CN111097068B

CN111097068B - Bionic hydroxyapatite powder/gelatin/sodium alginate composite 3D printing support and preparation method thereof

Info

Publication number: CN111097068B
Application number: CN201911421585.3A
Authority: CN
Inventors: 王迎军; 徐东; 杜昶
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-09-21
Anticipated expiration: 2039-12-31
Also published as: CN111097068A

Abstract

The invention discloses a biomimetic hydroxyapatite powder/gelatin/sodium alginate composite 3D printing support and a preparation method thereof. The preparation method of the biomimetic 3D scaffold includes: (1) preparation of hydroxyapatite powder with biomimetic hierarchical structure; (2) preparation of hydroxyapatite powder/gelatin/sodium alginate composite slurry; (3) biomimetic Preparation of 3D printed scaffolds. The present invention utilizes 3D printing technology, and the prepared mechanical/inorganic composite three-dimensional printing scaffold has controllable external dimensions and good biocompatibility, and fully simulates the composition and multi-level structure of natural bone tissue, and endows the material with controllable mechanical strength The micro-nano multi-level structure of the composite scaffold has high osteogenic activity and osteoinductivity, and the multi-level porous structure can effectively induce the ingrowth of tissues and blood vessels, and promote the regeneration and repair of bone tissue.

Description

Bionic hydroxyapatite powder/gelatin/sodium alginate composite 3D printing support and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a bionic apatite-based powder/gelatin/sodium alginate composite 3D printing support and a preparation method thereof.

Background

With the improvement of living standard and the increase of aging population, people have a rapidly increasing demand for bone defect repair caused by diseases such as trauma, bone tumor excision and bone tissue lesion. Autologous bone grafting is considered as the gold standard for treating bone defects, but the source of the autologous bone grafting is limited, the graft body is difficult to shape, and the implantation part is easy to be affected. The allogenic bone is easy to obtain, and the clinical use of the allogenic bone is greatly limited because the allogenic bone is easy to cause disease transmission, inactivation in the material treatment process, ethical struggle and the like. The development of novel bone tissue repair materials brings new hopes for solving bone defect repair.

Natural bone is a highly complex and excellent biomechanical tissue. Bone is the hardest connective tissue of the human body, primarily because its extracellular matrix is mineralized to impart rigidity and strength thereto. Bone tissue is composed primarily of bone cells and bone matrix, which in turn includes the inorganic component of 2/3 and the organic component of 1/3. The inorganic component is mainly hydroxyapatite crystals, which are the most important mineral in bone and can increase the rigidity and strength of bone. The organic component mainly comprises I-type collagen fiber, and can enhance the toughness of bone tissue. The characteristics of inorganic and organic combination can not only keep the flexibility of the skeleton, but also have strong bearing capacity.

The biopolymer materials such as collagen, chitosan, gelatin, alginate, polycaprolactone, polylactic acid and the like have good biocompatibility, controllable degradability, strong mechanical strength and plasticity, and are widely applied to the field of biomedical materials. The hydroxyapatite has chemical composition similar to that of natural bone tissue, excellent biocompatibility and bone regeneration promoting activity. The conventional preparation methods of the hydroxyapatite powder include a chemical deposition method, a hydrothermal method, a sol-gel method and the like. Biological functional small molecules, natural macromolecules, surfactants and the like are commonly used as a regulating matrix for biomimetic synthesis of HA crystal microstructures.

Natural bone tissue has seven-level complex structures from the nanoscale to the macroscopic level, which endows it with powerful mechanical properties and biological functions. Therefore, the novel biomaterial with the micro/nano multilevel structure simulates the composition, the structure and the performance of natural bone tissues, and has important research value and clinical application prospect. The hydroxyapatite particles with multilevel micro/nano structures, high specific surface area and excellent biocompatibility can be obtained by a biomimetic synthesis technology. The HA micro-nano particles can be used as protein, gene and drug carriers, support cell spreading and promote osteogenic differentiation.

The topology of the biomaterial can enhance protein adsorption, promote cell adhesion, migration, cell proliferation, osteogenic differentiation and osseointegration. Further studies have shown that biomaterials with hierarchical pore structures or hierarchical architectures can achieve superior relevant biological activities than biomaterials with single scale features. For example, the hierarchical pore structure of biological materials helps promote nutrient diffusion, protein adsorption, cell adhesion, vascular ingrowth, and osteogenesis induction; the multi-stage structure of the biomaterial has more advantages in stimulating the biological stress and cell morphology on the cell surface, promoting cell migration and cell differentiation, and the like.

Mao, Q.Li, D.Li, Y.Tan, Q.Che,3D porous polys (epsilon-carprolone)/58S bioactive glass-sodium alloy/gelatin hybrid coatings prepared by a modified molding method for bone tissue engineering, Materials & Design 160(2018)1-8. this study uses sodium alginate and gelatin as printing inks to promote bone tissue repair by loading bioglass, but the alkaline environment generated by the bioglass degradation process is not conducive to the growth of stem cells. Therefore, the 3D printing bracket which has better biocompatibility and simultaneously has the performance of promoting bone and bone induction and is compounded by organic and inorganic materials is developed, and has important application prospect.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a bionic hydroxyapatite powder/gelatin/sodium alginate composite 3D printing support and a preparation method thereof.

Aiming at the defects and shortcomings of the existing bone repair material in design performance, the invention provides a bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing support and a preparation method thereof.

The 3D scaffold is prepared by blending hydroxyapatite particles with a bionic structure, gelatin and sodium alginate to obtain printing slurry, and crosslinking the printing slurry with calcium ions and genipin respectively to obtain a formed three-dimensional scaffold. The bionic hydroxyapatite particles have high specific surface area and a multi-stage structure, so that the rheological property and the printing stability of the slurry are effectively improved, and the micro-nano structure of the bionic hydroxyapatite particles has high osteogenic activity and osteoinductivity; the organic/inorganic composite scaffold simulates the components and structure of the bone, gives controllable mechanical strength to the material, and can effectively induce the tissue and blood vessel to grow in and promote the regeneration and repair of the bone tissue.

The invention also aims to provide a preparation method of the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing bracket.

The purpose of the invention is realized by at least one of the following technical solutions.

The invention provides a preparation method of a bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing bracket, which comprises the following steps:

(1) will (NH4)₂PO₄Adding into water, mixing to obtain (NH4)₂PO₄A solution; then the (NH4)₂PO₄Adjusting the pH value of the solution to 5.8-6.2 to obtain a solution a;

(2) preparing the bionic hydroxyapatite powder with the hierarchical structure: mixing Ca (NO)₃)₂·4H₂Adding O into the solution a in the step (1), uniformly stirring, and fully dissolving to obtain a solution b; adding sodium citrate into the solution b, stirring uniformly, fully dissolving to obtain a mixture, transferring the mixture into a high-pressure reaction kettle with a polytetrafluoroethylene lining, heating for treatment, centrifuging to obtain a precipitateCentrifugally washing, and freeze-drying to obtain Hydroxyapatite (HA) powder;

(3) preparing hydroxyapatite powder/gelatin/sodium alginate composite slurry: adding gelatin into water, and mixing uniformly to obtain gelatin solution; adding the hydroxyapatite powder obtained in the step (2) into a gelatin solution, uniformly stirring to obtain a compound solution, then adding sodium alginate into the compound solution, uniformly stirring, and ultrasonically removing bubbles to obtain printing slurry (hydroxyapatite particles/gelatin/sodium alginate compound slurry);

(4) preparing a bionic 3D printing support: loading the printing slurry obtained in the step (3) into a charging barrel for printing, setting the external size and the internal structure of a support material with the assistance of a computer, debugging the extrusion pressure of printing and the traction speed after extrusion, and performing 3D printing to obtain a blank; and soaking the embryo body in a calcium ion solution to enable the hydroxyapatite particle/gelatin/sodium alginate composite scaffold to be subjected to rapid crosslinking, taking out, soaking in a genipin solution to further perform crosslinking, taking out, soaking in a sodium glutamate solution to remove redundant crosslinking agent, taking out, and finally soaking in deionized water to obtain the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold.

Preferably, in the step (1), the (NH4)₂PO₄The pH of the solution was adjusted to 6.0.

Further, step (1) is (NH4)₂PO₄The concentration of the solution is 1-40 mM.

Preferably, step (1) is performed using (NH4)₂PO₄The concentration of the solution was 24 mM.

Further, the Ca (NO) of step (2)₃)₂·4H₂The molar volume ratio of O to the solution a is 1.5-60 mmol/L; the Ca (NO)₃)₂·4H₂The molar ratio of O to sodium citrate is 4-8: 1; the temperature of the heating treatment is 180 ℃, and the time of the heating treatment is 2-12 h.

Preferably, the temperature of the heat treatment in the step (2) is 180 ℃, and the time of the heat treatment is 3 h.

Further, the concentration of the gelatin solution in the step (3) is 0.05-0.2 g/mL; the mass ratio of the hydroxyapatite powder to the gelatin in the step (3) is 1:3-3: 1.

Preferably, the mass ratio of the hydroxyapatite powder to the gelatin in the step (3) is 1:1.5-1.5: 1.

In the printing sizing agent in the step (3), the concentration of the sodium alginate is 0.02-0.08 g/mL.

Preferably, in the printing paste of step (3), the concentration of sodium alginate is 0.03 g-0.06/mL.

In the step (3), the quality of the hydroxyapatite powder is as follows: the total mass ratio of the gelatin to the sodium alginate is 10:1-2: 1.

Preferably, the raw material form of the hydroxyapatite, the gelatin and the alginate in the step (3) is particles.

Further, in the step (3), the gelatin solution is preferably an aqueous solution of gelatin, and is preferably deionized water, purified water, distilled water or ultrapure water as a solvent.

Preferably, the stirring time in the step (3) is 0.5-24 hours, the stirring temperature is 30-60 ℃, and the bubbles are removed by ultrasonic treatment for 1-4 hours after the full mixing.

Further, in the compound solution in the step (3), the concentration of the hydroxyapatite powder is 0.15-0.30 g/mL.

Preferably, in the compound solution in the step (3), the concentration of the hydroxyapatite powder is 0.2-0.25 g/mL.

Further, the extrusion pressure of the 3D printing in the step (4) is 0.5-5.0bar, and the drawing speed is 5-50 mm/s.

Preferably, the extrusion pressure of the 3D printing in the step (4) is 1.0-4.0 bar.

Preferably, the drawing speed of the 3D printing in the step (4) is 5-20 mm/s.

Further, the shape of the blank in the step (4) is cylindrical; the inner structure of the blank body is a layered spinning fiber layer, the diameter of the filaments is 0.2-0.8mm, the distance between the spinning fibers on the same layer is 0.2-1.2mm, the fibers between layers are 30-90 degrees, and a communicated polygonal three-dimensional network pore structure is formed inside the blank body. The external dimensions (diameter and height) of the embryo body can be designed according to the actual needs.

Preferably, the shape of the blank in the step (4) is cylindrical; the inner structure of the blank body is a layered spinning fiber layer, and the diameter of the silk is 0.3-0.5 mm; the thickness of each spinning fiber layer in the inner part is 0.24-0.4 mm; the distance between the spinning fiber yarns on the same layer is 0.4-0.5 mm; the filaments between the layers are at 90 deg..

Further, the calcium ion solution in the step (4) is a solution obtained by uniformly mixing calcium salt and water, wherein the calcium salt is calcium chloride; the concentration of the calcium ion solution is 0.2-0.6M, and the time for soaking the embryo body in the calcium ion solution is 0.05-1 h; during the soaking process, the sodium alginate molecules are quickly crosslinked with calcium ions.

Further, the concentration of the genipin solution in the step (4) is 2-10mg/mL, and the time for soaking the embryo body in the genipin solution is 1-5 days;

preferably, the embryo body in the step (4) is soaked in the genipin solution for 2-4 d. In the soaking process, groups on molecules of the gelatin and the sodium alginate are fully crosslinked to form a three-dimensional crosslinking network.

Further, the concentration of the sodium glutamate solution in the step (4) is 4-20mg/mL, and the time for soaking the embryo body in the sodium glutamate solution is 1-5 days. When the embryo body is soaked in the sodium glutamate solution, the excessive cross-linking agent on the embryo body can be removed.

Preferably, the embryo body in the step (4) is soaked in the sodium glutamate solution for 2-4 days.

Further, the embryo body in the step (4) is soaked in water for 1-5 days. The embryo body is soaked in water, and the cross-linking agent in the network in the embryo body can be removed.

Preferably, the embryo body in the step (4) is soaked in water for 2-4 d.

Further, in the step (4), when the embryo body is soaked in the sodium glutamate solution, the sodium glutamate solution is replaced every 4-12 hours.

Preferably, in the step (4), when the embryo body is soaked in the sodium glutamate solution, the sodium glutamate solution is replaced every 12 hours.

Further, in the step (4), when the embryo body is soaked in water, the water is changed every 4-12 hours.

Preferably, in step (4), the water is changed every 12 hours while the embryo body is immersed in the water.

Preferably, the water in step (4) is deionized water.

The invention provides a bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing support prepared by the preparation method, which is round in external shape, wherein the thickness of each layered spinning fiber layer in the support is 0.15-0.65mm, the distance between the spinning fibers on the same layer is 0.2-1.2mm, and the fiber yarns between the layers are 30-90 degrees, so that a polygonal three-dimensional network pore structure with communicated internal parts is formed.

Preferably, the thickness of each layered spinning fiber layer inside the 3D printing support is 0.32 mm.

The principle of the support printing preparation method provided by the invention is as follows:

the addition of the gelatin enables the prepared composite slurry to have temperature sensitivity, the temperature of the charging barrel is controlled to be 27-35 ℃ during printing, the temperature of the receiving platform is 0 ℃, and the environmental temperature is about 20 ℃, so that the printed fiber yarns can be quickly solidified and stabilized on the low-temperature receiving platform. And after printing, soaking the solidified stent into a calcium ion solution to quickly crosslink and solidify, and then further crosslinking amino groups and carboxyl groups in the stent by using a genipin solution to form a three-dimensional crosslinking network to obtain the final molded stent.

Compared with the prior art, the invention has the following advantages and effects:

(1) the method utilizes the printing technology to prepare the hydroxyapatite powder/gelatin/sodium alginate composite 3D printing support with a bionic multistage structure, has controllable external dimension and internal pore structure and good biocompatibility, fully simulates the components and the multistage structure of natural bone tissues, and endows the material with controllable mechanical strength;

(2) in the preparation method provided by the invention, the rheological property and stability of the slurry are obviously improved by adding the hydroxyapatite powder with a specific hierarchical structure; the microstructure of the powder is fully exposed in the degradation process of the printed composite scaffold, so that the osteogenic activity and the osteoinduction can be remarkably enhanced;

(3) the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing support provided by the invention has a multi-stage communicated pore structure such as micropores, mesopores and macropores, and can effectively promote protein adsorption and nutrient exchange, induce tissue and blood vessel growth, and promote bone tissue regeneration and repair.

Drawings

Fig. 1 is an SEM image of the hydroxyapatite powder of a hierarchical structure prepared in example 1;

fig. 2 is a rheological comparison graph of the multistage-structured hydroxyapatite powder/gelatin/sodium alginate composite slurry and the gelatin/sodium alginate composite slurry prepared in example 2;

fig. 3 is a front SEM image of the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold obtained in example 8;

fig. 4 is an element distribution diagram of the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold obtained in example 8;

fig. 5 is a cross-sectional SEM image of the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold obtained in example 8;

fig. 6 is a confocal microscope image of live and dead staining of mouse mesenchymal stem cells on the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold obtained in example 8.

Detailed Description

The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.

Example 1

Preparing hydroxyapatite powder with a hierarchical structure:

configured at 24mM (NH4)₂PO₄The pH of the solution was adjusted to 6.0 to obtain a solution a. 40mmol of Ca (NO)₃)₂·4H₂And adding the O into the solution a, fully dissolving, and uniformly stirring to obtain a solution b. And then adding 6mmol of sodium citrate into the solution b, and stirring the mixture vigorously to obtain a mixed solution. And transferring the mixed solution into a high-pressure reaction kettle with a polytetrafluoroethylene lining, and reacting for 3 hours at 180 ℃. Centrifugally washing, freezing and drying to obtain hydroxyapatite powder (HA particles) with a hierarchical structure, wherein the morphology of the hydroxyapatite powder is shown in figure 1 under the observation of a scanning electron microscope. The HA particles have a surface micro-nano hierarchical structure and a hollow structure, and the specific surface area is as high as 106.7m²/g。

Example 2

The preparation method of the hydroxyapatite powder/gelatin/sodium alginate composite 3D printing bracket comprises the following steps:

(1) preparing hydroxyapatite powder/gelatin/sodium alginate composite slurry: adding 5g of gelatin granules into 50mL of deionized water, and stirring the mixture fully and magnetically to obtain a gelatin solution with the concentration of 0.1 g/mL; then 5g of the HA particles obtained in example 1 were added and stirred until the HA particles were uniformly dispersed in the gelatin solution; adding 2.2g of sodium alginate into the solution, fully and uniformly stirring, and ultrasonically removing bubbles to prepare printing slurry;

(2) preparing a bionic 3D printing support: the method comprises the steps of filling hydroxyapatite particle/gelatin/sodium alginate composite slurry (namely the printing slurry) into a charging barrel, enabling the diameter of a needle to be 0.4mm, designing the outer dimension of a support material to be a circle with the diameter of 10mm and the height of 2mm, enabling the printing height of each layer to be 0.32mm, enabling the distance between fiber yarns on the same layer to be 0.5mm, enabling the fiber yarns between the layers to be 90 degrees, enabling the extrusion pressure to be 2.5bar, enabling the traction speed to be 10mm/s after solution extrusion, and printing the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D support to obtain a blank of the 3D support.

Example 3

(1) preparing hydroxyapatite powder/gelatin/sodium alginate composite slurry: adding 5g of gelatin granules into 50mL of deionized water, and fully and magnetically stirring to obtain a gelatin solution with the concentration of 0.1 g/mL; then 7.5g of the HA particles obtained in example 1 were added and stirred until the HA particles were uniformly dispersed in the gelatin solution; adding 1.8g of sodium alginate into the solution, fully and uniformly stirring, and ultrasonically removing bubbles to prepare printing slurry;

(2) preparing a bionic 3D printing support: the method comprises the steps of filling hydroxyapatite particle/gelatin/sodium alginate composite slurry (namely the printing slurry) into a charging barrel, enabling the diameter of a needle to be 0.4mm, designing the outer dimension of a support material to be a circle with the diameter of 10mm and the height of 2mm, enabling the printing height of each layer to be 0.32mm, enabling the distance between fiber yarns on the same layer to be 0.5mm, enabling the fiber yarns between the layers to be 90 degrees, enabling the extrusion pressure to be 3.5bar, enabling the traction speed to be 8mm/s after solution extrusion, and printing the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D support to obtain a blank of the 3D support.

Example 4

(1) preparing hydroxyapatite powder/gelatin/sodium alginate composite slurry: adding 7.5g of gelatin particles into 50mL of deionized water, and fully and magnetically stirring to obtain a gelatin solution with the concentration of 0.15 g/mL; then 5g of the HA particles obtained in example 1 were added and stirred until the HA particles were uniformly dispersed in the gelatin solution; adding 3g of sodium alginate into the solution, fully and uniformly stirring, and ultrasonically removing bubbles to prepare printing slurry;

(2) preparing a bionic 3D printing support: the method comprises the steps of filling hydroxyapatite particle/gelatin/sodium alginate composite slurry (namely the printing slurry) into a charging barrel, enabling the diameter of a needle to be 0.4mm, designing the outer dimension of a support material to be a circle with the diameter of 10mm and the height of 2mm, enabling the printing height of each layer to be 0.32mm, enabling the distance between fiber yarns on the same layer to be 0.5mm, enabling the fiber yarns between the layers to be 90 degrees, enabling the extrusion pressure to be 2.0bar, enabling the traction speed to be 10mm/s after solution extrusion, and printing the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D support to obtain a blank of the 3D support.

Example 5

And (3) soaking the embryo body obtained by printing in the embodiment 2 in 0.45M calcium ion solution for 0.25h to rapidly crosslink sodium alginate in the hydroxyapatite/gelatin/sodium alginate composite to obtain the soaked crosslinked scaffold.

Example 6

The cross-linked scaffold after soaking described in example 5 was soaked in genipin solution of 5mg/mL for 3d for further sufficient cross-linking to obtain a stable scaffold.

Example 7

The stable scaffolds described in example 6 were soaked with 10mg/mL sodium glutamate solution for 3d and the solution was changed every 12h to obtain scaffolds from which excess cross-linking agent was removed.

Example 8

And (3) soaking the scaffold obtained in the embodiment 7 and from which the excessive cross-linking agent is removed in deionized water for 3D, changing the solution every 12h, and freeze-drying the obtained scaffold material to obtain the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold.

Example 9

The printing slurry obtained in the step (1) in the embodiment 2 is subjected to an oscillation temperature scanning measurement method to study the deformation characteristic of the slurry; the flow characteristics of the slurry were studied using a rotametry method. The rheology diagram of the printing paste obtained in step (1) in example 2 is shown in fig. 2. Part a of fig. 2 is a line graph showing storage modulus and loss modulus of the printing paste, and part B of fig. 2 is a line graph showing fluidity analysis of the printing paste at a temperature of 30 ℃; in FIG. 2, Gel/ALG is gelatin/sodium alginate slurry without HA particles, and Gel/ALG-HA is gelatin/sodium alginate slurry with HA particles;

part A of figure 2 shows that the gelatin/sodium alginate slurry has a gel temperature of 26 ℃, when the temperature is higher than 26 ℃, the loss modulus is far higher than the storage modulus, and the material is mainly subjected to viscous deformation and is in a liquid state; the gelatin/sodium alginate composite slurry of the micro-nano hierarchical structure HA particles HAs the gel temperature of 28 ℃, when the temperature is higher than 28 ℃, the storage modulus and the loss modulus are equivalent, and the material is in a semi-solid state, namely a gel state; section B of fig. 2 shows that the viscosity of the slurry with and without the HA particle group is lower when the shear rate is higher, facilitating the extrusion of the slurry from the printer needle; when the shear rate is low, the viscosity of the HA particle-containing composite slurry is high, which is beneficial to forming the slurry on the receiving plate, and the viscosity of the slurry without HA particles is low, which is not beneficial to forming the slurry on the receiving plate.

Example 10

Observing the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing support obtained in the embodiment 8 under a scanning electron microscope; scanning electron microscopy is used for shooting the front side of the bionic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing support obtained in the example 8, and the result is shown in figure 3; the section of the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold obtained in example 8 was photographed by a scanning electron microscope, and the result is shown in fig. 5; the element distribution and composition of the scaffold were analyzed by scanning electron microscopy spectroscopy, and as shown in fig. 4, it was shown that the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold obtained in example 8 contains elements of three main raw materials, calcium, phosphorus, nitrogen, oxygen, carbon, and the like.

Example 11

The biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing support obtained in example 8 was subjected to irradiation sterilization, and then placed in a well plate, mouse mesenchymal stem cell suspension was planted on the composite support material to allow the cells to sufficiently adhere for 1 hour, a complete culture medium was added to immerse the support, the well plate was placed in a constant temperature incubator at 37 ℃, the complete culture medium in the well plate was aspirated away after 3 days of culture, a staining solution for cell death was added, incubation was performed in the constant temperature incubator at 37 ℃ for 0.5 hour, the staining solution for cell death was aspirated away, and the growth of mouse mesenchymal stem cells on the 3D support material was observed with a confocal laser microscope, as shown in fig. 6. The cells have good proliferation state on the scaffold material, and the three-dimensional space provides sufficient spreading growth space for osteoblasts. Under the in vivo implantation environment, the scaffold can be degraded in a programmed manner, and the degradability of gelatin is fully utilized; the HA powder with the micro-nano structure is exposed and released, so that the bone regeneration and bone induction can be effectively promoted, the bone promoting property of the graded micro-nano structure material can be effectively exerted, and the cascading biological effect of the HA powder and the bone inducing property in the bone tissue regeneration process is the outstanding advantage of the invention. In addition, the macroporous structure is also favorable for nutrient supply and vascular ingrowth, thereby synergistically promoting the regeneration of bone tissues.

The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.

Claims

1. a preparation method of biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing support, is characterized in that, comprises the following steps:

(1) Add (NH4) ₂ PO ₄ into water and mix evenly to obtain (NH 4 ) ₂ PO ₄ solution; then adjust the pH of the (NH 4 ) ₂ PO ₄ solution to 5.8-6.2 to obtain solution a; Ca(NO ₃ ) ₂ ·4H ₂ O was added to the solution a, stirred uniformly to obtain solution b; then sodium citrate was added to solution b, stirred uniformly, heated for heating, centrifuged to collect the precipitate, washed by centrifugation, and freeze-dried , to obtain hydroxyapatite powder;

(2) adding gelatin into water, mixing evenly to obtain a gelatin solution; adding the hydroxyapatite powder described in step (1) into the gelatin solution, stirring evenly, to obtain a composite solution, and then adding to the composite solution Sodium alginate, stir evenly, ultrasonically remove air bubbles, and obtain printing paste;

(3) Loading the printing paste described in step (2) into a barrel used for printing, and performing 3D printing to obtain an embryo body; immersing the embryo body in a calcium ion solution, taking it out, and immersing it in a genipin solution, Take out, soak in sodium glutamate solution, take out, and finally soak in water to obtain the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printed scaffold.

2 . The method for preparing a biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold according to claim 1, wherein the concentration of the (NH ₄ ) ₂ PO ₄ solution described in step (1) is 1-40mM; the molar volume ratio of the Ca(NO ₃ ) ₂ ·4H ₂ O to solution a is 1.5-60: 1 mmol/L; the Ca(NO ₃ ) ₂ ·4H ₂ O and sodium citrate The molar ratio is 4-8:1.

3. The preparation method of the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold according to claim 1, wherein the temperature of the heat treatment is 180°C, and the time of the heat treatment is 2 -12h.

4. The preparation method of the biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold according to claim 1, wherein the concentration of the gelatin solution in step (2) is 0.05-0.2g/ mL; the mass ratio of the hydroxyapatite powder and gelatin described in step (2) is 1:3-3:1; in the printing paste described in step (2), the concentration of sodium alginate is 0.02-0.08g /mL.

5. The method for preparing a biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold according to claim 1, wherein in the composite solution of step (2), the hydroxyphosphate The concentration of limestone powder is 0.15-0.30g/mL.

6. The preparation method of biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing support according to claim 1, wherein the extrusion pressure of the 3D printing in step (3) is 0.5-5.0 bar, the pulling speed is 5-50mm/s.

7. The preparation method of biomimetic hydroxyapatite particles/gelatin/sodium alginate composite 3D printing support according to claim 1, wherein the shape of the embryo body in step (3) is cylindrical; the described The internal structure of the embryo body is a layered spinning fiber layer, the diameter of the silk is 0.2-0.8mm, the spacing between the spinning fibers in the same layer is 0.2-1.2mm, and the fibers between the layers are 30 mm. -90°, forming a connected polygonal three-dimensional network pore structure inside.

8. The preparation method of biomimetic hydroxyapatite particles/gelatin/sodium alginate composite 3D printing support according to claim 1, wherein the calcium ion solution in step (3) is that calcium salt and water are mixed evenly To obtain a solution, the calcium salt is calcium chloride; the concentration of the calcium ion solution is 0.2-0.6M, and the time for soaking the embryo body in the calcium ion solution is 0.05-1h; the concentration of the genipin solution It is 2-10mg/mL, and the time that the embryo body is soaked in the genipin solution is 1-5d; the concentration of the sodium glutamate solution is 4-20mg/mL, and the embryo body is soaked in the sodium glutamate solution. The time of the solution is 1-5d; the time of soaking the embryo body in water is 1-5d.

9. The method for preparing a biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold according to claim 1, wherein in step (3), the embryo body is soaked in sodium glutamate When in the solution, the sodium glutamate solution is changed every 4-12h; when the embryo body is soaked in water, the water is changed every 4-12h.

10. A biomimetic hydroxyapatite particle/gelatin/sodium alginate composite 3D printing scaffold prepared by the preparation method of any one of claims 1-9.