CN121401482A - A self-crosslinking composite porous microsphere in contact with blood and its preparation method - Google Patents

Abstract

A composite porous microsphere capable of self-crosslinking when meeting blood and a preparation method thereof comprise a first microsphere (1) which is prepared by compositing a biodegradable polymer material containing a first active functional group with inorganic active ceramic powder, and a second microsphere (2) which is prepared by compositing a biodegradable polymer material containing a second active functional group with inorganic active ceramic powder, wherein the first active functional group and the second active functional group can perform specific chemical reaction under the condition of blood existence to form a covalent bond (3), so that the crosslinking between the first microsphere and the second microsphere is realized, a stable and integrated three-dimensional porous bracket is formed in situ, the implant migration is effectively prevented, and the excellent osteogenic activity is realized.

Description

Composite porous microsphere capable of self-crosslinking in blood and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and in particular relates to an injectable, degradable and self-crosslinking porous composite microsphere capable of being subjected to blood contact and a preparation method thereof.

Background

Bone defects are common diseases in clinical fields such as orthopaedics, oral and maxillofacial surgery, neurosurgery and the like, and effective repair and functional reconstruction are always important challenges in clinic. The ideal bone repair material has good biocompatibility, bone conductivity/bone inducibility and biodegradability, can adapt to the form of irregular bone defects, and provides temporary mechanical support.

At present, the research and application of bone repair materials are mainly divided into the following categories:

a. autologous bone grafting, considered as "gold standard", has good osteogenic potential and no immunological rejection. But the source is limited, secondary injury to the donor area is caused, and the bone extraction amount is limited.

B. Allograft/xenograft, although solving the source problem, has the problems of potential disease transmission risk, immune rejection reaction, bone formation efficacy decay and the like.

C. Metallic materials (such as titanium alloys) provide strong mechanical support, but have a modulus of elasticity that does not match that of natural bone, may cause a "stress shielding" effect that impedes bone healing, and are often permanent implants that may require secondary surgical removal.

D. Bioceramic materials (e.g. hydroxyapatite HA, beta-tricalcium phosphate beta-TCP) have excellent bone conductivity and biocompatibility, but their inherent brittleness and degradation rate are difficult to control, limiting their individual application.

In recent years, injectable porous microparticle systems (e.g., microspheres based on polylactic-co-glycolic acid (PLGA), chitosan, gelatin, etc.) have shown great potential in bone tissue engineering. The material can be implanted in a minimally invasive mode, perfectly fits irregular bone defects, and the three-dimensional porous structure of the material can provide space for cell adhesion, proliferation and new bone ingrowth and can be used as a controlled release carrier of growth factors (such as BMP-2).

However, the prior injectable porous microparticle technology has a critical defect in clinical application that loose microparticles are easily migrated and dispersed under the action of body fluid (such as blood and tissue fluid) flushing, muscle contraction or external force after implantation, and cannot stably reside at a bone defect part to form a complete and uniform three-dimensional scaffold. This has the following serious consequences:

The repairing effect is poor, the effective filling of the defect area is reduced due to the displacement of the particles, a continuous and stable climbing bracket can not be provided for the new bone tissue, and the quality and the speed of bone repairing are seriously influenced.

Complications are caused by the fact that particles migrating to surrounding soft tissues (e.g. muscles, neurovascular) may trigger a granulomatous response of foreign matter or chronic inflammation.

In order to solve the problem of particle migration, some strategies are proposed in the prior art, but all have obvious defects:

external crosslinking agents are used, for example, after implantation of alginate microspheres, calcium chloride solution is sprayed to crosslink and cure. The method has complicated operation, the crosslinking reaction is not easy to control, and the introduced external chemical reagent can bring about biocompatibility risks and can have adverse effects on the activity of the co-carried active factors (such as growth factors and cells).

Prefabricating the formed bracket, namely prefabricating the material into a rigid bracket matched with the defect shape. The method completely loses the minimally invasive advantage and shape self-adaptation capability of the injectable microspheres, and gaps can be generated due to mismatching in the implantation process, so that the integration effect is affected.

Therefore, there is an urgent need in the art to develop a novel injectable bone repair material that can not only retain the excellent properties of porous microspheres, but also be quickly and autonomously stabilized after implantation in vivo, thereby fundamentally solving the migration problem. Based on the above, the invention provides a composite porous microsphere system capable of automatically generating crosslinking in a blood environment, which aims to realize intelligent conversion from loose particles to an integrated bracket and provides a better solution for clinical bone defect repair.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a composite porous microsphere capable of automatically crosslinking when meeting blood, which can form a stable porous bracket in situ, prevent migration and promote bone tissue repair.

Another object of the present invention is to provide a method for preparing the composite porous microsphere.

It is still another object of the present invention to provide an application of the composite porous microsphere in preparing bone defect repair material.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

In a first aspect, the present invention provides a blood-self-crosslinkable composite porous microsphere system, comprising:

The first microsphere is prepared by compounding a degradable high polymer material containing a first active functional group and porous ceramic powder;

the second microsphere is prepared by compounding a degradable high polymer material containing a second active functional group and porous ceramic powder;

The first active functional group and the second active functional group can perform specific chemical reaction under the condition of blood existence to form covalent bonds or coordination bonds, so that the crosslinking between the first type of microspheres and the second type of microspheres is realized.

Preferably, the first active functional group is amino, the second active functional group is aldehyde, or the first active functional group is mercapto, the second active functional group is alkene or acrylate, or the first active functional group is tetrazine, and the second active functional group is trans-cyclooctene.

Preferably, the degradable high polymer material is selected from one or more of chitosan, gelatin, collagen, oxidized sodium alginate, oxidized dextran, polylactic acid-glycolic acid copolymer (PLGA) and polylactic acid (PLA).

Preferably, the porous ceramic powder is selected from one or more of beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) and Bioactive Glass (BG).

Preferably, the particle size range of the first type of microspheres and the second type of microspheres is 500-5000 microns, the porosity is 50-80%, and the pore diameter is 50-500 microns.

Preferably, the first and second types of microspheres are further loaded with a bone growth promoting factor and/or a drug, wherein the growth factor is bone morphogenic protein-2 (BMP-2), and the drug is an antibiotic or an anti-inflammatory drug.

In a second aspect, the invention provides a preparation method of the composite porous microsphere, which comprises the following steps:

1. Preparation of first-class microspheres:

a. dissolving a degradable polymer containing a first active functional group in a solvent to form a solution A;

b. Dispersing porous ceramic powder in the solution A to obtain mixed slurry A;

c. The mixed slurry A is prepared into microspheres by an emulsification-solvent volatilization method, a spray drying method or a microfluidic technology, and the microspheres are washed and freeze-dried to obtain the first porous composite microspheres.

2. Preparation of the second type of microspheres:

a. dissolving a degradable polymer containing a second active functional group in a solvent to form a solution B;

b. Dispersing porous ceramic powder in the solution B to obtain mixed slurry B;

c. And preparing the mixed slurry B into microspheres by an emulsification-solvent volatilization method, a spray drying method or a microfluidic technology, and washing and freeze-drying to obtain the second type porous composite microspheres.

3. And (3) optionally loading the active factors, namely adding growth factors or medicines into the solution obtained in the step 1a or 2a, or loading the active factors on the prepared microspheres by an adsorption method.

In a third aspect, the present invention provides the use of the composite porous microsphere system described above in the preparation of bone defect repair materials, tissue engineering scaffolds, or drug delivery systems.

The invention has the beneficial effects that:

1. In-situ self-crosslinking, after the microsphere system contacts blood, active functional groups on the surfaces of the two types of microspheres can quickly generate efficient and specific chemical reactions (such as Schiff base reaction and click chemical reaction), covalent crosslinking among the microspheres is realized within a few seconds to a few minutes, and a stable and integrated three-dimensional porous bracket is formed in situ, so that implant migration is effectively prevented.

2. Excellent osteogenesis activity, which is achieved by compounding beta-TCP and other biological ceramics with the microspheres, provides good bone conductivity and biological activity, and simultaneously, the porous structure is beneficial to cell ingrowth and nutrition transportation.

3. The micro-wound and personalized adaptation is that the microsphere can be implanted by a common injector or a double-barrel injector, the operation is simple and the wound is small. The flow state can be perfectly matched with any irregularly shaped bone defect.

4. The function is diversified, the porous structure of the microsphere can be used as a warehouse to load and control the release of growth factors (such as BMP-2) or medicines, so that the treatment and repair integration is realized.

5. The biological safety is high, the selected materials are all degradable biocompatible materials, the crosslinking reaction is a bio-friendly reaction, and degradation products can be absorbed or metabolized by human bodies.

Description of the drawings:

FIG. 1 is a schematic structural diagram of a composite porous microsphere

FIG. 2 is a schematic view showing the effect of the composite porous microspheres on filling bone defect sites

FIG. 3 is a composite porous microsphere scanning electron microscope image

FIG. 4 is a physical view of a first type of microspheres simulating a bone-packing defect site

Detailed Description

Example 1 preparation of chitosan/oxidized sodium alginate-beta-TCP composite microsphere 1 based on Schiff base reaction first class of microspheres (1) (amino microsphere-chitosan/beta-TCP microsphere):

raw materials of chitosan (the deacetylation degree is more than or equal to 90 percent, the viscosity is 100-200 mPa.s), beta-tricalcium phosphate (beta-TCP, the particle size is less than or equal to 5 mu m) and glacial acetic acid.

The steps are as follows:

(1) 2.0g of chitosan powder was dissolved in 100ml of 1% by mass aqueous glacial acetic acid solution, and magnetically stirred for 4 hours until complete dissolution, to obtain a transparent colloidal solution A.

(2) 1.0G of beta-TCP powder was slowly added to the above solution A, and the mixture was subjected to ultrasonic treatment at 500W for 30 minutes in an ice-water bath to sufficiently and uniformly disperse the powder, thereby obtaining a mixed slurry A.

(3) Slurry a was transferred to a spray dryer for granulation. The inlet air temperature is set to be 130 ℃, the outlet air temperature is set to be 80 ℃, the feeding pump speed is set to be 5mL/min, and the aperture of the nozzle is set to be 0.5mm.

(4) The dried microspheres were collected, washed three times with absolute ethanol and deionized water to remove residual acetic acid, and then freeze-dried at-50 ℃ under 0.1Pa for 24 hours to obtain white first-class porous microspheres 1.

2. Preparation of a second type of microspheres (2) (aldehyde-based microspheres-oxidized sodium alginate/beta-TCP microspheres):

Raw materials of the composition comprise sodium alginate, sodium periodate (NaIO ₄), beta-TCP and calcium chloride (CaCl ₂).

The preparation of oxidized sodium alginate comprises dissolving 5g sodium alginate in 500mL deionized water, adding 2.5g sodium periodate under dark condition, and magnetically stirring at room temperature for reaction for 6h. After the reaction was completed, the reaction was terminated by adding an excessive amount of ethylene glycol. And (3) putting the solution into a dialysis bag with the molecular weight cut-off of 8000-14000, dialyzing with deionized water for 3 days, and freeze-drying to obtain oxidized sodium alginate.

The steps are as follows:

(1) 2.0g of oxidized sodium alginate (about 50% of the degree of oxidation) was dissolved in 100mL of deionized water and stirred until completely dissolved, to obtain solution B.

(2) 1.0G of beta-TCP powder is added into the solution B, and the mixed slurry B is obtained by ultrasonic dispersion uniformly in the same way as the above method.

(3) Slurry B was added dropwise to 100mL of a 2% by mass CaCl ₂ solution at a rate of 20mL/h using a microinjection pump while gently stirring at 200rpm, and solidified for 30 minutes to preliminary ball formation.

(4) The microspheres are filtered off, washed thoroughly with deionized water and then freeze-dried to give a second class of porous microspheres 2.

3. Performance test:

The morphology characterization is that the Scanning Electron Microscope (SEM) observation shows that the two types of microspheres are spherical with porous surfaces and regular morphology, the particle size is mainly distributed between 100 and 300 mu m, and the interior of the microsphere is provided with a pore channel structure which is mutually communicated. In vitro crosslinking verification that equal mass of the first type of microspheres and the second type of microspheres are physically mixed uniformly in a culture dish. 2mL of fresh goat blood was then added dropwise to simulate an in vivo environment. It can be observed that the loose microspheres lose fluidity rapidly within 60-90S, forming an integral hydrogel block, indicating that rapid and effective autonomous cross-linking between the microspheres is achieved by schiff base reaction.

The microsphere morphology and the state after the crosslinking reaction are shown in FIG. 4

Example 2 PLGA-based composite microspheres based on click chemistry

1. Synthesis of functionalized PLGA:

Preparation of thiolated PLGA (PLGA-SH) 10gPLGA (LA: GA=75:25) was dissolved in 100mL anhydrous dimethyl sulfoxide (DMSO), and an excess of 1.5 equivalents of cystamine dihydrochloride and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) were added and reacted under nitrogen at room temperature for 24 hours. After the reaction is finished, adding excessive Dithiothreitol (DTT) to reduce disulfide bonds, dialyzing and freeze-drying to obtain PLGA-SH.

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

2. Preparation of first class of microspheres (PLGA-SH/bioactive glass microspheres):

(1) 1.0gPLGA-SH was dissolved in 10mL of Dichloromethane (DCM) as the oil phase.

(2) 0.3G of bioactive glass (BG, particle size <10 μm) was dispersed in the oil phase.

(3) The oil phase was poured into 100mL of an aqueous phase containing 2% polyvinyl alcohol (PVA) and emulsified at 5000rpm for 2min to form a W/O emulsion.

(4) The emulsion was slowly poured into 400mL of 0.5% PVA solution and magnetically stirred for 6h to evaporate the DCM completely.

(5) The microspheres were collected, washed by centrifugation and freeze-dried.

3. Preparation of a second class of microspheres (PLGA-Acr/bioactive glass microspheres):

The preparation process is the same as 2, except that the polymer is replaced by PLGA-Acr.

4. Performance test:

(1) Crosslinking test two PLGA microspheres were mixed in equal amounts, added dropwise with PBS solution containing photoinitiator (Irgacure 2959), and irradiated under 365nm ultraviolet light for 30s. The mixture rapidly crosslinked to an elastic solid. In an environment that does not use ultraviolet light, but only mimics body fluids, the crosslinking reaction is completed slowly over a period of minutes.

(2) Cell experiments premouse osteoblasts (MC 3T 3-E1) were co-cultured with the cross-linked microsphere scaffolds. The results of live dying staining and CCK-8 testing show that the material has no cytotoxicity, and cells can be well adhered and proliferated on the surface of a bracket and in pores.

Example 3 gelatin/hydroxyapatite composite microspheres based on enzyme catalyzed Cross-linking

1. Preparation of first class of microspheres (enzyme-loaded gelatin/hydroxyapatite microspheres):

(1) 2.0g of gelatin (form A) was dissolved in 50mL of PBS at 50℃to give solution A.

(2) 0.5G of hydroxyapatite (HA, nanorod) and 50mg of microbial transglutaminase (mTGase) were added, and the mixture was gently stirred and dispersed uniformly.

(3) Solution a was made into microspheres by spray drying (inlet air temperature 90 ℃) and collected rapidly.

2. Preparation of a second type of microspheres (matrix gelatin/hydroxyapatite microspheres):

(1) 2.0g of gelatin was dissolved in 50mL of PBS at 50℃to give solution B.

(2) 0.5G HA and 0.1g of a short peptide containing glutamine and lysine (as a preferred substrate for mTGase) were added and dispersed with gentle stirring.

(3) Microspheres were also made by spray drying.

3. Crosslinking mechanism and test:

The mechanism is that when the two kinds of microspheres are mixed and contacted with blood, mTG enzyme wrapped in the first kind of microspheres catalyzes the formation of epsilon- (gamma-glutamyl) lysine covalent bond between the glutamine residue and lysine residue in gelatin and short peptide molecules on the surface of the second kind of microspheres under the activation of blood moisture and Ca ²⁺ (cofactor of mTG enzyme, existing in blood), so as to realize crosslinking.

Rheology test two types of microspheres were mixed with a small amount of blood and placed in a rheometer. The time-sweep pattern showed that the storage modulus (G') of the mixture rapidly increased from initial 10Pa to 1000Pa in 10 minutes, confirming its rapid in situ gelation ability.

The three embodiments respectively represent three different technical paths of bionic chemistry, click chemistry and biological enzyme catalysis, and the composite porous microsphere capable of being automatically crosslinked in the environment of blood or body fluid is successfully prepared. The microspheres can effectively overcome the defects of the prior art, form a stable three-dimensional bracket in situ at the bone defect part, and have good clinical application prospect.

The foregoing is a description of the principles of the invention, and it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the true scope of which is defined by the legal protection scheme.

Claims (10)

1. A composite porous microsphere that can self-crosslink upon contact with blood and its preparation method, characterized in that it comprises: 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, wherein the first active functional group and the second active functional group are a functional group pair capable of undergoing click chemistry or Schiff base reaction. 3. The composite porous microsphere system according to claim 2, wherein the functional group pairs are selected from any combination of the following: 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, wherein the biodegradable polymer material is selected from one or more of 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, wherein 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 it comprises 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, wherein 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.