Blood-meeting self-crosslinkable composite porous microspheres and preparation method thereof

Abstract

The application discloses a blood-contacting self-crosslinking composite porous microsphere and a preparation method thereof. The composite porous microsphere comprises: first microspheres (1) which are prepared by compounding chitosan containing amino functional groups and beta-tricalcium phosphate powder and have a porous structure; and second microspheres (2) which are prepared by compounding oxidized sodium alginate containing aldehyde functional groups and beta-tricalcium phosphate powder and have a porous structure. When the first microspheres and the second microspheres are contacted in the presence of blood, a Schiff base chemical reaction occurs between the amino groups and the aldehyde groups, a covalent bond (3) is formed, crosslinking between the microspheres is realized, a stable and integrated three-dimensional porous scaffold is formed in situ, migration of an implant is effectively prevented, and excellent osteogenic activity is achieved. The application further discloses a preparation method of the composite porous microsphere and application of the composite porous microsphere in bone defect repair materials, tissue engineering scaffolds or drug controlled release carriers.

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. Currently, the research and application of bone repair materials can be mainly divided into the following categories: 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 restricted. b. Allogeneic / xenograft bone transplantation: Although it solves the source problem, it has potential risks of disease transmission, immune rejection, and decreased osteogenic efficacy. 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. 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. 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). 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: 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. Complications: Particles that migrate to surrounding soft tissues (such as muscles, nerves, and blood vessels) may trigger foreign body granulomatous reactions or chronic inflammation. To address the problem of particle migration, existing technologies have proposed several strategies, but all have significant shortcomings: 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). 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. 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

[0003] 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. Another object of the present invention is to provide a method for preparing the above-mentioned composite porous microspheres. 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.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: 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: The first type of microspheres is made of chitosan containing amino functional groups and β-tricalcium phosphate powder, and the first type of microspheres has a porous structure. The second type of microspheres is made of sodium alginate containing aldehyde functional groups and β-tricalcium phosphate powder, and the second type of microspheres have a porous structure. In the presence of blood, the amino functional groups and aldehyde functional groups can undergo a Schiff base chemical reaction to form covalent bonds, thereby achieving cross-linking between the first type of microspheres and the second type of microspheres.

[0005] In one specific embodiment of the present invention, the degree of deacetylation of the chitosan is ≥90%, and the viscosity is 100-200 mPa·s; the degree of oxidation of the oxidized sodium alginate is 40%-60%.

[0006] In one specific embodiment of the present invention, the particle size of the β-tricalcium phosphate powder is ≤5μm.

[0007] In one specific embodiment of the present invention, the particle size range of the first type of microspheres and the second type of microspheres is 100-300 μm, the porosity is 50%-80%, and the pore size is 5-500 μm.

[0008] In one specific embodiment of the present invention, the first type of microspheres and the second type of microspheres are further loaded with bone growth factor and / or drug, wherein the growth factor is bone morphogenetic protein-2 (BMP-2) and the drug is an antibiotic or an anti-inflammatory drug.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned composite porous microspheres, comprising the following steps: Preparation of the first type of microspheres: a. Dissolve chitosan in an acidic aqueous solution to form solution A; b. Disperse β-tricalcium phosphate powder in solution A to obtain mixed slurry A; c. The mixed slurry A is spray-dried into microspheres, and after washing and freeze-drying, the first type of porous composite microspheres are obtained.

[0010] Preparation of the second type of microspheres: a. Dissolve sodium oxidized alginate in deionized water to form solution B; b. Disperse β-tricalcium phosphate powder in solution B to obtain mixed slurry B; c. The mixed slurry B is prepared into microspheres by emulsification-ion crosslinking curing method, and after washing and freeze drying, the second type of porous composite microspheres are obtained.

[0011] (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.

[0012] In the preparation method of the present invention, the inlet air temperature of the spray drying method is 120-140℃ and the outlet air temperature is 70-90℃; the emulsification-ion crosslinking curing method includes: adding slurry B dropwise into a solution containing CaCl2 for curing, wherein the mass fraction of the CaCl2 solution is 1%-3%.

[0013] 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.

[0014] The beneficial effects of this invention are: 1. In-situ self-crosslinking: After the microsphere system of the present invention comes into contact with blood, the amino and aldehyde functional groups on the surface of the two types of microspheres can rapidly undergo Schiff base reaction, achieving covalent crosslinking between microspheres within 60-90 seconds, forming a stable, integrated three-dimensional porous scaffold in situ, effectively preventing implant migration. 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. 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. 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. 5. High biocompatibility: The selected materials (chitosan, sodium alginate, β-TCP) are all biodegradable and biocompatible materials. The cross-linking reaction is a bio-friendly reaction, and the degradation products can be absorbed or metabolized by the human body. Attached Figure Description Figure 1 Schematic diagram of the structure of composite porous microspheres Figure 2 Schematic diagram of the effect of composite porous microspheres filling bone defects. Figure 3 Scanning electron microscope image of composite porous microspheres Figure 4 Image of a simulated bone defect filling site using type I and II microspheres Detailed Implementation

[0015] Example 1: Chitosan / sodium oxidized alginate-β-TCP composite microspheres based on Schiff base reaction 1. Preparation of the first type of microspheres (1) (amino microspheres-chitosan / β-TCP microspheres): Raw materials: chitosan (degree of deacetylation ≥90%, viscosity 100-200 mPa·s), β-tricalcium phosphate (β-TCP, particle size ≤5μm), glacial acetic acid. step: (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. (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. (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 5 mL / min, and the nozzle orifice diameter to 0.5 mm. (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 0.1 Pa for 24 h to obtain white first-class porous microspheres 1. 2. Preparation of the second type of microspheres (2) (aldehyde-based microspheres-sodium oxidized alginate / β-TCP microspheres): Raw materials: sodium alginate, sodium periodate (NaIO4), β-TCP, calcium chloride (CaCl2). 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. step: (1) Dissolve 2.0g of sodium alginate oxide (oxidation degree about 50%) in 100 mL of deionized water and stir until completely dissolved to obtain solution B. (2) Add 1.0g of β-TCP powder to solution B and disperse it evenly by ultrasonication as above to obtain mixed slurry B. (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. (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. 3. Performance Testing: Morphological characterization: Scanning electron microscopy (SEM) observation showed that both types of microspheres were spherical with porous surfaces and regular shapes, with particle sizes mainly distributed between 100-300 μm and interconnected pore structures inside. In vitro cross-linking verification: In a culture dish, equal masses of type I and type II 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. The morphology and post-crosslinking state of the microspheres are shown in the figure above. Figure 4

[0016] 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, characterized in that, include: The first type of microspheres is made of chitosan containing amino functional groups and β-tricalcium phosphate powder, and the first type of microspheres has a porous structure; The second type of microspheres is made of sodium alginate containing aldehyde functional groups and β-tricalcium phosphate powder, and the second type of microspheres has a porous structure. The amino and aldehyde functional groups are configured such that when the first type of microspheres and the second type of microspheres come into contact in the presence of blood, they can undergo a Schiff base chemical reaction to form covalent bonds, thereby achieving cross-linking between the first type of microspheres and the second type of microspheres.

2. The composite porous microspheres according to claim 1, characterized in that, The chitosan has a degree of deacetylation ≥90% and a viscosity of 100-200 mPa·s; the oxidized sodium alginate has an oxidation degree of 40%-60%.

3. The composite porous microspheres according to claim 1, characterized in that, The particle size of the β-tricalcium phosphate powder is ≤5μm.

4. The composite porous microspheres according to claim 1, characterized in that, The particle size range of the first type of microspheres and the second type of microspheres is 100-300 μm, the porosity is 50%-80%, and the pore size range is 5-500 μm.

5. The composite porous microspheres 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; the bioactive factor is bone morphogenetic protein-2 (BMP-2), and the drug is an antibiotic or an anti-inflammatory drug.

6. A method for preparing composite porous microspheres as described in any one of claims 1-5, characterized in that, Includes the following steps: a. Preparation of the first type of microspheres: Chitosan was dissolved in an acidic aqueous solution and mixed with β-tricalcium phosphate powder to form slurry A, which was then spray-dried to form porous microspheres; b. Preparation of the second type of microspheres: Dissolve sodium oxidized alginate in deionized water and mix it with β-tricalcium phosphate powder to form slurry B. Then, porous microspheres are prepared by emulsification-ion crosslinking curing method. c. The microspheres obtained in steps a and b are washed and freeze-dried to obtain dried composite porous microspheres.

7. The method according to claim 6, characterized in that, In step a, the inlet air temperature of the spray drying method is 120-140℃, and the outlet air temperature is 70-90℃.

8. The method according to claim 6, characterized in that, In step b, the emulsification-ion crosslinking curing method includes: adding slurry B dropwise into a solution containing CaCl2 for curing, wherein the mass fraction of the CaCl2 solution is 1%-3%.

9. The application of a blood-reactive composite porous microsphere as described in any one of claims 1-5 in the preparation of tissue engineering scaffolds or drug-controlled release carriers.

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