Preparation method of gas-jet mineralized collagen-hydrogel microspheres

Abstract

The invention provides a preparation method of gas-jet mineralized collagen-hydrogel microspheres, and relates to the technical field of biological materials, and the preparation method comprises the following steps: dissolving collagen in a phosphate solution, dropwise adding the phosphate solution and a CaCl2 solution into a continuously stirred alkaline solution together, reacting for 12-48 hours, centrifugally collecting a precipitate, washing with water, centrifuging, and freeze-drying to obtain mineralized collagen; the method comprises the following steps: dissolving mineralized collagen and sodium alginate in deionized water, spraying the solution into a CaCl2 solution through a gas spraying spinning head by adopting a gas spraying technology, collecting microspheres, and repeatedly cleaning by adopting deionized water to obtain the gas spraying mineralized collagen-hydrogel microspheres. Mineralized collagen is encapsulated and loaded through microspheres, so that interferences of tissue fluid are isolated, and non-specific aggregation is reduced; optimizing the ratio of mineralized collagen, and directionally inducing stem cells to differentiate; the diameter of the microsphere is accurately controlled by regulating and controlling gas injection parameters to adapt to different wounds; active ingredients are controlled according to needs through a microsphere cross-linked network, and the multi-stage requirements of subcutaneous repair are met.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a method for preparing air-sprayed mineralized collagen-hydrogel microspheres. Background Art

[0002] Mineralized collagen is a nanocomposite structure formed by the combination of collagen and hydroxyapatite. It has excellent biocompatibility and mechanical properties similar to natural bone. Therefore, mineralized collagen is often used as an implant in the body. In the prior art, mineralized collagen is mainly produced by chemical cross-linking methods, such as the preparation method of a biomimetic mineralized collagen scaffold disclosed in Publication No. CN107929812B, and the preparation method of mineralized collagen based on dynamic cross-linking and the resulting mineralized collagen disclosed in Publication No. CN118286511B.

[0003] The prior art CN110787319A points out that microspheres made of collagen-loaded hydroxyapatite can be used for subcutaneous repair, which can stimulate collagen regeneration, improve facial sagging, and improve facial aging; mineralized collagen is a nano-chemical cross-linked complex composed of collagen and hydroxyapatite. Its porous structure is conducive to cell attachment, nutrient delivery and metabolic waste discharge. At the same time, the collagen in mineralized collagen can be biologically recognized in the body and enzymatic hydrolysis can induce cells to attach to the surface of mineralized collagen. Summary of the Invention

[0004] In response to the problems of uneven aggregation of mineralized collagen, poor microenvironment adaptability, and insufficient drug controlled release capacity in the existing technology, the present invention provides a method for preparing air-sprayed mineralized collagen-hydrogel microspheres. The present invention uses hydrogel microsphere encapsulation technology to stably load mineralized collagen into a three-dimensional cross-linked network, blocking direct interference from tissue fluid, significantly reducing the nonspecific aggregation of mineralized collagen, and maintaining the stability of ion release; by optimizing the mineralized collagen ratio, the subcutaneous repair efficiency is optimized; by adjusting the air-spray parameters, the diameter of the hydrogel microspheres is controlled to adapt to the needs of shallow to deep wound repair; based on the porous structure of the microspheres, high loading efficiency of mineralized collagen and drugs is achieved, and the release behavior is dynamically regulated by the cross-linked network to meet the functional requirements of subcutaneous repair in multiple stages.

[0005] To achieve the above objectives, the present invention provides the following technical solutions: A method for preparing air-sprayed mineralized collagen-hydrogel microspheres comprises the following steps: (1) Dissolve collagen in phosphate solution at room temperature; (2) adding the solution obtained in step (1) and the CaCl2 solution dropwise into a continuously stirred alkaline solution to form a collagen mixture, reacting for 12-48 hours, collecting the precipitate by centrifugation, washing the precipitate with water 3-4 times, centrifuging, and then freeze-drying to obtain mineralized collagen; (3) dissolving the mineralized collagen and sodium alginate obtained in step (2) in deionized water, wherein the concentration of the mineralized collagen in the solution is 0.5-4 wt %, and the concentration of the sodium alginate is 1-3 wt %; (4) The solution obtained in step (3) is sprayed into a continuously stirred CaCl2 solution through an air-jet spinning head using an air-jet technology, and the microspheres are collected and repeatedly washed with deionized water to obtain air-jet mineralized collagen-hydrogel microspheres.

[0006] Preferably, the collagen in step (1) is one or more of collagen or recombinant collagen; and the collagen concentration is 0.5 to 5 mg / mL.

[0007] Preferably, the phosphate solution in step (1) comprises one or more of potassium dihydrogen phosphate or sodium dihydrogen phosphate.

[0008] Preferably, the alkaline solution in step (2) is a phosphate buffer solution whose pH is adjusted to 8.0-9.0 using ammonia water.

[0009] Preferably, in the collagen mixture in step (2), the molar ratio of calcium ions to phosphate ions is 1:1.6-2; and the molar ratio of collagen to phosphate is 15-30:1.

[0010] Preferably, in the step (3), the concentration of mineralized collagen in the solution is 0.5-1.5 wt%, and the concentration of sodium alginate is 1 wt%.

[0011] Preferably, the air pressure of the air spraying technology in step (4) is 0.05-0.35 MPa, and the gas used is air.

[0012] Preferably, the injection flow rate of the solution in step (4) is 100-200 μl / min; the syringe nozzle is a 20-24G needle; and the distance between the syringe nozzle and the CaCl2 solution receiving surface is 5-20 cm.

[0013] More preferably, it is characterized in that the concentration of the CaCl2 solution in step (4) is 1-5wt%, and the stirring speed is 200-300rpm.

[0014] Preferably, the particle size of the air-sprayed mineralized collagen-hydrogel microspheres obtained in step (4) is 10-500 μm.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) Through hydrogel microsphere encapsulation technology, mineralized collagen is evenly dispersed and stably fixed in a three-dimensional cross-linked network, effectively blocking contact with tissue fluid, significantly reducing the aggregation of mineralized collagen, maintaining the uniformity and continuity of calcium and phosphorus ion release, and avoiding cell function inhibition caused by excessive local concentration; (2) By optimizing the ratio of mineralized collagen and simulating the ion concentration of subcutaneous tissue, it promotes the directional differentiation of subcutaneous stem cells, accelerates subcutaneous tissue regeneration and inhibits scar hyperplasia; (3) By adjusting the air-spraying process parameters, microspheres of different diameters can be flexibly prepared. Small-sized microspheres are suitable for shallow penetration and rapid repair, while large-sized microspheres provide mechanical support and sustained drug release for deep defects, thus achieving multi-level adaptation of subcutaneous repair. (4) The porous network structure of the microspheres significantly improves the loading efficiency of mineralized collagen and drugs. At the same time, the dynamic response characteristics of the sodium alginate cross-linked network enable the staged and controlled release of mineralized collagen to match the multi-stage requirements of subcutaneous repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention provides a schematic flow chart of the preparation method.

[0017] Figure 2 This is a scanning electron microscope image of the mineralized collagen prepared using the scheme of the present invention.

[0018] Figure 3 The figure is a scanning electron microscope image of air-sprayed mineralized collagen-hydrogel microspheres prepared by spraying at an air pressure of 0.1 MPa using the scheme of the present invention.

[0019] Figure 4 This is an optical microscope image of air-sprayed mineralized collagen-hydrogel microspheres prepared by spraying at an air pressure of 0.35 MPa using the solution of the present invention.

[0020] Figure 5 The figure is a scanning electron microscope image of air-sprayed mineralized collagen-hydrogel microspheres prepared by spraying at an air pressure of 0.35 MPa using the scheme of the present invention.

[0021] Figure 6 This is an optical microscope image of air-sprayed mineralized collagen-hydrogel microspheres prepared by spraying at an air pressure of 0.01 MPa.

[0022] Figure 7 This is an optical microscope image of air-sprayed mineralized collagen-hydrogel microspheres prepared by spraying at an air pressure of 0.40 MPa. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to specific embodiments.

[0024] Example 1 like Figure 1As shown in the flow chart, this embodiment provides a method for preparing air-sprayed mineralized collagen-hydrogel microspheres, which specifically includes the following steps: (1) Dissolve collagen in phosphate solution at room temperature; (2) adding the solution obtained in step (1) and the CaCl2 solution dropwise into a continuously stirred alkaline solution to form a collagen mixture, reacting for 12-48 hours, collecting the precipitate by centrifugation, washing the precipitate with water 3-4 times, centrifuging, and then freeze-drying to obtain mineralized collagen; (3) dissolving the mineralized collagen and sodium alginate obtained in step (2) in deionized water, wherein the concentration of the mineralized collagen in the solution is 0.5-4 wt %, and the concentration of the sodium alginate is 1-3 wt %; (4) The solution obtained in step (3) is sprayed into a continuously stirred CaCl2 solution through an air-jet spinning head using an air-jet technology, and the microspheres are collected and repeatedly washed with deionized water to obtain air-jet mineralized collagen-hydrogel microspheres.

[0025] In some preferred embodiments, the collagen in step (1) is one or more of collagen or recombinant collagen; and the collagen concentration is 0.5 to 5 mg / mL. The technical effect is that, by selecting type I collagen that is highly compatible with human tissue and combining it with a specific concentration range, the collagen molecules are ensured to be fully extended to expose mineralization active sites, providing a uniform template basis for subsequent mineralization reactions, while maintaining the fluidity of the solution to adapt to process requirements.

[0026] In some preferred embodiments, the phosphate solution in step (1) comprises one or more of potassium dihydrogen phosphate or sodium dihydrogen phosphate. The technical effect is that, by selecting a specific phosphate system, the concentration and release rate of phosphate ions can be precisely controlled, thereby promoting the directional deposition of the biomimetic mineral phase in the mineralized collagen and enhancing the structural stability and bioactivity of the material.

[0027] In some preferred embodiments, the alkaline solution in step (2) is a phosphate buffer solution adjusted to a pH of 8.0-9.0 using aqueous ammonia. The technical effect is that the alkaline environment is gently adjusted by aqueous ammonia, thereby preventing damage to the collagen structure caused by strong alkalis. At the same time, suitable conditions are provided for the calcium-phosphate coprecipitation reaction, ensuring close integration of the mineral phase and collagen fibers in the mineralized collagen.

[0028] In some preferred embodiments, the molar ratio of calcium ions to phosphate ions in the collagen mixture in step (2) is 1:1.6 to 2; and the molar ratio of collagen to phosphate is 15 to 30:1. The technical effect is that by optimizing the calcium-phosphorus ratio and the collagen-phosphate ratio, the mineral deposition rate and the collagen network accommodation capacity are balanced, a uniformly distributed nanoscale mineral phase is formed, and the stem cell differentiation-inducing ability of the mineralized collagen is enhanced.

[0029] In some preferred embodiments, the concentration of mineralized collagen in the solution in step (3) is 0.5-1.5 wt %, and the concentration of sodium alginate is 1 wt %. The technical effect is that, by limiting the concentration ratio of mineralized collagen to sodium alginate, the viscoelasticity of the mixed solution is ensured to be compatible with the air-spraying process, while maintaining the structural integrity and loading uniformity of the cross-linked microspheres.

[0030] In some preferred embodiments, the air pressure of the air spraying technique in step (4) is 0.05-0.35 MPa, and the gas used is air. The technical effect is that by optimizing the air pressure parameters to control the shear force and droplet dispersion during microsphere formation, combined with air as a carrier gas, precise control of microsphere size and process safety are achieved.

[0031] In some preferred implementation cases, the injection flow rate of the solution in step (4) is 100-200 μl / min; the injector nozzle is a 20-24G needle; and the distance between the injector nozzle and the CaCl2 solution receiving surface is 5-20 cm. The technical effect is that by limiting the matching relationship between the injection flow rate and the needle specification, the droplet size and dispersion uniformity during microsphere formation are coordinated and regulated. Combined with the optimization of the nozzle-receiving surface distance, the microspheres are ensured to maintain sphericity and surface smoothness during the crosslinking and curing process, while adapting to the process stability requirements of different scale production.

[0032] In some more preferred embodiments, the concentration of the CaCl2 solution in step (4) is 1-5 wt%, and the stirring speed is 200-300 rpm. The technical effect is that, by regulating the synergistic effect of the crosslinker concentration and the stirring speed, a gradient distribution of the crosslinking density on the surface of the microspheres is achieved, balancing the mechanical strength and swelling properties of the microspheres, while avoiding adhesion between the microspheres, and ensuring particle size uniformity and batch reproducibility.

[0033] In some preferred implementation cases, the particle size of the air-sprayed mineralized collagen-hydrogel microspheres obtained in step (4) is 10-500 μm. The technical effect is that by precisely controlling the particle size range of the microspheres, the functional requirements of different wound repair scenarios can be adapted. Small-particle microspheres are suitable for shallow wound penetration and rapid repair, while large-particle microspheres provide mechanical support and sustained drug release for deep defects, achieving multi-level adaptation for subcutaneous repair.

[0034] Example 2 This embodiment provides a method for preparing air-sprayed mineralized collagen-hydrogel microspheres, which specifically includes the following steps: (1) Dissolve 0.5 g of collagen in a solution containing 167 mM potassium dihydrogen phosphate at room temperature and mix well; (2) The solution obtained in step (1) and a 100 mM CaCl2 solution were added dropwise to a continuously stirred alkaline solution to form a collagen mixture, and the reaction was carried out for 24 hours. The alkaline solution was a phosphate buffer solution whose pH value was adjusted to 8-9 with ammonia water; (3) centrifuging the collagen mixture obtained in step (2) after reacting for 24 hours to collect the precipitate, repeatedly washing the precipitate with water and centrifuging it three times, and then freeze-drying it to obtain mineralized collagen; (4) preparing a mineralized collagen / sodium alginate mixed solution by first adding the mineralized collagen obtained in step (3) to deionized water at a solid content of 1 wt %, and ultrasonically dispersing the solution for 10 minutes to ensure that the solution is fully dissolved; then adding sodium alginate powder to a solid content of 1 wt % of sodium alginate in the mixed solution, and continuing stirring until the solution is completely dissolved; (5) preparing mineralized collagen-hydrogel microspheres by air-jet technology, sucking the solution obtained in step (4) with a syringe, connecting an air-jet spinning head, and spraying the solution into a continuously stirred 2 wt% CaCl2 solution at an air pressure of 0.10 MPa, thereby obtaining air-jet hydrogel microspheres by calcium ion diffusion cross-linking; wherein the air-jet spinning head specification is 20G, the flow rate of the mineralized collagen / sodium alginate mixed solution is 150 μL / min, the distance from the nozzle to the receiving solution is 16 cm, and the gas used in the air-jet technology is air; (6) collecting the hydrogel microspheres in step (5), and then washing them with deionized water 2-3 times to remove the residual CaCl2 solution to obtain air-sprayed mineralized collagen-hydrogel microspheres; (7) The obtained air-sprayed mineralized collagen-hydrogel microspheres were subjected to gradient ethanol dehydration and sealed and stored at -20°C for later use.

[0035] The mineralized collagen obtained in step (2) was observed and photographed under a scanning electron microscope. Figure 2 As shown. Figure 2 It can be found that collagen combines with minerals to achieve intrafiber mineralization. The minerals in the mineralized collagen are evenly deposited inside the collagen fibers to form an intrafiber mineralized structure. This structural feature shows that the minerals and collagen are combined at the molecular level through a biomimetic mineralization mechanism, rather than a simple physical mixing. Intrafiber mineralization not only gives the material a hierarchical structure similar to that of natural bone matrix and enhances its mechanical stability, but also improves the binding ability and sustained release efficiency of calcium and phosphate ions by exposing active sites on the surface of collagen fibers. In addition, the orderly arrangement of the mineral phase along the long axis of the fiber can simulate the topological characteristics of the natural extracellular matrix and provide physical guidance signals for cell adhesion and directional migration. This result verifies that the mineralization strategy of the present invention can effectively coordinate the interaction between the organic-inorganic phase interface and avoid the common mineral agglomeration phenomenon in traditional mineralized collagen.

[0036] The mineralized collagen obtained in step (2) was subjected to energy dispersive X-ray spectroscopy analysis to determine the types and contents of the elements therein. The molar ratio of calcium to phosphorus in the mineralized collagen was determined to be 1.67:1, which is close to the ideal ratio in the natural subcutaneous cell matrix. This feature confirms that the mineral phase is directionally deposited in a biomimetic form during the mineralization process to form a crystal structure with biological activity. The precise regulation of the calcium-phosphorus ratio provides an adaptive microenvironment for the stem cell differentiation process by simulating the ion release behavior of the natural cell matrix. In addition, the high uniformity of the element distribution indicates that the mineral phase and the collagen fibers have achieved uniform compounding at the molecular level, avoiding the risk of cytotoxicity caused by local ion concentration imbalance, while giving the material degradation kinetics matching natural subcutaneous cells, laying the foundation for the stable loading and controlled release of active ingredients in the subsequent microsphere construction.

[0037] The air-sprayed mineralized collagen-hydrogel microspheres prepared in this example were observed and photographed under a scanning electron microscope. The results are shown in the figure below. Figure 3 As shown, the diameter of the microspheres is 170 μm.

[0038] Example 3 This embodiment provides a method for preparing air-sprayed mineralized collagen-hydrogel microspheres, which specifically includes the following steps: (1) Dissolve 0.5 g of collagen in a solution containing 167 mM potassium dihydrogen phosphate at room temperature and mix well; (2) The solution obtained in step (1) and a 100 mM CaCl2 solution were added dropwise to a continuously stirred alkaline solution to form a collagen mixture, and the reaction was carried out for 24 hours. The alkaline solution was a phosphate buffer solution whose pH value was adjusted to 8-9 with ammonia water; (3) centrifuging the collagen mixture obtained in step (2) after reacting for 24 hours to collect the precipitate, repeatedly washing the precipitate with water and centrifuging it three times, and then freeze-drying it to obtain mineralized collagen; (4) preparing a mineralized collagen / sodium alginate mixed solution by first adding the mineralized collagen obtained in step (3) to deionized water at a solid content of 1 wt %, and ultrasonically dispersing the solution for 10 minutes to ensure that the solution is fully dissolved; then adding sodium alginate powder to a solid content of 1 wt % of sodium alginate in the mixed solution, and continuing stirring until the solution is completely dissolved; (5) preparing mineralized collagen-hydrogel microspheres by air-jet technology, sucking the solution obtained in step (4) with a syringe, connecting an air-jet spinning head, and spraying the solution into a continuously stirred 2 wt% CaCl2 solution at an air pressure of 0.35 MPa, thereby obtaining air-jet hydrogel microspheres by calcium ion diffusion cross-linking; wherein the air-jet spinning head specification is 20G, the flow rate of the mineralized collagen / sodium alginate mixed solution is 150 μL / min, the distance from the nozzle to the receiving solution is 16 cm, and the gas used in the air-jet technology is air; (6) collecting the hydrogel microspheres in step (5), and then washing them with deionized water 2-3 times to remove the residual CaCl2 solution to obtain air-sprayed mineralized collagen-hydrogel microspheres; (7) The obtained air-sprayed mineralized collagen-hydrogel microspheres were subjected to gradient ethanol dehydration and sealed and stored at -20°C for later use.

[0039] The air-sprayed mineralized collagen-hydrogel microspheres prepared in this example were observed and photographed under an optical microscope. Figure 4 As shown, through Figure 4 It can be found that the air-sprayed mineralized collagen-hydrogel microspheres prepared in this example are regular spherical with a diameter of 10-60 μm. The details of the obtained hydrogel microspheres were observed using a scanning electron microscope. The results are as follows Figure 5 As shown in the figure, it can be found that when the injection pressure increases from 0.10 MPa to 0.35 MPa, the diameter of the microspheres decreases from 170 μm to 15 μm.

[0040] The above results show that the air-sprayed mineralized collagen-hydrogel microspheres prepared by the present invention have a highly uniform spherical morphology and a controllable particle size range. The regular spherical structure ensures the uniform dispersion and stable residence of the microspheres in the local wound surface, avoiding local stress concentration or tissue friction damage caused by irregular shape; and the microsphere size precisely controlled by the air-spraying parameters can not only achieve efficient penetration through the capillary network, but also match the size of the wound repair cells, promoting cell encapsulation of the microspheres and intracellular delivery of active ingredients. This result is due to the coordinated optimization of the air-spraying parameters during the preparation process, especially the dynamic balance between the shear force of the high-pressure gas on the droplets and the CaCl2 cross-linking rate in the air-spraying technology, which enables the droplets to form a spherical shape before solidification. At the same time, the concentration ratio design of mineralized collagen and sodium alginate ensures that the viscoelasticity of the mixed solution is adapted to the microsphere forming process, avoiding deformation or rupture of the microspheres due to poor rheological properties of the mixed solution.

[0041] Example 4 This embodiment provides a method for preparing air-sprayed mineralized collagen-hydrogel microspheres, which specifically includes the following steps: (1) Dissolve 0.5 g of collagen in a solution containing 167 mM potassium dihydrogen phosphate at room temperature and mix well; (2) The solution obtained in step (1) and a 100 mM CaCl2 solution were added dropwise to a continuously stirred alkaline solution to form a collagen mixture, and the reaction was carried out for 24 hours. The alkaline solution was a phosphate buffer solution whose pH value was adjusted to 8-9 with ammonia water; (3) centrifuging the collagen mixture obtained in step (2) after reacting for 24 hours to collect the precipitate, repeatedly washing the precipitate with water and centrifuging it three times, and then freeze-drying it to obtain mineralized collagen; (4) preparing a mineralized collagen / sodium alginate mixed solution by first adding the mineralized collagen obtained in step (3) to deionized water at a solid content of 1 wt %, and ultrasonically dispersing the solution for 10 minutes to ensure that the solution is fully dissolved; then adding sodium alginate powder to a solid content of 1 wt % of sodium alginate in the mixed solution, and continuing stirring until the solution is completely dissolved; (5) preparing mineralized collagen-hydrogel microspheres by air-jet technology, sucking the solution obtained in step (4) with a syringe, connecting an air-jet spinning head, and spraying the solution into a continuously stirred 2 wt% CaCl2 solution at an air pressure of 0.16 MPa, thereby obtaining air-jet hydrogel microspheres by calcium ion diffusion cross-linking; wherein the air-jet spinning head specification is 20G, the flow rate of the mineralized collagen / sodium alginate mixed solution is 150 μL / min, the distance from the nozzle to the receiving solution is 16 cm, and the gas used in the air-jet technology is air; (6) collecting the hydrogel microspheres in step (5), and then washing them with deionized water 2-3 times to remove the residual CaCl2 solution to obtain air-sprayed mineralized collagen-hydrogel microspheres; (7) The obtained air-sprayed mineralized collagen-hydrogel microspheres were subjected to gradient ethanol dehydration and sealed and stored at -20°C for later use.

[0042] Comparative Example 1 The difference between this comparative example and Example 3 is that the air pressure of the air spraying operation in this comparative example is 0.01 MPa.

[0043] This comparative example provides a method for preparing air-sprayed mineralized collagen-hydrogel microspheres, which specifically comprises the following steps: (1) Dissolve 0.5 g of collagen in a solution containing 167 mM potassium dihydrogen phosphate at room temperature and mix well; (2) The solution obtained in step (1) and a 100 mM CaCl2 solution were added dropwise to a continuously stirred alkaline solution to form a collagen mixture, and the reaction was carried out for 24 hours. The alkaline solution was a phosphate buffer solution whose pH value was adjusted to 8-9 with ammonia water; (3) centrifuging the collagen mixture obtained in step (2) after reacting for 24 hours to collect the precipitate, repeatedly washing the precipitate with water and centrifuging it three times, and then freeze-drying it to obtain mineralized collagen; (4) preparing a mineralized collagen / sodium alginate mixed solution by first adding the mineralized collagen obtained in step (3) to deionized water at a solid content of 1 wt %, and ultrasonically dispersing the solution for 10 minutes to ensure that the solution is fully dissolved; then adding sodium alginate powder to a solid content of 1 wt % of sodium alginate in the mixed solution, and continuing stirring until the solution is completely dissolved; (5) preparing mineralized collagen-hydrogel microspheres by air-jet technology, sucking the solution obtained in step (4) with a syringe, connecting an air-jet spinning head, and spraying the solution into a continuously stirred 2 wt% CaCl2 solution at an air pressure of 0.01 MPa, thereby obtaining air-jet hydrogel microspheres by calcium ion diffusion cross-linking; wherein the air-jet spinning head specification is 20G, the flow rate of the mineralized collagen / sodium alginate mixed solution is 150 μL / min, the distance from the nozzle to the receiving solution is 16 cm, and the gas used in the air-jet technology is air; (6) collecting the hydrogel microspheres in step (5), and then washing them with deionized water 2-3 times to remove the residual CaCl2 solution to obtain air-sprayed mineralized collagen-hydrogel microspheres; (7) The obtained air-sprayed mineralized collagen-hydrogel microspheres were subjected to gradient ethanol dehydration and sealed and stored at -20°C for later use.

[0044] The air-sprayed mineralized collagen-hydrogel microspheres prepared in this comparative example were observed and photographed under an optical microscope. The results are as follows: Figure 6 As shown in the figure, the microsphere diameter is 800 μm. This indicates that under low-pressure conditions, the gas shear force is insufficient, the droplets cannot be fully broken up, and large-sized microspheres are formed. Due to their large particle size, these microspheres have difficulty penetrating deep into the wound surface, and insufficient surface cross-linking can lead to a loose structure. These microspheres are prone to disintegration in the tissue fluid or sudden release of mineralized collagen, disrupting local ion balance and reducing repair efficiency.

[0045] Comparative Example 2 The difference between this comparative example and Example 3 is that the gas pressure of the gas spraying operation in this comparative example is 0.40 MPa.

[0046] This comparative example provides a method for preparing air-sprayed mineralized collagen-hydrogel microspheres, which specifically comprises the following steps: (1) Dissolve 0.5 g of collagen in a solution containing 167 mM potassium dihydrogen phosphate at room temperature and mix well; (2) The solution obtained in step (1) and a 100 mM CaCl2 solution were added dropwise to a continuously stirred alkaline solution to form a collagen mixture, and the reaction was carried out for 24 hours. The alkaline solution was a phosphate buffer solution whose pH value was adjusted to 8-9 with ammonia water; (3) centrifuging the collagen mixture obtained in step (2) after reacting for 24 hours to collect the precipitate, repeatedly washing the precipitate with water and centrifuging it three times, and then freeze-drying it to obtain mineralized collagen; (4) preparing a mineralized collagen / sodium alginate mixed solution by first adding the mineralized collagen obtained in step (3) to deionized water at a solid content of 1 wt %, and ultrasonically dispersing the solution for 10 minutes to ensure that the solution is fully dissolved; then adding sodium alginate powder to a solid content of 1 wt % of sodium alginate in the mixed solution, and continuing stirring until the solution is completely dissolved; (5) preparing mineralized collagen-hydrogel microspheres by air-jet technology, sucking the solution obtained in step (4) with a syringe, connecting an air-jet spinning head, and spraying the solution into a continuously stirred 2 wt% CaCl2 solution at an air pressure of 0.40 MPa, thereby obtaining air-jet hydrogel microspheres by calcium ion diffusion cross-linking; wherein the air-jet spinning head specification is 20G, the flow rate of the mineralized collagen / sodium alginate mixed solution is 150 μL / min, the distance from the nozzle to the receiving solution is 16 cm, and the gas used in the air-jet technology is air; (6) collecting the hydrogel microspheres in step (5), and then washing them with deionized water 2-3 times to remove the residual CaCl2 solution to obtain air-sprayed mineralized collagen-hydrogel microspheres; (7) The obtained air-sprayed mineralized collagen-hydrogel microspheres were subjected to gradient ethanol dehydration and sealed and stored at -20°C for later use.

[0047] The air-sprayed mineralized collagen-hydrogel microspheres prepared in this comparative example were observed and photographed under an optical microscope. The results are as follows: Figure 7 As shown, the microspheres exhibit irregular spherical shapes. This suggests that high pressure causes the droplet ejection velocity to be too rapid, leading to collision deformation before cross-linking and curing. Simultaneously, excessive shear forces disrupt the viscoelastic balance of the mineralized collagen-alginate network, causing an abnormal increase in microsphere surface roughness and even fracture defects. These structural defects can trigger abnormal aggregation of immune cells and increase the risk of inflammatory responses. Furthermore, the irregular surface hinders directional cell migration and orderly ECM deposition, inhibiting tissue regeneration.

[0048] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; unless otherwise specified, the methods used in the present invention are conventional methods in the art. The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, alteration, or equivalent transformation of the above embodiment based on the technical essence of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing air-sprayed mineralized collagen-hydrogel microspheres, characterized in that: The following steps are involved: (1) Dissolve collagen in phosphate solution at room temperature; (2) adding the solution obtained in step (1) and the CaCl2 solution dropwise into a continuously stirred alkaline solution to form a collagen mixture, reacting for 12-48 hours, collecting the precipitate by centrifugation, washing the precipitate with water 3-4 times, centrifuging, and then freeze-drying to obtain mineralized collagen; (3) dissolving the mineralized collagen and sodium alginate obtained in step (2) in deionized water, wherein the concentration of the mineralized collagen in the solution is 0.5-4 wt %, and the concentration of the sodium alginate is 1-3 wt %; (4) The solution obtained in step (3) is sprayed into a continuously stirred CaCl2 solution through an air-jet spinning head using an air-jet technology, and the microspheres are collected and repeatedly washed with deionized water to obtain air-jet mineralized collagen-hydrogel microspheres.

2. The preparation method according to claim 1, characterized in that The collagen in step (1) is one or more of collagen or recombinant collagen; the collagen concentration is 0.5 to 5 mg / mL.

3. The preparation method according to claim 1, characterized in that The phosphate solution in step (1) comprises one or more of potassium dihydrogen phosphate or sodium dihydrogen phosphate.

4. The preparation method according to claim 1, characterized in that The alkaline solution in step (2) is a phosphate buffer solution whose pH is adjusted to 8.0-9.0 using ammonia water.

5. The preparation method according to claim 1, characterized in that In the step (2), the molar ratio of calcium ions to phosphate ions in the collagen mixture is 1:1.6-2; and the molar ratio of collagen to phosphate is 15-30:

1.

6. The preparation method according to claim 1, characterized in that In the step (3), the concentration of mineralized collagen in the solution is 0.5-1.5 wt %, and the concentration of sodium alginate is 1 wt %.

7. The preparation method according to claim 1, characterized in that The air pressure of the air spraying technique in step (4) is 0.05-0.35 MPa, and the gas used is air.

8. The preparation method according to claim 1, characterized in that The injection flow rate of the solution in step (4) is 100-200 μl / min; the syringe nozzle is a 20-24G needle; and the distance between the syringe nozzle and the CaCl2 solution receiving surface is 5-20 cm.

9. The preparation method according to any one of claims 1, 7 or 8, characterized in that The concentration of the CaCl2 solution in step (4) is 1-5wt%, and the stirring speed is 200-300rpm.

10. The preparation method according to claim 1, characterized in that The particle size of the air-sprayed mineralized collagen-hydrogel microspheres obtained in step (4) is 10-500 μm.

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