A 3D-printed scaffold with enhanced bioactivity and in vitro mineralization capacity, its preparation method and application

Abstract

This invention discloses a 3D-printed scaffold with improved bioactivity and in vitro mineralization capacity, its preparation method, and its applications. The scaffold is prepared by layer-by-layer extrusion 3D printing using an ink comprising biphasic calcium phosphate (composed of octacalcium phosphate (OCP) and amorphous calcium phosphate (ACP)), sodium alginate, and a dispersant. This invention uses a mixture of OCP and ACP to form biphasic calcium phosphate (BCP) as the inorganic phase component of the 3D composite scaffold, and sodium alginate (SA) as the organic phase component. Simultaneously, the organic substance sodium alginate (SA) can simulate a blood clot as a temporary matrix, providing mechanical stability and traction for cells migrating from adjacent tissues. Furthermore, it provides a stable environment for new bone replacement during the gradual degradation of the scaffold by cells, offering significant advantages for subsequent scaffold implantation, attracting cell migration, and inducing mineralization.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering, and specifically relates to a 3D printed scaffold with improved bioactivity and in vitro mineralization capacity, its preparation method and application. Background Technology

[0002] Bone is a hard tissue inside or on the surface of the human body, playing a variety of important roles in supporting, protecting, and regulating metabolism. It is a vital tissue in the human body. In terms of composition, bone consists of 30-40% collagen, 50-60% calcium orthophosphate (primarily hydroxyapatite (HA), and 10% water. Structurally, bone, from the outside in, consists of the periosteum, compact bone, cancellous bone, and medullary cavity. Compact bone comprises multiple ring-shaped lamellar layers, between which are numerous bone units composed of concentric bony rings surrounding Haversian canals. Blood vessels traverse these Haversian canals, forming an interconnected and orderly network that provides nutrients to the bone tissue and facilitates the transport of metabolic products and the transmission of information. Looking deeper into the microstructure, fine needle-like hydroxyapatite particles, 10-20 nm in size, are tightly bound and arranged in an orderly fashion along the long axis of collagen fibers, forming collagen fibers. These collagen fiber bundles then form layers of bone plates. This highly ordered arrangement is a crucial reason why bone tissue possesses high strength and toughness, enabling it to support the human body and facilitate its activities. Studies have shown that bone-related diseases account for one-fourteenth of all disease types, with bone defects being a common and frequently occurring form of bone injury. While small-scale defects in human bone tissue can heal themselves, larger defects (exceeding 2 cm) or more severe injuries (such as comminuted fractures) require bone fragment removal and additional external treatments.

[0003] For various bone injuries, when the body cannot heal itself and external assistance is needed, bone grafting is used as an adjunct therapy. Bone grafting can be mainly divided into autologous bone grafting and artificial bone grafting based on different bone sources. Autologous grafting is the ideal method due to its high immunocompatibility, but its widespread application is limited by factors such as the availability of bone and the trauma of secondary surgery. Due to the large demand for related bone, various artificial bone biomaterials are used in bone grafting. By combining this with 3D printing, various artificial biological scaffolds are printed to provide "personalized" and precise treatment for different patients and different affected areas. This application involves bone tissue engineering.

[0004] The development of bone tissue engineering began in the early 20th century when surgeons started experimenting with calcium phosphate and metals as bone graft materials; this stage belongs to the first generation of biomaterials. The 1960s saw the development of using bioactive materials for functional regeneration, primarily bioactive glass, bioceramics, and their composites with biodegradable or absorbable properties; this stage belongs to the second generation of biomaterials, and it was during this stage that the professional term "bone tissue engineering" was formally established. In the 21st century, further in-depth research revealed bioactive molecules such as proteins and peptides, which were used to modify scaffold materials to enhance their biological properties; this stage belongs to the third generation of biomaterials. With the continuous exploration and updating of bone tissue engineering materials, the emergence of numerous modification methods, and the development of additive manufacturing technologies such as 3D printing, new inspiration has been provided for designing more complex materials in bone tissue engineering.

[0005] Currently, commonly used bioscaffold materials for bone tissue engineering mainly include biopolymers, bioceramics, and metal biomaterials. Biopolymers are often divided into natural and synthetic polymers. Natural polymers, such as hyaluronic acid, silk fibroin, alginate, gelatin, and collagen, offer advantages such as good biocompatibility, low cost, and cross-linking ability. However, they often have poor mechanical properties and may exhibit immunogenicity. Synthetic biopolymers, such as PLA, PLGA, PCL, PEEK, and PU, offer advantages such as biodegradability, biocompatibility, and ease of molding. However, they may have issues such as toxic degradation products and low degradation efficiency. In addition, bioceramics are also frequently used as key materials in bone tissue engineering. Common bioceramics include β-tricalcium phosphate, octacalcium phosphate, amorphous calcium phosphate, and bioactive glass. They offer advantages such as biocompatibility, high compressive modulus, and osteoconductivity, but they also share the common problem of brittleness found in ceramic materials. Commonly used metallic biomaterials, such as titanium and its alloys, are often used to prepare bone bioscaffold materials. Although they have the advantages of high strength and low density, their non-degradability and bioinertness limit their widespread application. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a 3D-printed scaffold with enhanced bioactivity and in vitro mineralization capacity, along with its preparation method and applications. The bone repair scaffold of this invention exhibits excellent mineralization induction capabilities.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] The present invention provides an ink comprising biphasic calcium phosphate, sodium alginate and a dispersant, wherein the biphasic calcium phosphate is composed of octacalcium phosphate and amorphous calcium phosphate.

[0009] According to an embodiment of the present invention, the mass ratio of the biphasic calcium phosphate and sodium alginate is (4-6):(4-6), with exemplary ratios of 4:6, 5:6, and 6:4.

[0010] According to an embodiment of the present invention, in the biphasic calcium phosphate, the mass ratio of octacalcium phosphate to amorphous calcium phosphate is (0-4):(0-4) and is not 0; exemplary ratios are 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:4.

[0011] According to an embodiment of the present invention, the total mass ratio of the biphasic calcium phosphate and sodium alginate to the mass ratio of the dispersant is 1:(2-5), with exemplary ratios of 1:2, 1:3, 1:4, and 1:5.

[0012] According to an exemplary embodiment of the present invention, the biphasic calcium phosphate is prepared by mixing octacalcium phosphate and amorphous calcium phosphate.

[0013] According to an exemplary embodiment of the present invention, the octacalcium phosphate is prepared by co-precipitation after mixing a phosphorus source (such as ammonium dihydrogen phosphate), urea, a calcium source (such as calcium acetate monohydrate) with water. For example, the phosphorus source (such as ammonium dihydrogen phosphate) and urea are first dissolved in water to obtain a mixed solution, and then the mixed solution is added to a calcium source (such as calcium acetate monohydrate) solution.

[0014] Preferably, the molar ratio of phosphorus source, urea, calcium source and water is 12:9:30:(15000-30000).

[0015] Preferably, the coprecipitation reaction temperature is 80-95℃ and the reaction time is 2-3h.

[0016] According to an exemplary embodiment of the present invention, the amorphous calcium phosphate is prepared by mixing a phosphorus source (such as diammonium hydrogen phosphate), sodium hydroxide, a calcium source (such as calcium nitrate tetrahydrate) with water to obtain a mixed solution, and then preparing it by a co-precipitation method.

[0017] Preferably, the molar ratio of phosphorus source, sodium hydroxide, calcium source and water is 3:2:(4000-5000). Preferably, the mixture further includes adjusting the pH of the mixed solution to 10±0.5 with sodium hydroxide.

[0018] According to an embodiment of the present invention, the dispersant is water. Specifically, the dispersant is deionized water.

[0019] The present invention also provides a method for preparing the above-mentioned ink, the method comprising mixing biphasic calcium phosphate, sodium alginate and a dispersant to obtain the ink.

[0020] Specifically, the definition of the dispersant is the same as above.

[0021] According to an embodiment of the present invention, the biphasic calcium phosphate and sodium alginate have the same selection and dosage ratio as described above.

[0022] According to an embodiment of the present invention, the method for preparing the ink includes mixing octacalcium phosphate with amorphous calcium phosphate to obtain biphasic calcium phosphate, and then mixing biphasic calcium phosphate with sodium alginate, adding a dispersant and stirring to prepare the ink.

[0023] The present invention also provides a scaffold comprising biphasic calcium phosphate and sodium alginate. Preferably, the biphasic calcium phosphate is composed of octacalcium phosphate and amorphous calcium phosphate.

[0024] According to an embodiment of the present invention, the scaffold is prepared by layer-by-layer printing of the above-mentioned ink using an extrusion 3D printing method. Preferably, the scaffold further includes cross-linking and curing the printed scaffold in a CaCl2 solution.

[0025] According to an embodiment of the present invention, the scaffold is a biphasic calcium phosphate / sodium alginate 3D-printed scaffold with an interconnected porous structure that enhances bioactivity and in vitro mineralization capabilities.

[0026] The present invention also provides a method for preparing the above-mentioned scaffold, the method comprising: printing the above-mentioned ink layer by layer by an extrusion 3D printing method to prepare the scaffold.

[0027] According to an embodiment of the present invention, the preparation method further includes placing the printed scaffold in a CaCl2 solution for cross-linking and curing.

[0028] According to an embodiment of the present invention, the method for preparing the stent includes the following steps:

[0029] (1) Mix phosphorus source (such as ammonium dihydrogen phosphate), urea, calcium source (such as calcium acetate monohydrate) with water and use co-precipitation method to prepare octacalcium phosphate powder;

[0030] (2) Mix phosphorus source (such as diammonium hydrogen phosphate), sodium hydroxide, calcium source (such as calcium nitrate tetrahydrate) with water and use co-precipitation method to prepare amorphous calcium phosphate powder;

[0031] (3) Mix octacalcium phosphate powder and amorphous calcium phosphate to obtain biphasic calcium phosphate, then mix biphasic calcium phosphate and sodium alginate, add a dispersant (such as water) and stir to obtain ink.

[0032] (4) The ink is printed layer by layer using the extrusion 3D printing method, and the formed scaffold is placed in CaCl2 solution for cross-linking and curing.

[0033] According to an embodiment of the present invention, the method for preparing the scaffold may further include step (5): drying the printed and cured cross-linked scaffold to obtain a 3D printed bone repair scaffold with good in vitro mineralization ability and an interconnected porous structure.

[0034] Preferably, in step (1), the molar ratio of phosphorus source (such as ammonium dihydrogen phosphate), urea, calcium source (such as calcium acetate monohydrate) to deionized water is 12:9:30:(15000-30000).

[0035] Preferably, in step (1), the coprecipitation reaction temperature is 80-95℃ and the reaction time is 2-3h.

[0036] Preferably, in step (2), the molar ratio of phosphorus source (such as diammonium hydrogen phosphate), sodium hydroxide, calcium source (such as calcium nitrate tetrahydrate) to deionized water is 3:2:(4000-5000).

[0037] Preferably, step (2) further includes stirring the mixed solution. For example, the stirring temperature is room temperature, and the stirring time is 10-20 minutes.

[0038] Preferably, step (2) further includes drying the product obtained by the coprecipitation method (e.g., by freeze drying). For example, the freeze drying temperature is -(20-40)℃ and the time is 48-72h.

[0039] Preferably, in step (3), the mass ratio of biphasic calcium phosphate to sodium alginate is (4-6):(4-6). Further, the mass ratio of the powder after mixing biphasic calcium phosphate and sodium alginate to the dispersant (such as water) is 1:(2-5); exemplary ratios are 1:2, 1:3, 1:4, and 1:5.

[0040] Preferably, in step (3), the stirring temperature is room temperature and the stirring time is 2 minutes.

[0041] Preferably, in step (4), the infill density of the 3D printed scaffold is 40%-60%; for example, 40%, 50%, and 60%.

[0042] Preferably, in step (4), the concentration of the CaCl2 solution is 5-20 wt%, for example 5 wt%, 10 wt%, 15 wt%, or 20 wt%; the crosslinking curing time is 5-24 h, for example 12 h.

[0043] Preferably, in step (5), the drying time is 6-48 hours, for example 24 hours; the drying temperature is 40-70°C, for example 60°C.

[0044] According to an exemplary embodiment of the present invention, the method for preparing the stent includes the following steps:

[0045] (1) Mix ammonium dihydrogen phosphate, urea, calcium acetate and water in a molar ratio of 12:9:30:(15000-30000) and react at 80-95℃ for 2-3 hours using a co-precipitation method to obtain octacalcium phosphate powder;

[0046] (2) Mix diammonium hydrogen phosphate, calcium nitrate tetrahydrate and water in a molar ratio of 3:2:(4000-5000), adjust the pH of the solution to 10±0.5 with sodium hydroxide, and obtain amorphous calcium phosphate powder by co-precipitation at room temperature for 10-20 min.

[0047] (3) Mix octacalcium phosphate powder, amorphous calcium phosphate powder, sodium alginate and water at a mass ratio of 1:(2-5), stir at room temperature to obtain printing ink;

[0048] (4) Using extrusion 3D printing method, print ink layer by layer with a filling density of 40%-60%, and place the formed scaffold in 5-20wt% CaCl2 solution for cross-linking and curing for 5-24h, and then dry at 40-70℃ for 6-48h to obtain a biphasic calcium phosphate / sodium alginate 3D printed bone repair scaffold with good in vitro mineralization ability and interconnected porous structure.

[0049] The present invention also provides the use of the above-described scaffold in the preparation of angiogenesis-promoting scaffolds. For example, its use in the preparation of scaffolds for bone defect repair (osteoporotic fractures).

[0050] The present invention also provides a method for treating or alleviating bone defects, comprising providing the above-mentioned stent to an individual in need of such treatment.

[0051] The beneficial effects of this invention:

[0052] (1) This invention achieves a synergistic effect (1+1>2) by preparing bone tissue scaffolds using composite polymer materials and bioceramic materials with high osteoconductivity. Octacalcium phosphate (OCP) possesses excellent biodegradability, osteoconductivity, osteoinductive properties, and bioactivity. It can be converted into HA, an inorganic component of natural bone, in vivo and can stimulate osteocytes, thereby promoting bone remodeling. Therefore, it is considered a precursor phase for HA formation during bone mineralization and exists in human bones and teeth. Structurally, OCP consists of alternating layers of apatite and hydrated layers. The atomic arrangement of calcium and phosphate ions in the apatite layer is similar to that of HA, while the hydrated layer contains Ca... 2+ and PO4 3-The larger spacing allows for greater water of crystallization. Amorphous calcium phosphate (ACP) is a transitional phase in the formation of calcium phosphate in aqueous solution and is therefore considered another precursor for the formation of HA during bone mineralization. Its chemical composition depends primarily on the pH of the solution and the concentrations of calcium and phosphate ions in the mother liquor. Structurally, ACP may have a short-range apatite structure, but its crystal size is so small that it appears amorphous in X-ray diffraction, thus it is often found in pathological calcifications of soft tissues in vivo. Both OCP and ACP are precursor phases of HA. OCP transforms into HA, while ACP first crystallizes into an OCP-like phase and then transforms into HA. Both OCP and ACP are phosphate components that may exist in the human body and possess good osteoconductivity, osteoinductive properties, and bioactivity. Studies have shown that scaffolds prepared from a mixture of OCP and ACP powders exhibit better in vitro mineralization effects and better cell activity in MC3T3-E1 cells compared to scaffolds prepared from either powder alone. This invention uses a mixture of OCP and ACP to create biphasic calcium phosphate (BCP), which serves as the inorganic phase component of a 3D composite scaffold. Sodium alginate (SA) is used as a binder to effectively bond and shape the inorganic phase. Simultaneously, SA can mimic a blood clot as a temporary matrix, providing mechanical stability and traction for cells migrating from adjacent tissues. Furthermore, SA provides a stable environment for bone regeneration during the gradual degradation of the scaffold by cells. The biphasic calcium phosphate scaffold prepared by mixing ACP and OCP powders exhibits superior in vitro mineralization and better cell viability in MC3T3-E1 cells compared to monophasic calcium phosphate scaffolds prepared from either ACP or OCP alone.

[0053] (2) This invention uses 3D bioprinting (the general process of 3D printing mainly includes four steps: diagnosis, design, selection of bio-ink, printing, and 3D functional reconstruction) to provide precise treatment for different disease sites. For different sites, personalized designs are made in different ways to meet the needs of patients. At the same time, the extrusion printing method used in this invention has advantages such as low printing cost, wide applicability of materials, and suitable printing accuracy. Therefore, it also has great advantages for subsequent scaffold implantation and attracting cell migration. Attached Figure Description

[0054] Figure 1 SEM images of the surface of single-phase and dual-phase calcium phosphate scaffolds before and after mineralization.

[0055] Figure 2 In the middle (A) and (B), respectively, the live / dead staining images and cell proliferation results of MC3T3-E1 cells were obtained using monophasic calcium phosphate scaffolds and biphasic calcium phosphate scaffolds. Detailed Implementation

[0056] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0057] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0058] Example 1

[0059] A method for preparing a 3D-printed bone repair scaffold includes the following steps:

[0060] (1) Mix 0.012 mol NH4H2PO4 and 0.04 mol CO(NH2)2 with 400 mL of deionized water, and then stir magnetically at room temperature until completely dissolved to prepare solution A. Simultaneously, dissolve 0.0016 mol Ca(CH3COO)2·H2O in 400 mL of deionized water to prepare solution B. Then, add solution B dropwise to solution A and stir for 10 min to form solution C. Then, heat solution C in a 90 °C water bath and stir vigorously for 2 h. After the reaction is complete, filter the resulting precipitate and wash it three times with deionized water and twice with anhydrous ethanol. Dry the washed product at 60 °C for 24 h to obtain octacalcium phosphate powder.

[0061] (2) 0.0075 mol Ca(NO3)2·4H2O and 0.0005 mol (NH4)2HPO4 were mixed with 200 mL of deionized water, respectively, and then magnetically stirred at room temperature until completely dissolved to prepare solutions E and D. Solution D was then added dropwise to solution E to obtain a mixed solution, and the mixture was stirred for 2 min. The pH of the mixed solution was then adjusted to 10 with 2 mol / L sodium hydroxide solution, and stirred at room temperature for 15 min. After the reaction was complete, the precipitate was collected by centrifugation at 5000 rpm for 5 min, washed three times with deionized water and twice with anhydrous ethanol, and the collected product was freeze-dried at -20℃ for 48 h to obtain amorphous calcium phosphate powder.

[0062] (3) Mix 3g of octacalcium phosphate powder obtained in step (1), 1g of amorphous calcium phosphate powder obtained in step (2), 6g of sodium alginate powder and 30mL of deionized water evenly (where 3g of octacalcium phosphate and 1g of amorphous calcium phosphate are mixed to form biphasic calcium phosphate, and 4g of biphasic calcium phosphate and 6g of sodium alginate are mixed and dispersed in 30mL of deionized water), and stir evenly at room temperature for 2min to obtain 3D printing ink.

[0063] (5) Cylindrical models with dimensions of Φ15×5mm and Φ15×30mm were constructed using modeling software, and slicing files were exported using slicing software. The layer thickness was 0.6mm and the infill density was 50%. Then, ink was printed layer by layer using an extrusion 3D printer under the control of gcode to obtain a porous scaffold. After printing, a biphase calcium phosphate / sodium alginate 3D printed scaffold was obtained, which was crosslinked in a 10wt% CaCl2 solution for 12 hours until cured. The crosslinked scaffold was washed with deionized water and dried in a 60℃ oven for 24 hours, and named BCP / SA.

[0064] Comparative Example 1

[0065] A method for preparing a 3D-printed bone repair scaffold includes the following steps:

[0066] (1) To verify the effect of the interaction between octacalcium phosphate and amorphous calcium phosphate in biphasic calcium phosphate on the in vitro mineralization performance of the scaffold, a printing ink without amorphous calcium phosphate was prepared. 4g of octacalcium phosphate prepared in step (1) of Example 1 was mixed with 6g of sodium alginate, and the mixed powder was dispersed in 30mL of deionized water and stirred thoroughly to obtain the 3D printing ink.

[0067] (2) Cylindrical models with diameters of 15×5mm and 15×30mm were constructed using modeling software, and slicing files were exported using slicing software. The layer thickness was 0.6mm, and the infill density was 50%. Then, ink was printed layer by layer using an extrusion 3D printer under the control of gcode to obtain a porous scaffold. After printing, an octacalcium phosphate / sodium alginate scaffold was obtained. The octacalcium phosphate / sodium alginate scaffold was placed in a 10wt% CaCl2 solution for crosslinking for 12 hours until cured. The crosslinked scaffold was then washed with deionized water and dried in a 60℃ oven for 24 hours, and named OCP / SA.

[0068] Comparative Example 2

[0069] A method for preparing a 3D-printed bone repair scaffold includes the following steps:

[0070] (1) To verify the effect of the interaction between octacalcium phosphate and amorphous calcium phosphate in biphasic calcium phosphate on the in vitro mineralization performance of the scaffold, a printing ink without octacalcium phosphate was prepared. 4g of amorphous calcium phosphate prepared in step (2) of Example 1 was mixed with 6g of sodium alginate, and the mixed powder was dispersed in 30mL of deionized water and stirred thoroughly to obtain the 3D printing ink.

[0071] (2) Cylindrical models with dimensions of Φ15×5mm and Φ15×30mm were constructed using modeling software, and slicing files were exported using slicing software. The layer thickness was 0.6mm, and the infill density was 50%. Then, ink was printed layer by layer using an extrusion 3D printer under the control of gcode to obtain a porous scaffold. After printing, an amorphous calcium phosphate / sodium alginate scaffold was obtained. The amorphous calcium phosphate / sodium alginate scaffold was placed in a 10wt% CaCl2 solution for crosslinking for 12 hours until cured. The crosslinked scaffold was then washed with deionized water and dried in a 60℃ oven for 24 hours, and named ACP / SA.

[0072] Experimental Example 1

[0073] The scaffolds printed in Example 1, Comparative Example 1, and Comparative Example 2 were immersed in simulated body fluid (SBF) for 14 and 28 days, respectively, to compare the in vitro induced mineralization capacity among the groups. In short, each group of scaffolds was immersed in SBF solution at a solid-liquid ratio of 1 g / 100 mL (at 37°C), with the SBF solution replaced every three days. After 14 days (2 weeks) or 28 days (4 weeks), the immersed scaffolds were removed and dried at 60°C for 24 hours. The morphology of these three scaffold samples was then observed under a scanning electron microscope. The results are as follows: Figure 1 As shown. Experimental results show that, compared with Comparative Example 1 and Comparative Example 2, the scaffold prepared in Example 1 exhibits more obvious mineralization structure and earlier mineralization changes at different time periods.

[0074] The SBF solution was prepared as follows: the reagents were added to water in the order shown in Table 1. After all the reagents were completely dissolved, the solution was heated to 37°C. Finally, the pH was adjusted to 7.4 using HCl solution and the volume was increased to 1L.

[0075] Table 1. Reagents required to prepare 1 L of SBF solution

[0076]

[0077] Experiment Example 2

[0078] Cell viability of MC-3T3 cells cultured in the porous scaffold extracts prepared in Examples 1, 1, and 2 was determined using a Cell Counting Kit-8 (CCK-8) and a live / dead cell staining kit. The scaffold extracts were prepared according to ISO 10993-12 2017. In the experiments, MC-3T3 cells were cultured at 5 × 10⁻⁶ cells / mL. 3 cells / cm 2The density of the scaffolds prepared in Example 1, Comparative Example 1, and Comparative Example 2 was co-cultured with the extracts of the porous scaffolds prepared in Example 1, Comparative Example 1, and Comparative Example 2 in 96-well plates and placed in a cell culture incubator at 37°C and 5% CO2. After culturing in MEM-α medium for 1 day, 3 days, and 5 days, 10 μL of CCK-8 solution was added to each well, and after incubation for 2 hours, the optical density (OD) of each well was measured at 450 nm using a microplate reader. Cell viability was calculated as follows:

[0079] Cell viability = (OD) 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )

[0080] When cell viability is above 70%, the scaffold is considered non-cytotoxic; when cell viability is below 70%, the scaffold is considered cytotoxic.

[0081] Live / Dead fluorescence staining was performed on MC-3T3 cells cultured in extract medium for 3 days using the Adamas-life Cell Counting Kit-8, following the manufacturer's instructions. After staining, the cells were incubated at 37°C in the dark for 30 minutes. Fluorescent staining images of MC-3T3 cells were obtained using an inverted fluorescence microscope. The results are shown below. Figure 2 As shown. Figure 2 The experimental results showed that, after live / dead cell fluorescence staining, MC-3T3 cells co-cultured with the scaffold extract prepared in Example 1 for 3 days (3d) exhibited greater cell viability compared to Comparative Examples 1 and 2. The results also showed that on day 5 (5d), the cell viability of the porous scaffolds prepared in Example 1, Comparative Examples 1 and 2 reached (100.45±0.57)%, (98.52±0.83)%, and (97.85±2.24)%, respectively. This confirms that the scaffold prepared using the ink from Example 1 is non-toxic and exhibits better biocompatibility than the biological scaffolds prepared in Comparative Examples 1 and 2, demonstrating its potential as a bone repair scaffold.

[0082] The exemplary embodiments of the present invention have been described above. However, the scope of protection of this application is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A support, characterized in that, The scaffold is prepared by printing ink layer by layer using an extrusion 3D printing method. The ink includes biphase calcium phosphate, sodium alginate and dispersant. The biphasic calcium phosphate is composed of octacalcium phosphate and amorphous calcium phosphate; The mass ratio of biphasic calcium phosphate to sodium alginate is (4-6):(4-6). In the biphasic calcium phosphate, the mass ratio of octacalcium phosphate to amorphous calcium phosphate is (0-4):(0-4) and neither of them is 0; The total mass ratio of the biphasic calcium phosphate and sodium alginate to the mass ratio of the dispersant is 1:(2-5). The dispersant is water.

2. The bracket as described in claim 1, characterized in that, The method for preparing the ink includes mixing biphasic calcium phosphate, sodium alginate and a dispersant to obtain the ink.

3. The method for preparing the stent according to claim 1 or 2, characterized in that, The preparation method includes preparing the scaffold by printing the ink layer by layer using an extrusion 3D printing method.

4. The preparation method according to claim 3, characterized in that, Includes the following steps: (1) Phosphorus source, urea, calcium source and water are mixed and octacalcium phosphate powder is prepared by co-precipitation method; (2) Amorphous calcium phosphate powder was prepared by mixing phosphorus source, sodium hydroxide, calcium source and water and using co-precipitation method; (3) Mix octacalcium phosphate powder and amorphous calcium phosphate to obtain biphasic calcium phosphate, then mix biphasic calcium phosphate and sodium alginate, add dispersant water and stir to obtain ink; (4) Use the extrusion 3D printing method to print ink layer by layer, and place the formed scaffold in CaCl2 solution for cross-linking and curing.

5. The preparation method according to claim 4, characterized in that, In step (1), the phosphorus source is selected from ammonium dihydrogen phosphate.

6. The preparation method according to claim 4, characterized in that, In step (1), the calcium source is selected from calcium acetate monohydrate.

7. The preparation method according to claim 4, characterized in that, In step (2), the phosphorus source is selected from diammonium hydrogen phosphate; the calcium source is selected from calcium nitrate tetrahydrate.

8. The preparation method according to claim 4, characterized in that, In step (1), the molar ratio of phosphorus source, urea, calcium source and water is 12:9:30:(15000-30000). And / or, in step (1), the coprecipitation reaction temperature is 80-95℃ and the reaction time is 2-3 h; And / or, in step (2), the molar ratio of phosphorus source, calcium source and water is 3:2:(4000-5000); And / or, in step (3), the mass ratio of biphasic calcium phosphate to sodium alginate is (4-6):(4-6); And / or, the mass ratio of the powder after mixing biphasic calcium phosphate and sodium alginate to the dispersant is 1:(2-5).

9. The preparation method according to claim 4, characterized in that, In step (4), the infill density of the 3D printed scaffold is 40%-60%; And / or, in step (4), the concentration of the CaCl2 solution is 5-20 wt%; the cross-linking curing time is 5-24 h.

10. The use of the stent according to claim 1 or 2 and / or the stent prepared by the preparation method according to any one of claims 3-9 in the preparation of angiogenesis-promoting stents.

11. The application as described in claim 10, characterized in that, The application of the scaffold in the preparation of scaffolds for bone defect repair.

12. The application as described in claim 11, characterized in that, The application of the aforementioned scaffold in the preparation of scaffolds for osteoporotic fractures.

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