Composite porous microsphere capable of self-crosslinking when meeting blood and preparation method of composite porous microsphere

Abstract

The invention relates to a composite porous microsphere capable of self-crosslinking in the presence of blood and a preparation method thereof, the composite porous microsphere comprises: a first type of microsphere (1), which is prepared by compounding a biodegradable polymer material containing a first active functional group and inorganic active ceramic powder; the second type of microspheres (2) are prepared by compounding a biodegradable high polymer material containing a second active functional group and inorganic active ceramic powder; wherein the first active functional group and the second active functional group can be subjected to a specific chemical reaction in the presence of blood to form a covalent bond (3), so that cross-linking between the first type of microspheres and the second type of microspheres is realized, a stable and integrated three-dimensional porous scaffold is formed in situ, and implant migration is effectively prevented; the excellent osteogenic activity is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an injectable, biodegradable porous composite microsphere for bone tissue repair that can undergo autonomous cross-linking upon contact with blood, and its preparation method. Background Technology

[0002] Bone defects are common diseases in clinical fields such as orthopedics, oral and maxillofacial surgery, and neurosurgery, and their effective repair and functional reconstruction have always been major clinical challenges. Ideal bone repair materials should have good biocompatibility, osteoconductivity / osteoinductive properties, biodegradability, and be able to adapt to the morphology of irregular bone defects, providing temporary mechanical support.

[0003] Currently, the research and application of bone repair materials can be mainly divided into the following categories:

[0004] a. Autologous bone grafting: Considered the "gold standard," it has excellent osteogenic potential and no immune rejection. However, its sources are limited, it can cause secondary damage to the donor site, and the amount of bone harvested is limited.

[0005] b. Allogeneic / xenograft bone transplantation: Although it solves the source problem, it has potential risks of disease transmission, immune rejection, and decreased osteogenic efficacy.

[0006] c. Metallic materials (such as titanium alloys): provide strong mechanical support, but their elastic modulus does not match that of natural bone, which may lead to a "stress shielding" effect, hindering bone healing. They are mostly permanent implants and may require a second surgery to remove.

[0007] d. Bioceramic materials (such as hydroxyapatite HA, β-tricalcium phosphate β-TCP): have excellent osteoconductivity and biocompatibility, but their inherent brittleness and difficult-to-control degradation rate limit their application alone.

[0008] In recent years, injectable porous microparticle systems (such as microspheres based on polylactic-co-glycolic acid copolymer (PLGA), chitosan, gelatin, etc.) have shown great potential in bone tissue engineering. These materials can be implanted in a minimally invasive manner, perfectly conforming to irregular bone defects; their three-dimensional porous structure can provide space for cell adhesion, proliferation, and new bone ingrowth, and can also serve as controlled-release carriers for growth factors (such as BMP-2).

[0009] However, existing injectable porous microparticle technology has revealed a key drawback in clinical applications: after implantation, the loose microparticles are highly susceptible to migration and dispersion under the influence of bodily fluids (such as blood and tissue fluid), muscle contraction, or external forces, failing to stably reside at the bone defect site and form a complete, uniform three-dimensional scaffold. This leads to the following serious consequences:

[0010] Poor repair results: The displacement of microparticles leads to a reduction in the effective filler in the defect area, which cannot provide a continuous and stable scaffold for the new bone tissue, seriously affecting the quality and speed of bone repair.

[0011] Complications: Particles that migrate to surrounding soft tissues (such as muscles, nerves, and blood vessels) may trigger foreign body granulomatous reactions or chronic inflammation.

[0012] To address the problem of particle migration, existing technologies have proposed several strategies, but all have significant shortcomings:

[0013] Using external cross-linking agents: For example, after implanting alginate microspheres, a calcium chloride solution is sprayed to cross-link and solidify them. This method is cumbersome, the cross-linking reaction is difficult to control, and the introduced external chemical reagents may pose biocompatibility risks and may adversely affect the activity of co-loaded active factors (such as growth factors and cells).

[0014] Pre-fabricated scaffolds: The material is pre-fabricated into a rigid scaffold that matches the shape of the defect. This method completely loses the minimally invasive advantages and shape self-adaptation capabilities of injectable microspheres, and gaps may occur during implantation due to mismatch, affecting the integration effect.

[0015] Therefore, there is an urgent need in this field to develop a novel injectable bone repair material that retains the excellent properties of porous microspheres while rapidly and autonomously stabilizing after implantation, fundamentally solving the migration problem. Based on this, this invention proposes a composite porous microsphere system capable of automatically cross-linking in the blood environment, aiming to achieve an intelligent transformation from "loose microparticles" to "integrated scaffolds," providing a superior solution for clinical bone defect repair. Summary of the Invention

[0016] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composite porous microsphere that can automatically cross-link upon contact with blood. This microsphere can form a stable porous scaffold in situ, prevent migration, and promote bone tissue repair.

[0017] Another object of the present invention is to provide a method for preparing the above-mentioned composite porous microspheres.

[0018] Another object of the present invention is to provide the application of the above-mentioned composite porous microspheres in the preparation of bone defect repair materials.

[0019] To achieve the above objectives, the present invention adopts the following technical solution:

[0020] In a first aspect, the present invention provides a composite porous microsphere system that can self-crosslink upon contact with blood, characterized in that it comprises:

[0021] The first type of microspheres is made of a composite of biodegradable polymer materials containing first active functional groups and porous ceramic powder;

[0022] The second type of microspheres is made of biodegradable polymer materials containing second active functional groups and porous ceramic powder.

[0023] In this process, the first and second active functional groups can undergo specific chemical reactions in the presence of blood to form covalent or coordination bonds, thereby achieving cross-linking between the first and second types of microspheres.

[0024] Preferably, the first active functional group is an amino group and the second active functional group is an aldehyde group; or, the first active functional group is a mercapto group and the second active functional group is an olefin or acrylate group; or, the first active functional group is a tetrazine group and the second active functional group is trans-cyclooctene.

[0025] Preferably, the biodegradable polymer material is selected from one or more combinations of chitosan, gelatin, collagen, sodium alginate, dextran, polylactic acid-glycolic acid copolymer (PLGA), and polylactic acid (PLA).

[0026] Preferably, the porous ceramic powder is selected from one or more of the following: β-tricalcium phosphate (β-TCP), hydroxyapatite (HA), and bioactive glass (BG).

[0027] Preferably, the particle size range of the first type of microspheres and the second type of microspheres is 500-5000 μm, the porosity is 50%-80%, and the pore size is 50-500 μm.

[0028] Preferably, the first and second types of microspheres are further loaded with bone growth factors and / or drugs, wherein the growth factor is bone morphogenetic protein-2 (BMP-2) and the drug is an antibiotic or an anti-inflammatory drug.

[0029] Secondly, the present invention provides a method for preparing the above-mentioned composite porous microspheres, comprising the following steps:

[0030] 1. Preparation of Type I Microspheres:

[0031] a. Dissolve a biodegradable polymer containing a first active functional group in a solvent to form solution A;

[0032] b. Disperse porous ceramic powder in solution A to obtain mixed slurry A;

[0033] c. The mixed slurry A is made into microspheres by emulsification-solvent evaporation, spray drying or microfluidic technology, and then washed and freeze-dried to obtain the first type of porous composite microspheres.

[0034] 2. Preparation of the second type of microspheres:

[0035] a. Dissolve a biodegradable polymer containing a second active functional group in a solvent to form solution B;

[0036] b. Disperse porous ceramic powder in solution B to obtain mixed slurry B;

[0037] c. The mixed slurry B is made into microspheres by emulsification-solvent evaporation, spray drying or microfluidic technology, and then washed and freeze-dried to obtain the second type of porous composite microspheres.

[0038] 3. (Optional) Loading active factors: Add growth factors or drugs to the solution in step 1a or 2a, or load active factors onto the prepared microspheres by adsorption.

[0039] Thirdly, the present invention provides the application of the above-mentioned composite porous microsphere system in the preparation of bone defect repair materials, tissue engineering scaffolds or drug delivery systems.

[0040] The beneficial effects of this invention are:

[0041] 1. In-situ self-crosslinking: After the microsphere system of the present invention comes into contact with blood, the active functional groups on the surfaces of the two types of microspheres can rapidly undergo efficient and specific chemical reactions (such as Schiff base reaction and click chemical reaction), achieving covalent crosslinking between microspheres within seconds to minutes, forming a stable, integrated three-dimensional porous scaffold in situ, effectively preventing implant migration.

[0042] 2. Excellent osteogenic activity: The microspheres are combined with bioceramics such as β-TCP, providing good osteoconductivity and bioactivity; at the same time, the porous structure facilitates cell ingrowth and nutrient delivery.

[0043] 3. Minimally invasive and personalized fit: Microspheres can be implanted using a regular syringe or a dual-barrel syringe, making the procedure simple and minimally invasive. Their flow properties allow them to perfectly adapt to bone defects of any irregular shape.

[0044] 4. Diverse functions: The porous structure of microspheres can serve as a "warehouse" to load and control the release of growth factors (such as BMP-2) or drugs, achieving integrated treatment and repair.

[0045] 5. High biocompatibility: All selected materials are biodegradable and biocompatible, and the cross-linking reaction is a bio-friendly reaction. The degradation products can be absorbed or metabolized by the human body. Attached image description:

[0046] Figure 1 Schematic diagram of the structure of composite porous microspheres

[0047] Figure 2 Schematic diagram of the effect of composite porous microspheres filling bone defects.

[0048] Figure 3 Scanning electron microscope image of composite porous microspheres

[0049] Figure 4 Image of a simulated bone defect filling site using type I and II microspheres Detailed Implementation

[0050] Example 1: Chitosan / Sodium Oxygenated Alginate-β-TCP Composite Microspheres Based on Schiff Base Reaction 1. Preparation of Type I Microspheres (1) (Amino Microspheres-Chitosan / β-TCP Microspheres):

[0051] Raw materials: chitosan (degree of deacetylation ≥90%, viscosity 100-200 mPa·s), β-tricalcium phosphate (β-TCP, particle size ≤5μm), glacial acetic acid.

[0052] step:

[0053] (1) Dissolve 2.0g of chitosan powder in 100ml of 1% glacial acetic acid aqueous solution and stir magnetically for 4h until completely dissolved to obtain transparent colloidal solution A.

[0054] (2) Slowly add 1.0g of β-TCP powder to the above solution A, and sonicate it at 500W power for 30 minutes in an ice-water bath to make it fully and evenly dispersed, thus obtaining mixed slurry A.

[0055] (3) Transfer slurry A to a spray dryer for granulation. Set the inlet air temperature to 130℃, the outlet air temperature to 80℃, the feed pump speed to 5mL / min, and the nozzle orifice diameter to 0.5mm.

[0056] (4) Collect the dried microspheres and wash them three times each with anhydrous ethanol and deionized water to remove residual acetic acid. Then freeze-dry them at -50℃ and 0.1Pa for 24h to obtain white first-class porous microspheres 1.

[0057] 2. Preparation of the second type of microspheres (2) (aldehyde-based microspheres-sodium oxidized alginate / β-TCP microspheres):

[0058] Raw materials: sodium alginate, sodium periodate (NaIO4), β-TCP, calcium chloride (CaCl2).

[0059] Preparation of oxidized sodium alginate: 5g of sodium alginate was dissolved in 500mL of deionized water, and 2.5g of sodium periodate was added under light-protected conditions. The mixture was magnetically stirred at room temperature for 6 hours. After the reaction was completed, excess ethylene glycol was added to terminate the reaction. The solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed against deionized water for 3 days, and then freeze-dried to obtain oxidized sodium alginate.

[0060] step:

[0061] (1) Dissolve 2.0g of sodium alginate oxide (oxidation degree about 50%) in 100mL of deionized water and stir until completely dissolved to obtain solution B.

[0062] (2) Add 1.0g of β-TCP powder to solution B and disperse it evenly by ultrasonication as above to obtain mixed slurry B.

[0063] (3) Use a micro-injection pump to drop slurry B into 100 mL of 2% CaCl2 solution at a rate of 20 mL / h, while gently stirring at 200 rpm and solidifying for 30 minutes to form initial spheres.

[0064] (4) Filter out the microspheres, wash them thoroughly with deionized water, and then freeze-dry them to obtain the second type of porous microspheres 2.

[0065] 3. Performance Testing:

[0066] Morphological characterization: Scanning electron microscopy (SEM) observation showed that both types of microspheres were porous, regularly shaped spheres with particle sizes mainly distributed between 100-300 μm, and possessed interconnected pore structures. • In vitro cross-linking verification: In a culture dish, equal masses of the first and second types of microspheres were physically mixed uniformly. Then, 2 mL of fresh goat blood was added to simulate the in vivo environment. It was observed that the loose microspheres rapidly lost their fluidity within 60-90 seconds, forming a monolithic hydrogel block, indicating that rapid and effective autonomous cross-linking was achieved between the microspheres through the Schiff base reaction.

[0067] The morphology and post-crosslinking state of the microspheres are shown in the figure above. Figure 4

[0068] Example 2: PLGA-based composite microspheres based on click chemistry

[0069] 1. Synthesis of functionalized PLGA:

[0070] Preparation of thiolized PLGA (PLGA-SH): 10 g of PLGA (LA:GA = 75:25) was dissolved in 100 mL of anhydrous dimethyl sulfoxide (DMSO). An excess of 1.5 equivalents of cystamine dihydrochloride and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) was added, and the reaction was carried out under nitrogen protection at room temperature for 24 hours. After the reaction was complete, excess dithiothreitol (DTT) was added to reduce the disulfide bonds. The resulting product was dialyzed and freeze-dried to obtain PLGA-SH.

[0071] Preparation of acrylated PLGA (PLGA-Acr): 10 g of PLGA was dissolved in 100 mL of anhydrous DMSO, and an excess of 2.0 equivalents of acryloyl chloride and triethylamine was added. The reaction was carried out in an ice bath for 12 h. The reaction solution was precipitated, washed, and dried to obtain PLGA-Acr.

[0072] 2. Preparation of the first type of microspheres (PLGA-SH / bioactive glass microspheres):

[0073] (1) Dissolve 1.0g PLGA-SH in 10mL dichloromethane (DCM) as the oil phase.

[0074] (2) Disperse 0.3g of bioactive glass (BG, particle size <10μm) in the oil phase.

[0075] (3) Pour the oil phase into 100 mL of an aqueous phase containing 2% polyvinyl alcohol (PVA) and emulsify at 5000 rpm for 2 min to form a W / O emulsion.

[0076] (4) Slowly pour the emulsion into 400 mL of 0.5% PVA solution and stir magnetically for 6 h to allow the DCM to evaporate completely.

[0077] (5) Collect the microspheres, centrifuge and wash them, and freeze dry them.

[0078] 3. Preparation of the second type of microspheres (PLGA-Acr / bioactive glass microspheres):

[0079] The preparation method is the same as in 2, except that the polymer is replaced with PLGA-Acr.

[0080] 4. Performance Testing:

[0081] (1) Crosslinking test: Equal amounts of the two types of PLGA microspheres were mixed, and a PBS solution containing the photoinitiator (Irgacure 2959) was added. The mixture was then irradiated under 365 nm ultraviolet light for 30 s. The mixture rapidly crosslinked into an elastic solid. In a simulated body fluid environment without ultraviolet light, the crosslinking reaction was completed slowly within a few minutes.

[0082] (2) Cell experiments: Mouse pre-osteoblasts (MC3T3-E1) were co-cultured with the cross-linked microsphere scaffold. Live / dead staining and CCK-8 assay results showed that the material was non-cytotoxic, and cells could adhere well and proliferate on the scaffold surface and in the pores.

[0083] Example 3: Gelatin / hydroxyapatite composite microspheres based on enzyme-catalyzed cross-linking

[0084] 1. Preparation of the first type of microspheres (enzyme-loaded gelatin / hydroxyapatite microspheres):

[0085] (1) Dissolve 2.0g of gelatin (type A) in 50mL of PBS at 50℃ to obtain solution A.

[0086] (2) Add 0.5g hydroxyapatite (HA, nanorod) and 50mg microbial transglutaminase (mTGase), and gently stir to disperse evenly.

[0087] (3) Solution A was made into microspheres by spray drying (inlet air temperature 90℃) and collected quickly.

[0088] 2. Preparation of the second type of microspheres (matrix gelatin / hydroxyapatite microspheres):

[0089] (1) Dissolve 2.0g of gelatin in 50mL of PBS at 50℃ to obtain solution B.

[0090] (2) Add 0.5g HA and 0.1g short peptide containing glutamine and lysine (as a preferred substrate for mTGase), and gently stir to disperse evenly.

[0091] (3) Microspheres were also prepared by spray drying.

[0092] 3. Crosslinking mechanism and testing:

[0093] Mechanism: When the two types of microspheres mix and come into contact with blood, the mTG enzyme encapsulated in the first type of microspheres reacts with blood water and calcium... 2+ Under the activation of (a cofactor of mTG enzyme, present in the blood), it catalyzes the formation of ε-(γ-glutamyl)lysine covalent bonds between glutamine residues and lysine residues in gelatin and short peptide molecules on the surface of the second type of microspheres, thereby achieving cross-linking.

[0094] Rheological testing: The two types of microspheres were mixed with a small amount of blood and placed in a rheometer. Time-scan mode showed that the storage modulus (G') of the mixture rapidly increased from the initial ~10 Pa to ~1000 Pa within 10 minutes, confirming its rapid in-situ gelation ability.

[0095] The three embodiments above represent three different technical approaches: biomimetic chemistry, click chemistry, and bio-enzyme catalysis. All three successfully prepared composite porous microspheres that can automatically cross-link upon contact with blood or bodily fluids. These microspheres effectively overcome the shortcomings of existing technologies, forming stable three-dimensional scaffolds in situ at bone defect sites, and have promising clinical application prospects.

[0096] The above is a description of the principles of the present invention. For those skilled in the art, it will be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The actual protection scope of the present invention shall be determined by the legally defined protection boundaries.

Claims

1. A composite porous microsphere that can self-crosslink upon contact with blood and its preparation method, characterized in that, include: The first type of microspheres (1) is made of a biodegradable polymer material containing a first active functional group and an inorganic active ceramic powder composite; The second type of microspheres (2) is made of biodegradable polymer materials containing second active functional groups and inorganic active ceramic powder; Among them, the first active functional group and the second active functional group can undergo a specific chemical reaction in the presence of blood to form a covalent bond (3), thereby realizing the cross-linking between the first type of microspheres and the second type of microspheres.

2. The composite porous microsphere system according to claim 1, characterized in that, The first and second active functional groups are functional group pairs capable of undergoing click chemistry or Schiff base reactions.

3. The composite porous microsphere system according to claim 2, characterized in that, The functional group pair is selected from any of the following combinations: amino and aldehyde groups; Mercapto groups with olefin or acrylate groups; Tetraazine and trans-cyclooctene.

4. The composite porous microsphere system according to claim 1, characterized in that, The biodegradable polymer material is selected from one or more of the following: chitosan, gelatin, collagen, sodium alginate, dextran, polylactic acid-glycolic acid copolymer (PLGA), and polylactic acid (PLA).

5. The composite porous microsphere system according to claim 1, characterized in that, The inorganic active ceramic powder is selected from one or more of the following: β-tricalcium phosphate (β-TCP), hydroxyapatite (HA), and bioactive glass (BG).

6. The composite porous microsphere system according to claim 1, characterized in that, The particle size range of the first type of microspheres and the second type of microspheres is 50-5000 μm, the porosity is 50%-80%, and the pore size range is 5-500 μm.

7. The composite porous microsphere system according to claim 1, characterized in that, The porous structure of the first type of microspheres and / or the second type of microspheres is further loaded with bioactive factors and / or drugs; preferably, the bioactive factor is bone morphogenetic protein-2 (BMP-2), and the drug is an antibiotic or an anti-inflammatory drug.

8. A method for preparing a composite porous microsphere system as described in any one of claims 1-7, characterized in that, Includes the following steps: a. Preparation of the first type of microspheres: Dissolve the biodegradable polymer containing the first active functional group, mix it with inorganic active ceramic powder to form slurry A, and then form porous microspheres through a molding process; b. Preparation of the second type of microspheres: Dissolve the biodegradable polymer containing the second active functional group, mix it with inorganic active ceramic powder to form slurry B, and then form porous microspheres through a molding process; c. Post-process the microspheres obtained in steps a and b to obtain dried composite porous microspheres.

9. The method according to claim 8, characterized in that, The molding process is an emulsification-solvent evaporation method, a spray drying method, or a microfluidic technology.

10. The application of a blood-reactive composite porous microsphere as described in any one of claims 1-7 in the preparation of bone defect repair materials, tissue engineering scaffolds, or drug-controlled release carriers.

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