阅读说明：本技术 一种生物陶瓷微球及其制备方法和应用 (Biological ceramic microsphere and preparation method and application thereof ) 是由 林开利 万健羽 王旭东 于 2021-08-19 设计创作，主要内容包括：本申请公开了一种生物陶瓷微球及其制备方法和应用。以陶瓷粉末、海藻酸钠和钙源的混合水溶液作为分散相并以第一流动速率向下游流动；以油相作为第二流动相,以第二流动速率与所述分散相在交汇点汇合后形成反应液并向下游流动；以含有引发剂的油相作为外接流动相与反应液在所述交汇点的下游汇合后发生交联反应,得到含有微球的溶液；分离微球,煅烧后去除海藻酸钠得到生物陶瓷微球。得到的生物陶瓷微球孔隙率、粒径大小可控、粒径均一、且具有良好的生物活性,可以用作硬组织缺损修复材料和体外骨组织培养用细胞载体。(The application discloses a biological ceramic microsphere and a preparation method and application thereof. Taking a mixed water solution of ceramic powder, sodium alginate and a calcium source as a dispersion phase and flowing downstream at a first flow rate; taking the oil phase as a second flow phase, converging with the dispersed phase at a second flow rate at a convergence point to form a reaction liquid and flowing downstream; taking an oil phase containing an initiator as an external mobile phase, converging a reaction liquid at the downstream of the convergence point, and then carrying out a crosslinking reaction to obtain a solution containing microspheres; separating the microspheres, and removing sodium alginate after calcining to obtain the biological ceramic microspheres. The obtained biological ceramic microspheres have controllable porosity and particle size, uniform particle size and good biological activity, and can be used as hard tissue defect repair materials and cell carriers for in vitro bone tissue culture.)

1. A preparation method of biological ceramic microspheres is characterized in that mixed aqueous solution of ceramic powder, sodium alginate and a calcium source is used as a disperse phase and flows downstream at a first flow rate; taking the oil phase as a second flow phase, converging with the dispersed phase at a second flow rate at a convergence point to form a reaction liquid and flowing downstream; taking an oil phase containing an initiator as an external mobile phase, converging the reaction liquid at the downstream of the convergence point, and then carrying out a crosslinking reaction to obtain a solution containing microspheres; separating the microspheres, and removing the sodium alginate after calcining to obtain the biological ceramic microspheres.

2. The method for preparing bioceramic microspheres according to claim 1, wherein the oil phase is dimethicone.

3. The method of claim 1, wherein the ceramic powder comprises one or more of hydroxyapatite, β -tricalcium phosphate, calcium silicate, or akermanite; and/or, the calcium source comprises disodium calcium ethylenediaminetetraacetate; and/or, the initiator comprises acetic acid.

4. The preparation method of the bioceramic microspheres according to claim 3, wherein the particle size of the ceramic powder is 0.1-50 μm.

5. The preparation method of the bioceramic microspheres according to claim 1, wherein the weight volume percentage concentration of the ceramic powder is 3-20% based on the total volume of the dispersed phase; and/or the weight volume percentage concentration of the sodium alginate is 1-3%; and/or the weight volume percentage concentration of the calcium source is 1-3%.

6. The preparation method of the bioceramic microspheres according to claim 1, wherein the initiator is contained in an amount of 0.5-5% by volume based on the total volume of the external mobile phase.

7. The method for preparing bioceramic microspheres according to claim 1, wherein the first flow rate is 0.04-0.2 mL/min; and/or the second flow rate is 0.1-2 mL/min, and the flow rate of the external mobile phase is the same as the second flow rate.

8. The preparation method of the bioceramic microspheres according to claim 1, wherein the calcining temperature is 800-1300 ℃; and/or the calcining time is 2-4 hours.

9. A bioceramic microsphere prepared by the preparation method of any one of claims 1-7.

10. The bioceramic microsphere of claim 9, wherein the diameter of the bioceramic microsphere is 100-1000 μm; and/or the pore size of the biological ceramic microspheres is 0.1-0.6 μm.

11. The application of the biological ceramic microspheres prepared by the preparation method of any one of claims 1-8 in hard tissue defect repair materials and in-vitro cell culture carriers.

Technical Field

The application relates to the technical field of biological materials, in particular to a biological ceramic microsphere and a preparation method and application thereof.

Background

Bone defects are a common clinical manifestation, and the current traditional bone defect treatment methods comprise autologous bone transplantation and allogeneic bone transplantation, wherein the donor is limited and easily causes secondary injury, and the allogeneic bone transplantation easily causes host immune rejection. In the past decades, researchers have developed a large number of bone biomaterials, such as titanium metal and its composites, ceramic materials, natural polymers, and synthetic polymers. The ceramic material is mainly divided into a biological inert material and a biological active material. The bioactive material has good osteoinductive and mechanical properties, and various ions released by degradation can stimulate cells to generate new tissues and promote the formation of new bones.

It is known that human bone is composed of 65% of inorganic minerals (mainly hydroxyapatite) and 35% of organic components (various collagens), and therefore bioceramic materials similar to human bone components are often selected. Hydroxyapatite belongs to calcium-phosphorus-based biological ceramics, and the ceramics have good biocompatibility and mechanical properties but low biological activity. In recent years, calcium-silicon-based biomaterials are increasingly gaining attention due to their excellent bioactivity and biodegradability.

Bioactive materials are often applied to bone repair treatments in the form of particles, microspheres, blocks, or scaffolds. The granular ceramic lacks macropores and can only provide limited cell growth, and various edges and corners of the granular ceramic can cause damage to cells and tissues; conventional bulk ceramics are too brittle to be cut into special shapes to accommodate irregular bone defects, and lack of interconnected pore structures also limits the use of bulk ceramics in critical-sized bone defects. The microspheres have high specific surface area, have curved surfaces beneficial to flow, can fill various irregular bone defects in an injection mode, and can meet various clinical requirements. Li et al (Mater Sci Eng C.,2017,70:1200-1205) prepare 250-500 μm beta-TCP microspheres by a solvent volatilization method, the microspheres have good biocompatibility and cell adhesion, apatite deposits are deposited on the surfaces after being soaked in simulated body fluid, the expression of osteogenic genes can be promoted, and abundant bone-like structures are formed among the microspheres after the microspheres are implanted under the skin of a mouse for 8 weeks.

The traditional preparation methods of the biological ceramic microspheres include a spray drying method, a solvent volatilization method and a sol-gel method. However, the microspheres obtained by the method have wide particle size distribution range, and the phenomenon of tight packing of small-size spheres and large-size spheres occurs when filling is performed at the bone defect filling position, so that the pores among the microspheres become small, and further, cells, blood vessels and new bone tissues are difficult to grow. The micro-fluidic method is one of the methods for preparing microspheres with uniform particle size and good monodispersity, and the diameter of the microspheres is effectively regulated and controlled on a micro-fluidic chip channel by controlling the parameters such as solution concentration, the flow rate ratio of a continuous phase and a disperse phase and the like. At present, the micro-fluidic method for preparing the ceramic microspheres with uniform particle sizes is not reported.

Disclosure of Invention

The application provides a biological ceramic microsphere and a preparation method and application thereof, and the biological ceramic microsphere which has bioactivity and biodegradability, controllable particle size and porosity and uniform particle size can be obtained so as to meet the development requirement of an injectable bone repair biological material.

The application provides a preparation method of biological ceramic microspheres, which takes mixed aqueous solution of ceramic powder, sodium alginate and a calcium source as a disperse phase and flows downstream at a first flow rate; taking the oil phase as a second flow phase, converging with the dispersed phase at a second flow rate at a convergence point to form a reaction liquid and flowing downstream; taking an oil phase containing an initiator as an external mobile phase, converging the reaction liquid at the downstream of the convergence point, and then carrying out a crosslinking reaction to obtain a solution containing microspheres; separating the microspheres, and removing sodium alginate after calcining to obtain the biological ceramic microspheres.

Optionally, in some embodiments of the present application, the oil phase is dimethicone.

Optionally, in some embodiments of the present application, the ceramic powder comprises one or more mixtures of hydroxyapatite, β -tricalcium phosphate, calcium silicate, akermanite, or other bioceramic powders.

Optionally, in some embodiments of the present application, the calcium source comprises calcium disodium ethylenediaminetetraacetate (Ca-EDTA); the initiator comprises acetic acid.

Optionally, in some embodiments of the present disclosure, the particle size of the ceramic powder may be 0.1 to 50 μm, may also be 1 to 40 μm, and may also be 10 to 30 μm.

Optionally, in some embodiments of the present disclosure, the ceramic powder may have a weight volume percentage concentration of 3 to 20%, or 5 to 18%, or 10 to 15%, based on the total volume of the dispersed phase.

Optionally, in some embodiments of the present application, the weight volume percentage concentration of the sodium alginate may be 1 to 3%, or 1.3 to 2.7%, or 1.5 to 2.5%, based on the total volume of the dispersed phase.

Optionally, in some embodiments of the present disclosure, the calcium source may be present in a concentration of 1 to 3% by volume, or 1 to 2.5% by volume, or 2% by volume, based on the total volume of the dispersed phase.

Optionally, in some embodiments of the present disclosure, the initiator may be present in an amount of 0.5 to 5% by volume, 1 to 4% by volume, or 1.5 to 3% by volume based on the total volume of the external mobile phase.

Optionally, in some embodiments of the present application, the first flow rate may be 0.04-0.2 mL/min, or 0.05-0.15 mL/min, or 0.07-0.1 mL/min.

Optionally, in some embodiments of the present application, the second flow rate may be 0.1-2 mL/min, or 0.2-1.5 mL/min, or 0.5-1 mL/min.

Optionally, in some embodiments of the present application, the flow rate of the circumscribed mobile phase and the second flow rate are the same.

Optionally, in some embodiments of the present application, the calcination temperature may be 800 to 1300 ℃, 900 to 1200 ℃, or 1000 to 1100 ℃.

Optionally, in some embodiments of the present application, the calcination time may be 2 to 4 hours, or 2.3 to 3.7 hours, or 2.5 to 3.5 hours.

Correspondingly, the application also provides a biological ceramic microsphere, and the biological ceramic microsphere is prepared by adopting the preparation method.

Optionally, in some embodiments of the present application, the diameter of the bioceramic microsphere may be 100 to 1000 μm, may also be 200 to 900 μm, and may also be 300 to 800 μm.

Optionally, in some embodiments of the present application, the pore size of the bioceramic microsphere may be 0.1-0.6 μm, or 0.2-0.5 μm, or 0.3-0.4 μm.

In addition, the application also provides application of the biological ceramic microspheres in hard tissue defect repair materials and in-vitro cell culture carriers.

The application adopts a microfluidic method to prepare the biological ceramic microspheres, and has the following beneficial effects:

(1) the biological ceramic microspheres with uniform particle size are prepared by the preparation method. The diameter (100-1000 μm) of the ceramic microspheres can be regulated and controlled by adjusting the concentration of the ceramic powder, the first flow rate, the second flow rate, the model of the coaxial needle and other preparation process parameters. By adopting the method provided by the invention, porous materials with different characteristics can be prepared according to different requirements of different tissue injury repairs on the materials so as to meet the requirements of clinical application;

(2) in vitro cell experiments show that the biological ceramic microspheres can promote the proliferation and adhesion of osteoblasts. In vivo animal experiments show that rich bone samples are formed among the biological ceramic microspheres prepared by the method, so that new bone formation is well promoted, and the biological ceramic microspheres have excellent bone regeneration capacity;

(3) the process is simple and easy to implement and easy to popularize.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a schematic diagram of a method of making a bioceramic microsphere;

FIG. 2 is the viewing pictures (A) 5%, (B) 10%, (C) 15% of the bioceramic microspheres prepared under different ceramic powder concentration conditions in example one;

FIG. 3 is a graphical representation of the viewing of the bioceramic microspheres prepared under the different second flow rate conditions of example one (A)0.45mL/min, (B)0.6mL/min, and (C)0.75 mL/min;

FIG. 4 is a schematic representation of 17G, 22G and (B)14G, 18G of bio-ceramic microspheres prepared under different coaxial needle types in the first example;

FIG. 5 is a diagram of CCK-8 of the bio-ceramic microspheres prepared in the first example after being cultured with rat bone marrow mesenchymal stem cells;

FIG. 6 shows the cell adhesion of the bio-ceramic microspheres prepared in the first example after co-culture with rat bone marrow mesenchymal stem cells;

FIG. 7 is a graph of Micro-CT of bio-ceramic microspheres filled into rat femur fractures prepared in example one;

FIG. 8 is a graph of bone mass fraction of bio-ceramic microspheres prepared in example one filled into rat femoral fractures;

FIG. 9 is a perspective view of the bioceramic microspheres prepared in examples two and three.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The application provides a biological ceramic microsphere and a preparation method and application thereof. The following are detailed below. It should be noted that the following description of the embodiments is not intended to limit the preferred order of the embodiments.

The embodiment of the application provides a preparation method of a biological ceramic microsphere, as shown in fig. 1, an adopted microfluidic device comprises an injection pump 1, a polytetrafluoroethylene tube 2 and a coaxial needle 3, and the preparation method comprises the following steps:

(1) preparing 1-3% (w/v) sodium alginate aqueous solution, adding 3-20% (w/v) ceramic powder and 1-3% (w/v) Ca-EDTA, stirring overnight, and ultrasonically dispersing for 2 hours before use;

(2) taking ceramic powder/Ca-EDTA/sodium alginate aqueous solutions with different concentrations as a dispersion phase, enabling the ceramic powder/Ca-EDTA/sodium alginate aqueous solutions to flow downstream at a first flow rate of 0.04-0.2 mL/min, taking dimethyl silicone oil as a second flow phase, enabling the dimethyl silicone oil and the dispersion phase to converge at a second flow rate of 0.1-2 mL/min, and forming microsphere liquid drops with uniform particle sizes at an outlet of a coaxial needle 3;

(3) an externally-connected mobile phase of simethicone containing 0.5-5% (v/v) acetic acid is externally connected to the channel of the microsphere liquid drop, when Ca-EDTA in the microsphere liquid drop meets the acetic acid, calcium ions can be released, and the calcium ions can enable sodium alginate to be crosslinked immediately to form microspheres;

(4) washing the dimethyl silicone oil on the surface of the microsphere by using petroleum ether, and drying in a 60 ℃ drying oven. And finally, calcining the microspheres for 2-4 h at the temperature rising rate of 2 ℃/min to 800-1300 ℃, and removing sodium alginate of the microspheres to obtain the biological ceramic microspheres.

In some embodiments of the present application, the ceramic powder comprises hydroxyapatite [ Ca [ ]₁₀(PO₄)₆(OH)₂,HA]Beta-tricalcium phosphate (beta-TCP), calcium silicate (CaSiO)₃CS) or Akermanite (AKT); the ceramic powder has good bioactivity and biodegradability.

In some embodiments of the present application, the coaxial needle may be 17G, 22G or 14G, 18G, with a 17G inner diameter of 1.11mm and a 22G inner diameter of 0.42 mm; the 14G inner diameter was 1.64mm and the 18G outer diameter was 0.92 mm.

The following description is given in conjunction with specific embodiments.

In the following examples, the ceramic powder may be prepared by a chemical precipitation method, a sol-gel method, a high temperature solid phase method, or a commercially available powder material.

Sodium alginate, acetic acid, simethicone, ethylene diamine tetraacetic acid disodium calcium salt and the like are all of analytical grade; the water is deionized water.

The syringe pump model is LSP01-3A, LonggerPump.

The first embodiment,

The method for preparing the bioceramic microspheres of the embodiment comprises the following steps:

(1) dissolving 0.4g of sodium alginate and 0.4g of Ca-EDTA in 20mL of deionized water, stirring overnight, adding 3g of beta-tricalcium phosphate (beta-TCP) powder into the sodium alginate solution, and stirring for more than 2 hours;

(2) the micro-fluidic system consists of 17G and 22G coaxial needles, a polytetrafluoroethylene tube with the inner diameter of 1.2mm and the outer diameter of 1.6mm and an LSP01-3A injection pump, beta-TCP/Ca-EDTA/sodium alginate composite solution is used as a dispersion phase (water phase), simethicone is used as an oil phase, the microspheres are prepared by a water-in-oil method, the flow rate of the dispersion phase is 0.06mL/min, the flow rate of the oil phase is 0.3mL/min, and microsphere liquid drops with uniform particle size are formed at the outlet of the coaxial needles;

(3) in order to enable the microspheres to be solidified, a new channel is added on a polytetrafluoroethylene pipeline, simethicone and 1% (v/v) acetic acid are added, the flow rate is 0.3mL/min, and the acetic acid can enable Ca-EDTA to release Ca ions, so that sodium alginate is crosslinked to form the microspheres;

(4) and repeatedly cleaning and drying the obtained microspheres by using petroleum ether, heating to 1100 ℃ at the heating rate of 2 ℃/min, calcining, keeping the temperature for 2 hours, and cooling along with the furnace to obtain the biological ceramic microspheres.

The diameter of the prepared beta-TCP bioceramic microsphere is 455 +/-23 mu m.

As shown in fig. 2, the weight volume percentage concentrations of the ceramic powders were changed to (a) 5%, (B) 10%, (C) 15%, respectively; when the diameter distribution of the beta-TCP bioceramic microspheres prepared under the above different conditions was observed under the stereoscope of SZ2-ILST by OLYMP M S, Japan, it was found that the diameter of the bioceramic microspheres increased from 320. + -.21 μm to 455. + -.23 μm when the weight volume percentage concentration of the ceramic powder increased by 15%.

As shown in FIG. 3, the second flow rates were varied to (A)0.45mL/min, (B)0.6mL/min, and (C)0.75mL/min, respectively; when the distribution of the diameters of the beta-TCP bioceramic microspheres prepared under the above different conditions was observed under the stereoscope of SZ2-ILST by OLYMP M S, Japan, it was found that the diameters of the bioceramic microspheres decreased from 378 + -23 μm to 301 + -19 μm when the second flow rate was increased from 0.45mL/min to 0.75 mL/min.

As shown in fig. 4, the types of the coaxial needles are changed, and the types of the coaxial needles are respectively (A)17G, 22G and (B)14G, 18G, wherein the inner diameter of 17G is 1.11mm, and the inner diameter of 22G is 0.42 mm; the inner diameter of 14G is 1.64mm, and the outer diameter of 18G is 0.92 mm; when the distribution of diameters of the beta-TCP bioceramic microspheres prepared under the different conditions described above was observed under the SZ2-ILST stereoscope of OLYMP M S, Japan, it was found that the diameters of the bioceramic microspheres increased from 455. + -. 23 μm to 725. + -. 15 μm when the coaxial needle was changed from 17G, 22G to 14G, 18G, i.e., the inner diameter of the coaxial needle increased.

From the above results, when the weight volume percentage concentration of the ceramic powder is increased from 5% to 15%, the diameter of the bioceramic microspheres is increased from 320 ± 21 μm to 455 ± 23 μm; when the second flow rate is increased from 0.45mL/min to 0.75mL/min, the diameter of the bioceramic microspheres is reduced from 378 +/-23 microns to 301 +/-19 microns; when the coaxial needle change is changed from 17G, 22G to 14G, 18G, the diameter of the bioceramic microspheres is increased from 455 +/-23 μm to 725 +/-15 μm. The diameter of the bioceramic microspheres obtained in the embodiment can be controlled within the range of 100-1000 μm, and the particle size of the bioceramic microspheres is uniform.

The beta-TCP bioceramic microspheres with different diameters obtained in the embodiment are CO-cultured with rat bone marrow mesenchymal stem cells at 37 ℃ and 5% CO₂Culturing for 1, 4 and 7 days, and then performing CCK-8 cell proliferation assay, as shown in FIG. 5, after co-culturing beta-TCP bioceramic microspheres of 300 μm, 500 μm and 700 μm with rat bone marrow mesenchymal stem cells, there is cell proliferation at 7 days compared with 1 st and 4 th days; compared with a blank control without adding the beta-TCP biological ceramic microspheres, the beta-TCP biological ceramic microspheres with the diameters of 300 mu m and 500 mu m have obvious increase of cell proliferation after being co-cultured with rat bone marrow mesenchymal stem cells for 7 days, wherein the cell proliferation is the most obvious after the beta-TCP biological ceramic microspheres with the diameters of 300 mu m are co-cultured, and the beta-TCP biological ceramic microspheres with the diameters of above are addedThe results show that the bioceramic microspheres prepared in the embodiment can better promote cell proliferation.

The cell adhesion condition of the bioceramic microspheres with different diameters obtained in the present example and rat bone marrow mesenchymal stem cells after 24 hours of co-culture was observed through a microscope, as shown in fig. 6, the cells of the β -TCP bioceramic microspheres with 300 μm, 500 μm, and 700 μm were well adhered after co-culture with rat bone marrow mesenchymal stem cells, which shows that the bioceramic microspheres prepared in the present example can promote cell adhesion. The above results show that the ceramic microspheres of the present application have good biocompatibility, bioactivity and cell adhesion.

After the beta-TCP bioceramic microspheres with different diameters obtained in the embodiment are filled in the femoral defect of a rat, Micro-CT analysis shows that after 8 weeks, after the beta-TCP bioceramic microspheres with the diameters of 300 microns and 500 microns are filled in the femoral defect of the rat, rich new bones are formed between the microspheres as shown in figure 7, and the bone volume fraction is calculated relative to a control group which is not filled with the beta-TCP bioceramic microspheres, and after the beta-TCP bioceramic microspheres are filled, the new bone formation bone volume fraction (BV/TV, bone volume/tissue volume) is higher than that of the unfilled beta-TCP bioceramic microspheres, wherein the new bone formation body fraction is higher after the beta-TCP bioceramic microspheres with the diameters of 500 microns are filled. The biological ceramic microspheres of the embodiment have better osteogenesis effect.

The in vitro cell experiment and in vivo animal experiment show that the beta-TCP bioceramic microsphere is a biological material with good biological activity and capable of promoting osteoblast proliferation and accelerating bone healing.

Example II,

The method for preparing the bioceramic microspheres of the embodiment comprises the following steps:

(1) dissolving 0.4g of sodium alginate and 0.4g of Ca-EDTA in 20mL of deionized water, stirring overnight, adding 3g of Hydroxyapatite (HA) powder into the sodium alginate solution, and stirring for more than 2 hours;

(2) adopting a micro-fluidic system consisting of 17G and 22G coaxial needles, a polytetrafluoroethylene tube with the inner diameter of 1.2mm and the outer diameter of 1.6mm and an LSP01-3A injection pump, taking a beta-TCP/Ca-EDTA/sodium alginate composite solution as a dispersion phase (water phase) and dimethyl silicone oil as a continuous phase (oil phase), preparing microspheres by adopting a water-in-oil method, wherein the flow rate of the dispersion phase is 0.06mL/min, the flow rate of the continuous phase is 0.3mL/min, and microsphere droplets with uniform particle sizes are formed at the outlet of the coaxial needles;

(3) in order to enable the microspheres to be solidified, a new channel is added on a polytetrafluoroethylene pipeline, simethicone and 1% (v/v) acetic acid are added, the flow rate is 0.3mL/min, and the acetic acid can enable Ca-EDTA to release Ca ions, so that sodium alginate is crosslinked to form the microspheres;

(4) and repeatedly cleaning and drying the obtained microspheres by using petroleum ether, heating to 1100 ℃ at the heating rate of 2 ℃/min, calcining, keeping the temperature for 2 hours, and cooling along with the furnace to obtain the biological ceramic microspheres.

The obtained HA bioceramic microspheres were observed under the stereoscope of SZ2-ILST manufactured by OLYMP Mm S of Japan, and the obtained bioceramic microspheres had diameters of 450. + -. 30 μm as shown in FIG. 9A.

Example III,

The method for preparing the bioceramic microspheres of the embodiment comprises the following steps:

(1) dissolving 0.4g of sodium alginate and 0.4g of Ca-EDTA in 20mL of deionized water, stirring overnight, adding 3g of Calcium Silicate (CS) powder into the sodium alginate solution, and stirring for more than 2 hours;

(2) adopting a micro-fluidic system consisting of 17G and 22G coaxial needles, a polytetrafluoroethylene tube with the inner diameter of 1.2mm and the outer diameter of 1.6mm and an LSP01-3A injection pump, taking a beta-TCP/Ca-EDTA/sodium alginate composite solution as a dispersion phase (water phase) and dimethyl silicone oil as a continuous phase (oil phase), preparing microspheres by adopting a water-in-oil method, wherein the flow rate of the dispersion phase is 0.06mL/min, the flow rate of the continuous phase is 0.3mL/min, and microsphere droplets with uniform particle sizes are formed at the outlet of the coaxial needles;

(3) in order to enable the microspheres to be solidified, a new channel is added on a polytetrafluoroethylene pipeline, simethicone and 1% (v/v) acetic acid are added, the flow rate is 0.3mL/min, and the acetic acid can enable Ca-EDTA to release Ca ions, so that sodium alginate is crosslinked to form the microspheres;

(4) and repeatedly cleaning and drying the obtained microspheres by using petroleum ether, heating to 1100 ℃ at the heating rate of 2 ℃/min, calcining, keeping the temperature for 2 hours, and cooling along with the furnace to obtain the biological ceramic microspheres.

The obtained CS bioceramic microspheres were observed under the SZ2-ILST stereoscope of OLYMP M S, Japan, and the obtained bioceramic microspheres had a diameter of 480. + -. 31 μm as shown in FIG. 9B.

The biological ceramic microspheres prepared by the method have bioactivity, degradability, controllable diameter and porosity and uniform particle size, and have unique advantages when being used as hard tissue defect repair materials and cell scaffold materials for in vitro bone tissue culture.

The bioceramic microspheres provided by the embodiments of the present application, the preparation method and the application thereof are described in detail above, and the principles and the embodiments of the present application are explained herein by using specific examples, and the description of the above examples is only used to help understand the method and the core concept of the present application; meanwhile, for those skilled in the art, according to the idea of the present application, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present application.