release ability of BMP-2

[0119] The GelMA microspheres and the mSilMA microspheres respectively combined with BMP-2 are immersed in 5 mL of normal saline containing 0.1% bovine serum albumin, and incubated at 37°C, and the supernatant is collected at 1, 4, 7, 14, 21, 28, 35 days, respectively, and the content of BMP-2 in the supernatant is detected by using a BMP-2 enzyme-linked immunosorbent assay kit. The specific time release percentage of BMP-2 is: the cumulative release amount of BMP-2 in the supernatant / the total amount of BMP-2 combined with the microspheres in the time. The in vitro release capacity of BMP-2 of different microspheres in a certain time is shown in Table 2.

[0120] 3. Experimental results

[0121] The results of the above different experiments are shown in Table 1 and Table 2:

[0122] Table 1, the content of BMP-2 combined with different microspheres

[0123] Name Amount of BMP-2 bound BMP-2 protein loading mSilMA microspheres 44.34 ng / mg 88.68％ GelMA microspheres 20.85 ng / mg 41.70％

[0124] Table 2, the in vitro release capacity of BMP-2 of different microspheres in a certain time

[0125]

[0126] From Table 1 and Table 2, it can be seen that only when the SilMA microspheres with surface penetrating mesoporous structure are successfully prepared by using fibroin methacrylamide as the main component, the BMP-2 protein loading rate can reach 88.68%, and at the same time, the BMP-2 can be continuously released at a low amount, and about 2.5% of BMP-2 is accumulated and released in vitro for 4 weeks; and when other components are used, the effect of the present application cannot be achieved. Therefore, fibroin methacrylamide which can form mesopores is preferably used as the main component of the microspheres in this embodiment.

[0127] In Example 3, the drying methods for preparing dry SilMA are screened

[0128] In order to obtain the mSilMA with the best performance in Example 1, the drying method of the mineralized SilMA hydrogel microspheres in the preparation process of the mSilMA is screened in this embodiment, and the specific screening process is as follows (the rest of the operation process and experimental conditions not mentioned are the best conditions in Example 1):

[0129] I. Comparison of results under scanning electron microscope

[0130] The prepared mineralized SilMA hydrogel microspheres are dehydrated by using freeze-drying and critical point drying methods, respectively, and the obtained dry SilMA microspheres are observed by scanning electron microscope.

[0131] 1. Freeze-drying: The prepared mineralized SilMA hydrogel microspheres were directly pre-frozen in a refrigerator at -80℃ for 24h, and then dried by a freeze-drying machine. The dry-state non-porous SilMA microspheres were harvested after 48h.

[0132] 2. Critical point drying: The prepared mineralized SilMA hydrogel microspheres were immersed in 30%-50%-70%-90%-100% ethanol aqueous solution for gradient dehydration, with each ethanol concentration immersion time of 15min; then the critical point drying instrument was used for critical point drying, with drying time of 3h, to harvest the dry-state mesoporous mineralized silk fibroin methacrylamide microspheres (mSilMA).

[0133] Specific results are shown in Figure 3 , Figure 3 The surface morphology of the dry-state SilMA microspheres prepared by freeze-drying and critical point drying methods is shown. Figure 3 It can be seen from that the mSilMA prepared by the critical point drying method has a smooth and uniform microsphere surface, without adhesion phenomenon, and the microsphere surface has uniform mesopores; the uniform mesoporous structure is beneficial to drug loading and slow release, and can provide a more stable and controllable release environment for BMP-2 and other drugs, so as to better play the role of the drug and improve the treatment effect.

[0134] The dry-state SilMA microspheres prepared by the freeze-drying method not only cannot form mesopores, resulting in limited drug loading and slow release performance, but also have adhesion between the microspheres and a rough surface; such rough surface and adhesion phenomenon may affect the dispersibility and stability of the microspheres, and further affect the effect in actual application.

[0135] II. Comparison of BMP-2 protein loading rate and long-acting low-dose slow-release ability

[0136] 1. Comparison of BMP-2 protein loading rate

[0137] At the same time, in order to further verify the importance of the drying method (critical drying method) in the successful preparation of mSilMA with high protein loading rate and long-acting low-dose slow-release bone morphogenetic protein, 10mg of the following microspheres were weighed in this embodiment:

[0138] 1. Dry-state non-porous mineralized SilMA microspheres prepared by freeze-drying in step one;

[0139] 2. Dry-state mesoporous mineralized SilMA microspheres prepared by critical point drying (mSilMA microspheres prepared by the best preparation method in Example 1);

[0140] 3. Mineralized SilMA hydrogel microspheres without drying (the preparation method of the microspheres is specifically as the best preparation method in Example 1, only the drying step is reduced).

[0141] and 500 μl of 500 ng BMP-2 solution (solvent: physiological saline containing 0.1% bovine serum albumin) was added respectively, and then incubated at 4°C for 24 h. Then 5 mL of physiological saline containing 0.1% bovine serum albumin was added respectively, and then washed at room temperature for 3 times. Then 15 mL of washing solution was used for dry non-porous mineralized SilMA microspheres, dry mesoporous mineralized SilMA microspheres and mineralized SilMA hydrogel microspheres. The BMP-2 not combined with the microspheres in the washing solution was detected by using BMP-2 enzyme-linked immunoassay kit. The BMP-2 combined with the microspheres per milligram was calculated as follows: (original amount of BMP-2 added (500 ng) minus the content of BMP-2 in the washing solution) / total mass of the microspheres. The content of BMP-2 combined with different microspheres is shown in Table 3.

[0142] 2. Comparison of long-acting and low-dose sustained-release ability of BMP-2

[0143] As above, dry non-porous SilMA microspheres and mSilMA microspheres combined with BMP-2 respectively were soaked in 5 mL of physiological saline containing 0.1% bovine serum albumin, and then incubated at 37°C. The supernatant was collected at 1, 4, 7, 14, 21, 28 and 35 days respectively, and the content of BMP-2 in the supernatant was detected by using BMP-2 enzyme-linked immunoassay kit. The release percentage of BMP-2 at a specific time was calculated as follows: the cumulative release amount of BMP-2 in the supernatant at the time / the total amount of BMP-2 combined with the microspheres %. The in vitro release ability of BMP-2 of different microspheres at a specific time is shown in Table 4.

[0144] 3. Experimental results

[0145] The results of the above different experiments are shown in Table 3 and Table 4.

[0146] Table 3, content of BMP-2 combined with different microspheres

[0147] Different drying methods Amount of BMP-2 bound BMP-2 protein loading Dry nonporous mineralized SilMA microspheres (freeze-dried) 36.22 ng / mg 72.44％ Dry mesoporous mineralized SilMA microspheres (critical point dried) 44.34 ng / mg 88.68％ Mineralized SilMA hydrogel microspheres (not dried) 32.89 ng / mg 65.78％

[0148] Table 4, in vitro release ability of BMP-2 of different microspheres at a specific time

[0149]

[0150] From Table 3 and Table 4, it can be seen that only when the critical drying method is used, the SilMA microspheres with surface through mesoporous structure can be successfully prepared, the BMP-2 protein loading rate can reach 88.68%, and at the same time, the BMP-2 is continuously released in a low amount, and the cumulative release of BMP-2 in vitro is about 2.51% in 4 weeks; when other drying methods or no drying are used, the effects of the present application cannot be achieved, the BMP-2 protein loading rate is obviously reduced, and the in vitro controlled release of BMP-2 is also obviously decreased. Therefore, in the present embodiment, the critical point drying method is preferably used to prepare mSilMA.

[0151] Example 4: Screening of different mineralization methods in preparation of mineralized SilMA hydrogel microspheres

[0152] In order to obtain the mSilMA with the best performance in Example 1, different mineralization methods in the preparation process of the mineralized SilMA hydrogel microspheres are screened in the present embodiment, and the specific screening process is as follows (the rest of the operation process and experimental conditions not mentioned are the best conditions in Example 1):

[0153] 1. 10 times simulated body fluid method (10×SBF method): The simulated body fluid (SBF, Simulated body fluid) is the most commonly used solution for in vitro simulation at present, and is the basis for in vitro simulation experiments. It is designed based on the ion concentration of human plasma, and the purpose is to test the biological activity of the material. The principle of SBF is that the artificially synthesized material can connect with the bone by forming a bone-like apatite layer on the surface after being implanted into the human body; similarly, the artificial material can also form hydroxyapatite in the SBF with similar ion concentration as human plasma, which can indicate that the material has biological activity.

[0154] Prepare 500 mL of 10 times simulated body fluid: sodium chloride 1000 mmol / L, potassium chloride 5 mmol / L, calcium chloride dihydrate 25 mmol / L, magnesium chloride hexahydrate 5 mmol / L, sodium dihydrogen phosphate 10 mmol / L, pH = 7.2-7.4.

[0155] 2. Nanocluster method (PILP method): 2.15 g of polyacrylic acid (PAA), 1.08 g of polyaspartic acid (PASP), and 0.362 g of disodium hydrogen phosphate were weighed into a 100 mL beaker, 25 mL of ultrapure water was added, the rotor was put in, the tin foil was sealed, and stirring was carried out at room temperature overnight. A dark yellow viscous solution A was obtained.

[0156] Weigh 3 g of PASP, add 10 mL of double distilled water, and mix thoroughly.

[0157] Under magnetic stirring, add 0.1 mL of PASP solution to a beaker, then slowly add 2 mL of 0.1 mol / L CaCl2 solution. Next, slowly add 2 mL of solution A using a syringe, stirring until fully mixed. Then, slowly add 0.4 mL of 3 mol / L NaOH solution, stirring thoroughly until homogeneous. The prepared nanoclusters can be stored at 4°C for 2 weeks.

[0158] 3. Calcium chloride / phosphate-ammonia reaction method (Ca / P method): Prepare 500 mL of 1-fold calcium chloride / phosphate solution with a calcium chloride / phosphate molar ratio of 1.67, a calcium chloride concentration of 0.167 mol / L, a phosphate concentration of 0.1 mol / L, and an ammonia volume fraction of 15% (V / V).

[0159] Equal amounts of SilMA microspheres were soaked in the three mineralization solutions mentioned above for 24 hours. For the 10×SBF and PILP methods, after soaking for 24 hours, the microspheres were washed three times with water and collected directly. After dehydration by ethanol gradient, they were subjected to critical drying. For the Ca / P method, after soaking for 24 hours, the microspheres were filtered and the upper mineralization solution was discarded. Then, 15% (w / v) ammonia solution was added, and after reacting for 3 hours, the supernatant was discarded by filtration. The prepared mineralized microspheres were then washed three times with water, dehydrated by ethanol gradient, and subjected to critical drying.

[0160] Specific results are as follows Figure 4 As shown, Figure 4 The structure and content of calcium phosphate formed on the surface of SilMA microspheres using different mineralization methods are shown. From Figure 4 As can be seen, the Ca / P method enables the formation of a more uniform and denser calcium phosphate mineralization coating on the surface of SilMA microspheres. This more uniform and denser coating enhances the stability and mechanical strength of the microspheres, improves their compatibility and bonding with bone tissue, and helps to better promote bone regeneration and repair processes. Furthermore, the uniform and high-density coating can more effectively control the drug release rate, providing a more stable loading environment for the drug, thereby improving its therapeutic effect. Therefore, in this embodiment, the Ca / P method is preferred as the mineralization method for SilMA microspheres.

[0161] Example 5: Screening of different mineralization concentrations in the preparation of mineralized SilMA hydrogel microspheres.

[0162] To obtain the mSilMA with optimal performance as in Example 1, this example screened different mineralization concentrations in the Ca / P method during the preparation of mineralized SilMA hydrogel microspheres and investigated the effects of different mineralization concentrations on the SilMA microspheres and the surface calcium phosphate mineralization layer. The specific screening process is as follows (all other unmentioned operations and experimental conditions are the optimal conditions in Example 1):

[0163] First, the different mineralization concentrations in the Ca / P method are defined as follows:

[0164] 1 times Ca / P mineralization solution: the molar ratio of calcium chloride / phosphoric acid is 1.67, the concentration of calcium chloride is 0.167 mol / L, and the concentration of phosphoric acid is 0.1 mom / L;

[0165] 5 times Ca / P mineralization solution, namely, the concentrations of calcium chloride and phosphoric acid are both increased by 5 times: the concentration of calcium chloride is 0.835 mol / L, and the concentration of phosphoric acid is 0.5 mom / L;

[0166] 10 times Ca / P mineralization solution, namely, the concentrations of calcium chloride and phosphoric acid are both increased by 10 times: the concentration of calcium chloride is 1.67 mol / L, and the concentration of phosphoric acid is 1.0 mom / L;

[0167] The same mineralization step was used for mineralization and subsequent critical point drying: 20% SilMA microspheres were soaked in the above three mineralization solutions for 24 hours, then filtered and the upper mineralization solution was discarded; then 15% (W / V) ammonia solution was added, and after 3 hours of reaction, the supernatant was filtered and discarded, then the prepared mineralized microspheres were washed with water 3 times, dehydrated with ethanol gradient, and then subjected to critical drying.

[0168] The specific results are shown in Figure 5 , Figure 5 The effects of three concentrations of Ca / P mineralization solution on the structure of 20% SilMA microspheres and the surface mineralized coating were demonstrated, and it can be seen that increasing the concentration of Ca / P mineralization solution while keeping other conditions unchanged will lead to the destruction of the microsphere structure, and the calcium phosphate coating still maintains a needle-like structure, and the coating density increases with the increase of the concentration of Ca / P mineralization solution. Since the 5 times and 10 times Ca / P mineralization solution has caused obvious damage to the microsphere structure, therefore, in this embodiment, 1 times calcium phosphate mineralization solution is preferred, under which the complete structure of the microspheres is retained, and a relatively uniform needle-like calcium phosphate coating exists on the surface of the 20% SilMA microspheres, and at the same time, the mesoporous channels of the microspheres are not completely blocked by the needle-like calcium phosphate coating.

[0169] Meanwhile, based on the 1 times Ca / P mineralization method, nitrogen physical adsorption tests were also conducted on the dry mesoporous 20% SilMA microspheres (referred to as 20% SilMA) and the dry mesoporous mineralized 20% SilMA microspheres (referred to as 20% mSilMA) prepared in this embodiment, and the pore size distributions of the two were compared, so as to further understand the influence of mineralization treatment on the pore size of the microspheres, and to evaluate the changes in the physical properties of the microspheres caused by the mineralization process. The specific results are shown in A of Figure 6 , B of Figure 6 and C of Figure 6 , B of Figure 6 and Figure 6The surface average pore size of 20% mSilMA microspheres in C can be seen to be about 10 nm, while the average pore size of 20% SilMA is about 19 nm (as shown in Figure 6 A and Figure 6 C in FIG. 1C) can be seen. Therefore, it can be known that the 1-fold Ca / P mineralization liquid concentration is more appropriate, and the mineralization treatment can make the pore size of the microspheres smaller. Within a suitable range, the smaller surface pore size of the microspheres has the following advantages: (1) better controlled release performance: a smaller pore size can more effectively control the release speed and release amount of drugs or bioactive molecules, so that they can be continuously and stably released for a longer period of time, thereby improving the treatment effect; (2) enhanced stability: a smaller pore size can make the microsphere structure more stable, reduce the influence of the external environment on the internal loaded substances, and improve the stability of the microspheres during storage and use; (3) improved selectivity: it can more selectively allow specific size molecules to pass through or be adsorbed, thereby achieving more precise drug or bioactive molecule delivery; (4) reduced burst release phenomenon: reducing the sudden mass release of drugs or bioactive molecules in the initial stage makes the release of drugs or bioactive molecules more uniform and controllable.

[0170] Therefore, it can be known that in the present application, the mineralization treatment is an essential step. Only by mineralizing the dry mesoporous SilMA microspheres and using the 1-fold Ca / P mineralization method, can mSilMA with a complete structure and a uniform mineralized coating be prepared, so as to achieve the best effect of optimizing the physical properties and BMP-2 controlled release performance of the microspheres, and provide a more ideal material selection for the application in the field of bone repair and the like.

[0171] Example 6 Necessity of the mineralization step for the BMP-2 in-vitro controlled release ability of the mSilMA of the present application

[0172] In this embodiment, the surface morphology of the 20% mSilMA microspheres (dry mesoporous mineralized microspheres) prepared by the 1-fold Ca / P mineralization method and the 20% SilMA microspheres (dry mesoporous microspheres) was further compared, and the characteristic elements of the surface mineralized coating of the 20% mSilMA microspheres were detected and verified, as shown in A of FIG. 1A. Figure 7

[0173] At the same time, in order to verify the necessity of mineralization for the preparation of the mSilMA of the present application, the following microspheres were also taken in this embodiment:

[0174] 1. 20% mSilMA microspheres (microspheres prepared by the best preparation method in Example 1);

[0175] 2. 20% SilMA microspheres (dry mesoporous and not mineralized, and the remaining steps are the same as the best preparation method in Example 1);

[0176] ​3. Microspheres prepared by other mineralization methods: 10 times simulated body fluid method in Example 4;

[0177] 4. Microspheres prepared by other mineralization methods: nanocluster method in Example 4;

[0178] 5. Microspheres prepared by other mineralization concentration: 5 times Ca / P mineralization solution in Example 5;

[0179] 6. Microspheres prepared by other mineralization concentration: 10 times Ca / P mineralization solution in Example 5;

[0180] The differences in the BMP-2 in-vitro controlled release ability of the microspheres prepared by the above different methods were compared and tested respectively, and the BMP-2 in-vitro release detection steps were as follows:

[0181] (1) Comparison of BMP-2 protein loading rate: the above different microspheres were weighed respectively, 500 μl of 500 ng BMP-2 solution (solvent: physiological saline containing 0.1% bovine serum albumin) was added respectively, 4°C incubation was performed for 24 h, then 5 mL of physiological saline containing 0.1% bovine serum albumin was added respectively, room temperature washing was performed for 3 times, then 15 mL of washing liquid could be collected for the above 6 different microspheres respectively. BMP-2 enzyme-linked immunoassay kit was used to detect the BMP-2 in the washing liquid which was not combined with the microspheres. The combined BMP-2 was equal to the original added amount (500 ng) minus the content of BMP-2 in the washing liquid.

[0182] (2) Comparison of long-acting low-dose sustained release ability of BMP-2: the above microspheres combined with BMP-2 respectively were soaked in 5 mL of physiological saline containing 0.1% bovine serum albumin, 37°C incubation was performed, the supernatant was collected at 1, 4, 7, 14, 21, 28, 35 days respectively, and BMP-2 enzyme-linked immunoassay kit was used to detect the content of BMP-2 in the supernatant. The specific time release percentage of BMP-2 was: the cumulative release amount of BMP-2 in the supernatant / the total amount of BMP-2 combined with the microspheres % in the time.

[0183] The results of the above different experiments were specifically shown in Table 5, Table 6 and B in Table 7 as follows: Figure 7

[0184] Table 5, BMP-2 content combined with different microspheres

[0185] Different mineralization methods / conditions Amount of BMP-2 bound BMP-2 protein loading 20% mSilMA microspheres 44.34 ng / mg 88.68％ 20% SilMA microspheres (dry mesoporous non-mineralized) 30.67 ng / mg 61.74％ SilMA-1 microspheres (10 times simulated body fluid method) 32.17 ng / mg 64.34％ SilMA-2 microspheres (nanocluster method) 31.29 ng / mg 61.29％ SilMA-3 microspheres (5 times Ca / P mineralization solution) 40.74 ng / mg 81.47％ SilMA-4 microspheres (10 times Ca / P mineralization solution) 42.19 ng / mg 84.38％

[0186] Table 6, BMP-2 in-vitro release ability of different microspheres in a certain time

[0187]

[0188] ​From Table 5 and Table 6, it can be known that only when the mineralization method is Ca / P method, and the mineralization concentration is 1 times Ca / P mineralization solution, the complete mSilMA microspheres with surface through mesoporous pore structure can be successfully prepared, the BMP-2 protein loading rate can reach 88.68%, and the BMP-2 can be continuously released at a low amount, and the cumulative release of BMP-2 is about 2.5% in vitro for 4 weeks; and when other mineralization methods, mineralization concentrations or no mineralization treatment are used, the effects of the present application cannot be achieved, the BMP-2 protein loading rates are obviously reduced, and the in vitro controlled release of BMP-2 is also obviously decreased.

[0189] At the same time, from the BMP-2 release in vitro of the mSilMA microspheres in Table 5 and Table 6, it can be known that the 20% mSilMA microspheres can continuously release BMP-2 at a low amount, and the cumulative release of BMP-2 is about 2% in vitro for 4 weeks, while the 20% SilMA microspheres cannot release BMP-2 for a long time, but reach a plateau in about 3 weeks, which may be because the adsorption / dissolution of mesopores to drugs reaches a balance to a certain extent, resulting in incomplete release. Figure 7

[0190] Therefore, in the present embodiment, the critical point drying method is preferably used to prepare the mSilMA.

[0191] Example 6: Screening of different SilMA solution concentrations in preparation of SilMA

[0192] In order to obtain the mSilMA with the best performance in Example 1, the concentration of the SilMA solution in the preparation process of the SilMA is screened in the present embodiment, and the specific screening process is as follows (the rest of the operation process and experimental conditions not mentioned are the best conditions in Example 1):

[0193] I. Preparation of microspheres with different concentrations

[0194] 1. The concentration of the SilMA solution is 10% (W / V%)

[0195] ​SilMA was dissolved in double distilled water, 25℃, to get 10%(W / V%) SilMA solution, a certain volume of LAP solution was added as the water phase, the final concentration was 0.25%(W / V%). The oil phase was composed of liquid paraffin and span 80, the mixing ratio was 1:20; the water phase was added dropwise into the oil phase, the oil-water ratio was 5:1, 25℃, stirring at 400rpm, ultraviolet irradiation was carried out during stirring. After 10min, stop stirring and irradiation, stand, microspheres precipitated at the bottom of the beaker. Discard the supernatant (oil phase), 50% isopropanol aqueous solution was washed 3 times, 75% isopropanol aqueous solution was washed 2 times, 80% isopropanol aqueous solution was washed 1 time; a large amount of water was washed 3 times, stand, microspheres precipitated at the bottom of the beaker. Discard the supernatant (water), filter to harvest microspheres. 10% SilMA hydrogel microspheres were soaked in 30%-50%-70%-90%-100% ethanol aqueous solution for gradient dehydration, each ethanol concentration soaking time was 15min; then critical point drying instrument was used for critical point drying, drying time was 3 hours, 10% dry state mesoporous silk fibroin methacrylamide microspheres (referred to as 10% SilMA) were harvested.

[0196] 2, the concentration of SilMA solution was 20%(W / V%)

[0197] SilMA was dissolved in double distilled water, 25℃, to get 20%(W / V%) SilMA solution, a certain volume of LAP solution was added as the water phase, the final concentration was 0.25%(W / V%). The oil phase was composed of liquid paraffin and span 80, the mixing ratio was 1:20; the water phase was added dropwise into the oil phase, the oil-water ratio was 5:1, 25℃, stirring at 400rpm, ultraviolet irradiation was carried out during stirring. After 10min, stop stirring and irradiation, stand, microspheres precipitated at the bottom of the beaker. Discard the supernatant (oil phase), 50% isopropanol aqueous solution was washed 3 times, 75% isopropanol aqueous solution was washed 2 times, 80% isopropanol aqueous solution was washed 1 time; a large amount of water was washed 3 times, stand, microspheres precipitated at the bottom of the beaker. Discard the supernatant (water), filter to harvest microspheres. 20% SilMA hydrogel microspheres were soaked in 30%-50%-70%-90%-100% ethanol aqueous solution for gradient dehydration, each ethanol concentration soaking time was 15min; then critical point drying instrument was used for critical point drying, drying time was 3 hours, 20% dry state mesoporous silk fibroin methacrylamide microspheres (referred to as 20% SilMA) were harvested.

[0198] 3, the concentration of SilMA solution was 30%(W / V%)

[0199] SilMA was dissolved in double-distilled water at 25°C to obtain a 30% (w / v) SilMA solution. A certain volume of LAP solution was added as the aqueous phase, resulting in a final concentration of 0.25% (w / v). The oil phase consisted of liquid paraffin and Span 80 in a 1:20 ratio. The aqueous phase was added dropwise to the oil phase at a 5:1 oil-to-water ratio. The mixture was stirred at 400 rpm at 25°C while being irradiated with UV light. After 10 minutes, stirring and irradiation were stopped, and the mixture was allowed to stand. The microspheres precipitated at the bottom of the beaker. The supernatant (oil phase) was discarded, and the mixture was washed three times with 50% isopropanol aqueous solution, twice with 75% isopropanol aqueous solution, and once with 80% isopropanol aqueous solution. The mixture was then washed three times with copious amounts of water. After standing, the microspheres precipitated at the bottom of the beaker. The supernatant (water) was discarded, and the microspheres were collected by filtration. 30% SilMA hydrogel microspheres were immersed in a gradient dehydration solution of 30%-50%-70%-90%-100% ethanol aqueous solution, with each ethanol concentration being immersed for 15 minutes; then, critical point drying was performed using a critical point dryer for 3 hours to obtain 30% dry mesoporous silk fibroin methacrylamide microspheres (abbreviated as 30% SilMA).

[0200] II. Results Analysis

[0201] The swelling properties and surface pore size of the dry mesoporous silk fibroin methacrylamide microspheres prepared using SilMA solutions of different concentrations were statistically analyzed. The specific results are as follows:

[0202] 1. Swelling performance test

[0203] Equal masses of 10%, 20%, and 30% dry mesoporous SilMA microspheres were weighed and soaked in an equal volume of phosphate buffer for 48 hours. The supernatant was filtered, and the microspheres were harvested and weighed. The swelling properties of mesoporous SilMA microspheres at different concentrations were preliminarily defined by the percentage increase in mass at each concentration.

[0204] Figure 8 The swelling properties of SilMA microspheres (A represents 10%, 20%, and 30%) are shown in the figure. Figure 8 As shown in A, 10% SilMA microspheres have the strongest water absorption and swelling capacity, and are prone to swelling and rupture within one week of soaking, losing their structural integrity. This indicates that their stability is insufficient and they are not suitable for subsequent processing. There is no significant difference in water absorption and swelling capacity between 20% and 30% SilMA microspheres.

[0205] 2. Statistical analysis of surface pore size

[0206] Based on the obtained scanning electron microscope images of the surface structure of mesoporous SilMA microspheres with different concentrations, the surface pore diameter of each concentration of mesoporous SilMA microspheres was measured and statistically analyzed using ImageJ software, and then statistically analyzed using GraphPad Prism software.

[0207] Figure 9 Scanning electron microscopy (SEM) results for 10%, 20%, and 30% mesoporous SilMA microspheres were obtained through statistical analysis of the SEM results. Figure 8 The result shown in B is from Figure 8 As shown in section B, the 10% SilMA microspheres have the largest surface pore size, with an average pore size of around 100 nm. The 20% SilMA microspheres have a surface pore size of around 20 nm, and the 30% SilMA microspheres have an average surface pore size of around 5 nm. Since the molecular diameter of bone morphogenetic protein is approximately 10 nm, only the 20% SilMA microspheres, with a surface pore size of around 20 nm, can both accommodate the entry of bone morphogenetic protein and, to some extent, limit its rapid release, thus contributing to better controlled-release effects. The 30% SilMA microspheres, with an average surface pore size of around 5 nm, are too small for bone morphogenetic protein, which has a molecular diameter of approximately 10 nm, hindering effective loading and subsequent release of the protein.

[0208] Therefore, considering the swelling properties and surface pore size distribution of SilMA microspheres with different concentrations, 20% SilMA microspheres exhibit a more balanced and ideal performance in terms of stability, loading of bone morphogenetic proteins, and controlled release. Thus, 20% SilMA microspheres are preferred for the next step of biomimetic mineralization.

[0209] Example 7: Screening of different preparation methods for SilMA

[0210] To obtain the mSilMA with the best performance in Example 1, different preparation methods were screened in this example. The specific screening process is as follows (all other unmentioned operations and experimental conditions are the optimal conditions in Example 1):

[0211] 1. Ultraviolet light crosslinking oil / water emulsification method (mechanical stirring): The specific preparation steps are shown in Example 1;

[0212] 2. Photocrosslinking 3D printing technology: SilMA is prepared using photocrosslinking 3D printing technology;

[0213] Specific results of SilMA obtained using different preparation methods are as follows: Figure 10 A and Figure 10 As shown in B, Figure 10 A in the image is a light micrograph of 20% SilMA hydrogel microspheres prepared by photocrosslinking oil / water emulsification (mechanical stirring); Figure 10 Image B in the image is a light micrograph of 20% SilMA hydrogel microspheres prepared by photocrosslinking 3D printing technology. Figure 10It can be known that the SilMA prepared by the ultraviolet light cross-linking oil / water emulsification method (mechanical stirring) has significant advantages in yield and sphericity compared with the microspheres prepared by the light cross-linking 3D printing technology; the microspheres prepared by the light cross-linking 3D printing technology are easy to adhere and have poor sphericity, and are mostly cylindrical.

[0214] In addition, the mechanical stirring method adopted by the present application can change the oil / water ratio, stirring speed and other parameters, thereby successfully preparing microspheres with a particle size range of 1 μm-1000 μm, but the light cross-linking 3D printing technology cannot print microspheres below 100 μm, and can only print microspheres of specific sizes, such as 200 μm, 400 μm, etc., in addition, the number of printed microspheres is limited by the volume of printing ink and the size of the printable stage.

[0215] Therefore, in the present embodiment, the ultraviolet light cross-linking oil / water emulsification method (mechanical stirring) is preferably used to prepare SilMA, which has the best yield, sphericity, particle size range controllability and other performances, and can better meet the needs of practical applications, and lays a solid foundation for subsequent preparation of mSilMA with excellent performance.

[0216] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A controlled release carrier, characterized by, The controlled release carrier is a ready-to-use mesoporous mineralized silk fibroin microsphere, which is prepared by first preparing SilMA hydrogel microspheres by means of ultraviolet light cross-linking oil / water emulsion method and mechanical stirring, and then mineralizing and drying the SilMA hydrogel microspheres, wherein the mineralization is a Ca / P method, and the drying is a critical drying method; the ready-to-use mesoporous mineralized silk fibroin microsphere has a mesoporous pore structure, a surface including a mineralized coating, and a main component having a fibrous structure; in the ultraviolet light cross-linking oil / water emulsion method, LAP solution is added to the SilMA solution as the water phase, and the water phase is stirred and mixed with the oil phase, wherein the final concentration of the LAP aqueous solution is 0.1%-0.5%; in the oil phase, the mass ratio of Span 80 to liquid paraffin is 1:10-1:

30.

2. The controlled release carrier of claim 1, wherein, The main component includes silk fibroin; the silk fibroin is silk fibroin methacrylamide.

3. The controlled release carrier of claim 2, wherein, The mineralized coating is needle-shaped calcium phosphate; the pore size of the mesoporous pore is 5-50 nm.

4. The controlled release carrier of claim 3, wherein, In the controlled release carrier, active proteins are loaded, and the active proteins include any one or more of BMP-2, VEGF, FGF, and TGF-β.

5. The controlled release carrier of claim 1, wherein, The mineralized coating is prepared by a Ca / P method, and the concentration of the mineralization solution is 1 times Ca / P mineralization solution, wherein the molar ratio of calcium chloride to phosphoric acid in the 1 times Ca / P mineralization solution is 1.5-2.

0.

6. The controlled release carrier of claim 1, wherein, The volume ratio of the oil phase to the water phase is 1:1-10:

1.

7. Use of a controlled release carrier for the manufacture of a preparation for improving bone repair ability or promoting bone regeneration, characterized in that, The controlled release carrier as claimed in any one of claims 1-6 is used to load active proteins, so as to prepare a preparation for improving bone repair ability or promoting bone regeneration.

8. Use of a controlled release carrier for the preparation of a formulation to increase the long-lasting low-dose sustained release ability of BMP-2 or to reduce the burst release ability of BMP-2, characterized in that, The controlled release carrier as claimed in any one of claims 1-6 is used to load active proteins, so as to prepare a preparation for improving long-acting low-dose sustained release ability of BMP-2 or reducing burst release ability of BMP-2.

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