3D printing biological material ink based on drug sustained-release poly-citrate material and preparation and printing methods of 3D printing biological material ink

Abstract

The invention discloses 3D printing biological material ink based on a drug sustained-release polycitrate material and a preparation method and a printing method of the 3D printing biological material ink, and relates to the technical field of 3D printing biologication.The 3D printing biological material ink is prepared from a medical polycitrate polymer, nano-hydroxyapatite and an active drug ingredient; the addition amount of the hydroxyapatite is 5-65% of the total mass of the obtained biological material ink; the active pharmaceutical ingredients are gastrodin, notoginsenoside, recombinant wnt3a protein and LL-37 peptide; when the active medicines are gastrodin and notoginsenoside, the addition amount of the active medicines is 4-12% of the total mass of the obtained biological material ink; when the used active drugs are recombinant wnt3a protein and LL-37 peptide, the addition amount of the active drugs is 0.001-0.002% of the total mass of the obtained biological material ink; and the particle size of the nano hydroxyapatite is 60-80 nm. According to the method, 3D printing process parameters are selected, post-treatment is adopted to adjust a cross-linked network structure of a poly-citrate polymer, biological material scaffolds with different mechanical properties and degradation properties are obtained, and stable and long-acting release of carried drugs is ensured.

Description

Technical Field

[0001] The present application relates to the field of 3D printing biotechnology, and in particular to a 3D printing biomaterial ink based on a drug sustained-release polycitrate material and a preparation and printing method thereof. Background Art

[0002] The regeneration and repair of large bone defects is still a severe challenge in clinical treatment. Bone tissue engineering is a common strategy for bone repair, and its key element lies in the transport and conduction function of the scaffold in the microenvironment. Reasonable composite structure design can not only provide good mechanical properties for the scaffold, but also facilitate the penetration between the inner layer structures and improve the overall transport capacity of the scaffold. Most of the porous scaffolds obtained by traditional preparation methods have defects such as single structure, poor pore penetration, and difficulty in satisfying both structural and mechanical properties, which limits the orderly spatial distribution of cell infiltration and new tissue. Therefore, it is of great significance to manufacture bone tissue engineering scaffolds with multi-cell delivery and complex morphology similar to natural bone tissue for bone tissue regeneration.

[0003] Additive Manufacturing (AM), also known as 3D printing technology, is a technology that uses materials to gradually accumulate to manufacture physical parts. The biggest advantage of biological 3D printing technology is that it uses computer-aided modeling to print different levels and gradients with printing equipment combined with special active inks. It can not only quickly manufacture tissues and organs that meet different personalized needs, but also finely control their microstructures, realize diversified module construction and highly interconnected system formation, and greatly alleviate the problem of tissue and organ shortage. 3D printing technology can accurately and quickly manufacture structures with multi-level characteristics similar to natural tissues. By effectively defining the pore size, porosity, spatial arrangement of pores, connectivity and other structural characteristics of three-dimensional scaffold materials, it provides a space for cell and tissue regeneration; it can also be designed as a carrier in a layered, gradient, and functionalized microspace to provide conditions for the fixed-point and sustained release of drugs or other bioactive substances, and is widely used in the manufacture of tissue engineering biological scaffolds. Therefore, the combination of biomaterials and 3D printing technology has huge clinical needs and scientific significance in the medical field.

[0004] Biomaterial inks are the key to 3D printing technology. They can simulate the complex structure of tissues in the body to temporarily replace functional defects and support their repair. For a long time, the stability and fluidity of biomaterials have been difficult to meet the high requirements of printing technology, and their application has been limited. Currently, there are few biomaterial inks available and they are expensive. Inks commonly used in 3D printing of large bone defects, such as metals and ceramics, face the release of metal corrosion products or mechanical mismatch, and the bone regeneration and repair effect is not ideal. Therefore, it is urgent to develop a biomaterial ink that can be used for 3D printing of fine and complex structures.

[0005] Polycitrate refers to a prepolymer of polycitrate obtained by reacting a multifunctional citric acid monomer with an aliphatic alcohol containing 3 to 16 carbon atoms. These prepolymers undergo different post-treatments, such as doping with different proportions of diisocyanate, adding polyurethane segments to the polyester cross-linked network, and obtaining a cross-linked amino-doped polyester network. This material perfectly combines the wide range of mechanical properties and biodegradability of polyurethane and has good biocompatibility.

[0006] Existing technologies such as CN202410504596.2 disclose that Gastrodin is added to a medical polycitrate polymer-nanohydroxyapatite (HA) complex to prepare a medical polycitrate material and a preparation method thereof. The material can be used for 3D printing, but the specific 3D printing process is not described in detail. At the same time, when the fine structure of the biomaterial is printed using the existing 3D printing process parameters, the printing capacity is single. In the existing 3D printing process, all materials must be melted at high temperature before they can be printed and formed. When the material is loaded with drugs that are sensitive to high temperature, such drugs are easily inactivated by high temperature. Since the biomaterial ratio is only based on the HA component in the load-bearing bone to determine the hydroxyapatite ratio, when used as a 3D printing ink, the application range of the printed product is small, and the resulting material cannot be directly used in the repair of other tissue defects that are different from the HA component of the load-bearing bone, such as cartilage tissue, which limits the use of the material.

[0007] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0008] In response to the above-mentioned technical problems, the present application provides a 3D printing biomaterial ink based on a drug-sustained-release polycitrate material and a preparation and printing method thereof. The ink can be used to produce a scaffold structure that meets the requirements of various implantation sites through the 3D printing process, and the obtained material can be loaded with temperature-sensitive drugs. The performance does not change after printing. The obtained scaffold structure can provide the channel structure required for cell and tissue growth, thereby increasing the available types of biomaterial inks for existing 3D printing.

[0009] The present application provides a 3D printing biomaterial ink based on a drug sustained-release polycitrate material, comprising: a medical polycitrate polymer, nanohydroxyapatite, and an active pharmaceutical ingredient; the amount of nanohydroxyapatite added is 5-65% of the total mass of the obtained biomaterial ink; The active pharmaceutical ingredients are gastrodin, notoginseng saponins, recombinant wnt3a protein, and LL-37 peptide; When the active drugs used are gastrodin and notoginseng saponin, the added amount is 4-12% of the total mass of the obtained biomaterial ink; When the active drug used is recombinant wnt3a protein or LL-37 peptide, the added amount is 0.001-0.002% of the total mass of the obtained biomaterial ink; The particle size of nanohydroxyapatite is 60~80nm.

[0010] The use of the biomaterial ink can effectively maintain the drug activity of the above-mentioned various active drug ingredients in the 3D printed stent.

[0011] Preferably, it includes: a solvent for adjusting the printing characteristics of the biomaterial ink; the solvent is at least one of 1,4-dioxane, N,N-dimethylformamide, dimethylacetamide, isopropanol, and dimethyl sulfoxide, and more preferably, the solvent is 1,4-dioxane; the amount of solvent added is 10~50 wt.% of the total mass of the biomaterial ink.

[0012] Another aspect of the present application also provides a method for preparing the above-mentioned biomaterial ink, comprising the following steps: After the medical polycitrate polymer is obtained by reaction, the medical polycitrate polymer and nano-hydroxyapatite are mixed to obtain a medical polycitrate polymer-nano-hydroxyapatite composite; The biomaterial ink is obtained by mixing or chemically grafting a medical polycitrate polymer-nano hydroxyapatite composite with an active pharmaceutical ingredient; Medical polycitrate polymer and nano-hydroxyapatite are mixed and stirred for 1 to 2 hours.

[0013] After adding active drug ingredients to the medical polycitrate polymer-nanohydroxyapatite composite, the mixture is reacted for 2 hours under the condition that the added drug is active, thereby obtaining a biomaterial ink.

[0014] Preferably, the medical polycitrate polymer and nano-hydroxyapatite are mixed and stirred for 1 to 2 hours.

[0015] Preferably, after adding active drugs gastrodin and notoginseng saponin components to the medical polycitrate polymer-nanohydroxyapatite composite, the mixture is reacted at 60° C. for 2 hours to obtain biomaterial ink.

[0016] Preferably, after adding active drug recombinant wnt3a protein and LL-37 peptide components to the medical polycitrate polymer-nanohydroxyapatite complex, the two components are uniformly mixed at room temperature to obtain biomaterial ink.

[0017] Another aspect of the present application further provides a 3D printing method of the above-mentioned biomaterial ink, comprising the following steps: Biomaterial ink is 3D printed at 0~140℃.

[0018] The material can be used in a wide range of 3D printing environments, and can produce scaffolds with clear microstructures under different temperature environments. And because the ink has good printing properties at different temperatures, the printing temperature can be set according to the drugs used, thereby achieving effective protection for different active drugs.

[0019] Preferably, the biomaterial ink is softened at 60-140°C for high-temperature melt extrusion printing, and the obtained scaffold is subjected to thermal cross-linking treatment; the thermal cross-linking parameters are drying at 35-40°C for 1 day, drying at 55-60°C for 1 day, drying at 70-80°C for 1 day, and drying at 100-120°C for 2 days; High temperature melt extrusion printing includes the following steps: After softening the biomaterial ink, a pneumatic extrusion nozzle with a pressure of 30~600k / Pa was used for printing. The printing parameters were set as follows: model height 10~15mm; printing layer height 0.30~0.40mm; printing speed 1~10mm / s; and the stent was printed according to the model slicing software Slic3r to generate the printing path.

[0020] In this method, the scaffold obtained by high-temperature melt extrusion printing is subjected to thermal cross-linking treatment, and the mechanical properties of the scaffold can be effectively improved after long-term high-temperature treatment. The bone scaffold obtained after treatment under different conditions can be subjected to thermal cross-linking treatment under different time-temperature conditions according to the characteristics of the specific implantation site. For example, by extending the time and increasing the temperature, a more stable and hard scaffold can be obtained for load-bearing defects, and conversely, a scaffold with good elasticity can be obtained for cartilage defects, etc., thereby obtaining a finished scaffold that meets the needs of use in different parts.

[0021] Preferably, it includes: room temperature and low temperature extrusion printing method; the room temperature and low temperature extrusion printing method includes the following steps: after 1,4-dioxane and biomaterial ink are mixed evenly in a volume ratio of 1:1, the biomaterial ink is not heated and a pneumatic extrusion nozzle with a pressure of 30~600k / Pa is used for printing, and the printing parameters are set as follows: model height is 10-15mm; printing layer height is 0.30~0.40mm; printing speed is 1~30mm / s; and the stent is obtained by slicing according to the model slicing software Slic3r to generate a printing path for printing.

[0022] Preferably, it includes: low-temperature extrusion printing; low-temperature extrusion printing includes the following steps: after dimethylacetamide and biomaterial ink are mixed evenly in a volume ratio of 1~2:1, a pneumatic extrusion nozzle with a pressure of 30~600k / Pa is used, and the nozzle temperature is set to 0~5℃, and the printing platform temperature is set to -20-0℃ for printing, and the printing parameters are set as follows: model height is 10-15mm; printing layer height is 0.30~0.40mm; printing speed is 10~30mm / s; according to the model slicing software Slic3r, the printing path is generated by slicing to print to obtain a bracket; the obtained bracket is subjected to thermal cross-linking treatment; the thermal cross-linking parameters are drying at 35~40℃ for 1d, drying at 55~60℃ for 1d, drying at 70~80℃ for 1d, and drying at 100~120℃ for 2d.

[0023] Preferably, it includes: a high temperature-low temperature combined printing method; the high temperature-low temperature combined printing method includes the following steps: After 1,4-dioxane and biomaterial ink were mixed evenly in a volume ratio of 1-2:1, they were softened at 60-80°C and printed using a pneumatic extrusion nozzle with a pressure of 30-600 k / Pa, and dropped onto a printing platform at -20-0°C. The printing parameters were set as follows: printing layer height 0.30-0.40 mm; printing speed 10-30 mm / s; and the scaffold was printed by slicing the model using the Slic3r software to generate the printing path.

[0024] Preferably, the temperature of the softened biomaterial ink is 40-140°C or 60-80°C.

[0025] Preferably, the thermal cross-linking parameters are drying at 40° C. for 1 day, drying at 60° C. for 1 day, drying at 80° C. for 1 day, and drying at 120° C. for 2 days. At this time, the compressive strain improvement effect of the stent is optimal.

[0026] Preferably, the obtained scaffold is used as a cortical bone repair scaffold.

[0027] The material is used to obtain a scaffold through 3D printing, and it is effective when used for load-bearing bones (cortical bones) or cartilage tissue.

[0028] The beneficial effects of this application include: 1) The 3D printing biomaterial ink based on drug-release polycitrate material provided in this application adds nano-hydroxyapatite in different proportions according to the content of inorganic components in the target tissue to be repaired; at the same time, after obtaining the prototype of the scaffold, different temperature-time conditions are flexibly adjusted for thermal cross-linking treatment, so that the finished scaffold acquires mechanical properties and degradation capabilities that are adapted to the target defect area, thereby expanding the application range of the material and realizing 3D printing using polycitrate glycol ester and hydroxyapatite as printing materials.

[0029] 2) The 3D printing biomaterial ink printing method based on drug sustained-release polycitrate material provided in this application is based on the property that polycitrate can obtain different mechanical properties and degradation properties after being treated at different temperatures and times. The 3D printing process parameters are selected to adjust the cross-linked network structure of the polycitrate polymer, obtain biomaterial scaffolds with different mechanical properties and degradation properties, ensure stable and long-term release of the loaded drugs, and stable biological activity promotes osteogenesis and angiogenesis to match the rate of tissue regeneration and repair.

[0030] 3) The 3D printing biomaterial ink based on drug sustained-release polycitrate material provided in this application can be prepared by adding a solvent to the synthesized polycitrate material to obtain a biomaterial ink with stable properties, mild printing conditions, and excellent fine structure preparation ability. The raw materials of this material are easy to obtain, the cost is low, the reaction is simple, the biocompatibility and stability are good, it can be mass-produced in industry, and it is suitable for popularization and use.

[0031] 4) The 3D printing biomaterial ink printing method based on drug-released polycitrate materials provided in this application prepares a scaffold with a large number of interconnected channel structures with suitable pore sizes, high porosity and stable structure. According to the state of the medical polycitrate biomaterial ink modified with different active drugs, the extrusion process (including nozzle diameter and printing temperature) and the adaptation platform used in the 3D printing process are flexibly selected to achieve the printing of materials with different properties, and the structure, porosity, pore size, i.e., penetration and other properties of the scaffold are regulated by simple methods to meet the needs of tissue engineering and can be used to repair tissue damage.

[0032] 5) The thermal cross-linking method of 3D printed biomaterial ink based on drug-release polycitrate material and the thermal cross-linking post-treatment method of tissue engineering implants with polycitrate material as the main body provided in this application are simple and easy to implement, and the mechanical and degradation properties of the treated scaffolds can be linearly gradient-controlled; the mechanical properties and degradation ability of the scaffolds can match the needs of different tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A digital model diagram of a biomimetic bone structure 3D printed with the biomaterial ink obtained in Example 1 of the present application; Figure 2 A path map for generating a slice of a biomimetic bone structure 3D printed with the biomaterial ink obtained in Example 1 of the present application; Figure 3 This is a physical picture of the biomimetic bone structure 3D printed with the biomaterial ink obtained in Example 1 of the present application; Figure 4 This is a digital model of a nose 3D printed with biomaterial ink obtained in Example 2 of the present application; Figure 5 A path map is generated for the slice of the 3D printed nose of the biomaterial ink obtained in Example 2 of the present application; Figure 6 This is a physical picture of a nose 3D printed with biomaterial ink obtained in Example 2 of the present application; Figure 7 This is a physical picture of the biomimetic bone structure 3D printed with the biomaterial ink obtained in Example 4 of the present application; Figure 8 This is a physical picture of the 3D printed structure of the biomaterial ink obtained in Example 7 of the present application; Fig. 9 A three-dimensional cross-sectional view of a three-dimensional image reconstructed after Micro-CT scanning of a 3D printed biomimetic bone structure of the biomaterial ink obtained in Example 3 of the present application; Fig.10 It is a cross-sectional front view of a three-dimensional image reconstructed after Micro-CT scanning of the biomimetic bone structure 3D printed with the biomaterial ink obtained in Example 3 of the present application; Fig.11 This application adopts the actual photo of the nose obtained by comparative example 1 (PCL6500 material 3D printing); Fig.12 This is a photo of the meniscus obtained by comparative example 2 (3D printing with PCL6500 material) in this application; Fig.13 This is a compressive stress-compressive strain relationship diagram of the ink material printed stent obtained in Example 1 of the present application; Line 1 in the figure is a stent obtained by sequentially drying at 40°C for 1d, drying at 60°C for 1d, drying at 80°C for 1d, and drying at 120°C for 2d; Line 2 in the figure is a stent obtained by sequentially drying at 40°C for 1d, drying at 60°C for 1d, drying at 80°C for 1d, and drying at 120°C for 1d; Line 3 in the figure is a stent obtained by sequentially drying at 40°C for 1d, drying at 60°C for 1d, and drying at 80°C for 1d; Fig.14 Schematic diagram of the printing platform used in each embodiment of the present application; 1 in the figure is a printing gun; 2 is a frame; 3 is a printing platform; 4 is a mobile bracket; Fig.15 It is a diagram of the hydrogen nuclear magnetic resonance spectrum of the material after thermal cross-linking post-treatment. Compared with the polycitrate material without the addition of drugs, the material with the addition of gastrodin shows characteristic peaks of gastrodin at positions 6.8-7.3 and 4.3-4.8; in the figure, A is a hydrogen nuclear magnetic resonance spectrum, wherein line 1 is the medical polycitrate polymer obtained in Example 1, and line 3 is the scaffold material obtained in Example 1 of the present application; B is a local enlarged view of point B in A; C is a local enlarged view of point C in A; it indicates that the active ingredients in the scaffold printed by the method provided in the present application are detectable, indicating that the scaffold can effectively retain the active characteristics of the added substance. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0035] Example Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0036] Example 1: Printing at 40~140℃ 1. Preparation of biomaterial ink: 1) At 140°C, 8.0 g of 1,8-octanediol and 9.6 g of citric acid were melted and reacted for one hour to obtain oligomer polyol pre-POC (polycitrate prepolymer).

[0037] 2) Add 24 ml of 1,4-dioxane as a solvent to every 12 g of oligomer polyol pre-POC, the amount of solvent added is 200 wt.% of the total mass of the polycitrate prepolymer, add 3 g of L-lysine diisocyanate, react at 60 ° C for 1 h, then add 0.2 mL of stannous octoate as a catalyst, stir for 2 h, and the reaction is completed to obtain 15 g of medical polycitrate polymer.

[0038] 3) Add 28 g of nano-hydroxyapatite (particle size 60-80 nm) to the medical polycitrate polymer, where the added nano-hydroxyapatite accounts for 65% of the mass ratio of the biomaterial ink, stir for 1 hour, and after stirring and mixing evenly, obtain 43 g of medical polycitrate polymer-nano-hydroxyapatite composite.

[0039] 4) Gastrodin was added to the medical polycitrate polymer-nanohydroxyapatite composite, where the gastrodin accounted for 4% of the total mass, i.e., 1.72 g of gastrodin was added, and the mixture was reacted at 60° C. for 2 h. After the reaction was completed, the residual solvent was removed to obtain the biomaterial ink.

[0040] 2. The printing material used for printing is: no solvent is added, the active drug gastrodin is modified by high-temperature melt printing of bone repair biomaterials, and the obtained biomaterial ink is in an oligomer state.

[0041] High temperature melt extrusion printing: 1) Place the biomaterial ink into the printer barrel and soften it at 40-140°C; 2) The pressure of the pneumatic extrusion nozzle is set to 30~600k / Pa; 3) Set the model height to 10~15mm; 4) The model printing layer height is 0.30~0.40mm; 5) Printing speed is 1~10mm / s; 6) Use the model slicing software Slic3r to slice and generate the printing path according to the following Figure 1~2 The digital model structure size and layer height of the target object shown in the figure generate a printing path with 25 to 50 layers. The material is stacked by adjusting the extrusion pressure to obtain the target structure as shown in the figure. Figure 3 As shown. Figure 3 It can be seen that the printed bone bionic structure scaffold is easy to form and maintains a stable fine structure.

[0042] The above printing temperature can avoid drug inactivation and stent deformation caused by too high temperature, and can also avoid equipment blockage and printing interruption caused by too low printing temperature.

[0043] 3. Post-treatment method of thermal cross-linking: Figure 3 The scaffold prototype shown is placed in a vacuum oven and dried at 40°C for 1 day, 60°C for 1 day, 80°C for 1 day, and 120°C for 2 days for thermal cross-linking. This operation can effectively improve the mechanical properties of the resulting scaffold and can be used as a scaffold for cortical bone repair.

[0044] Since the mechanical properties of the scaffolds did not change significantly after drying at 40℃ for 1d and 60℃ for 1d, the compressive stress test was not performed on them. The compressive stress-compressive strain performance test of the scaffolds obtained by drying at 40℃ for 1d, 60℃ for 1d, and 80℃ for 1d was performed. The results are as follows: Fig.13 Line 3 in the figure is marked as the 80℃24h segment; The compressive stress-compressive strain performance of the scaffolds dried at 40℃ for 1d, 60℃ for 1d, 80℃ for 1d, and 120℃ for 24h was tested. The results are as follows: Fig.13 Line No. 2 in the figure is marked as the 80℃24h+120℃24h segment; The compressive stress-compressive strain performance of the scaffolds dried at 40℃ for 1d, 60℃ for 1d, 80℃ for 1d, and 120℃ for 48h was tested. The results are as follows: Fig.13 Line 1 in the figure is marked as the 120℃48h segment.

[0045] By comparison Fig.13 It can be seen from the trend of line 3 that the thermal cross-linking post-treatment provided by the present application can effectively improve the mechanical properties of the stent. The stents made of the same material are post-treated under different time-temperature conditions. As the temperature increases and the time increases, the compressive stress of the stent under the same compressive strain is significantly increased, and the mechanical properties are greatly enhanced.

[0046] 0.5 g of the scaffold material obtained after the thermal cross-linking post-treatment was scraped off with a scraper, dissolved in a deuterated DMSO solution, and then subjected to nuclear magnetic resonance detection. The specific detection method is the same as the existing method and will not be repeated here.

[0047] The obtained hydrogen spectrum is as follows Fig.15 As shown, compared with the medical polycitrate polymer without the addition of drugs, after adding gastrodin to the above-mentioned biological scaffold material, the nuclear magnetic resonance detection results showed that the characteristic peaks of gastrodin appeared at the positions of 6.8-7.3 and 4.3-4.8; indicating that the active ingredients in the scaffold printed by the method provided in the present application are detectable, indicating that the scaffold can effectively retain the active characteristics of the added substance.

[0048] Example 2 60-80°C printing The difference from Example 1 is: 1. Preparation of biomaterial ink Raw materials: In step 2), the solvent is N,N-dimethylformamide. In step 3), the stirring time is 4 hours; the remaining operations not described in detail are the same as those in Example 1.

[0049] 2. Printing process: The printing material used for printing is: adding 10wt.% solvent to the biomaterial obtained by the reaction, and fully stirring and mixing for 1h to obtain the active drug gastrodin modified bone repair high-temperature melt printing biomaterial, and the obtained biomaterial ink is in an oligomer state.

[0050] High temperature melt extrusion printing: 1) Soften the biomaterial ink at 60-80°C; 2) The pressure of the pneumatic extrusion nozzle is set to 30~600k / Pa; 3) Set the model height to 10~15mm; 4) The model printing layer height is 0.30~0.40mm; 5) Printing speed is 1~10mm / s; 6) Use the model slicing software Slic3r to slice and generate the printing path according to the following Figures 4 and 5 The digital model structure size and layer height of the target object shown in the figure generate a printing path with 25 to 50 layers. The material is stacked by adjusting the extrusion pressure to obtain the target structure as shown in the figure. Figure 6 As shown. Figure 6 It can be seen that the printed stent is formed and maintains a stable fine structure. The remaining operations not described in detail are the same as those in Example 1.

[0051] 3. Post-treatment method of thermal cross-linking: Figure 6 The scaffold shown was sequentially dried at 35°C for 1 day, 55°C for 1 day, 70°C for 1 day, and 100°C for 2 days for thermal cross-linking, and the remaining operations not described in detail were the same as those in Example 1.

[0052] Example 3 60-80°C printing The difference from Example 1 is: 1. Preparation of biomaterial ink Raw materials: In step 2), the solvent is N,N-dimethylformamide. In step 3), the stirring time is 4 hours; in step 4), 5% of the total weight of the raw materials, i.e., 2.15 g of Chinese herbal medicine extract Panax notoginseng saponin, is added; the rest of the operations not described in detail are the same as those in Example 1.

[0053] 2. Printing process: The printing material used for printing is: 10wt.% solvent is added to the biomaterial obtained by the reaction, and the active drug Panax notoginseng saponin modified bone repair high temperature melt printing biomaterial is obtained by fully stirring and mixing for 1h. The obtained biomaterial ink is in an oligomer state. High temperature melt extrusion printing: 1) Soften the biomaterial ink at 60-80°C; 2) The pressure of the pneumatic extrusion nozzle is set to 30~600k / Pa; 3) Set the model height to 10~15mm; 4) The model printing layer height is 0.30~0.40mm; 5) Printing speed is 1~10mm / s; 6) Use the model slicing software Slic3r to slice and generate a printing path, and the printed bracket is formed and maintains a stable fine structure. The remaining operations not described in detail are the same as those in Example 1.

[0054] 3. Post-treatment method of thermal cross-linking: The obtained scaffold was dried at 35°C for 1 day, 55°C for 1 day, 70°C for 1 day, and 100°C for 2 days in sequence for thermal cross-linking. The remaining operations not described in detail were the same as those in Example 1. The stereoscopic cross-sectional view of the three-dimensional image reconstructed after Micro-CT scanning of the obtained bionic bone structure is shown in FIG. Fig. 9As shown, the sectional front view is as follows Fig.10 shown by Figures 9-10 It can be seen that after printing with this material under this condition, the internal structure of the obtained bracket is clear and complete, and the details can be clearly displayed to obtain the required mechanical properties.

[0055] Example 4 Normal temperature printing The difference from Example 1 is: 1. Raw materials for preparation of biomaterial ink: nano-hydroxyapatite with a particle size of 80 nm; In step 2), the solvent is dimethyl sulfoxide. In step 3), the stirring time is 4 hours. In step 4), 0.001% of the total mass of the raw materials, i.e., 0.00043 g of recombinant wnt3a protein (recombinant mouse Wnt3a protein product purchased from abcom, product number ab81484) is added. The remaining operations not described in detail are the same as those in Example 1.

[0056] 2. Printing process: The printing material used for printing is: adding 30wt.% solvent to the biomaterial obtained by the reaction, stirring and mixing for 1 hour to obtain the recombinant wnt3a protein-modified bone repair room temperature melt printing biomaterial, and the obtained biomaterial ink is in an oligomer state.

[0057] Normal temperature melt extrusion printing: Step 1) The biomaterial ink is not heated to soften, and the printing speed is 1-30 mm / s; Step 6) Use the model slicing software Slic3r to slice and generate the printing path according to the following Figure 1~2 The digital model structure size and layer height of the target object shown in the figure generate a printing path with 25 to 50 layers. The material is stacked by adjusting the extrusion pressure to obtain the target structure as shown in the figure. Figure 7 As shown. Figure 7 It can be seen that the printed stent is formed and maintains a stable fine structure. The remaining operations not described in detail are the same as those in Example 1.

[0058] Normal and low temperature printing To protect the activity of the drug, high temperature post-treatment is no longer used.

[0059] Example 5 Low temperature printing The difference from Example 1 is: 1. Raw materials for preparing biomaterial ink: the amount of nano-hydroxyapatite added is 65% of the total mass of the obtained biomaterial ink; the particle size of nano-hydroxyapatite is 60nm; In step 2), the solvent is dimethylacetamide. In step 3), the stirring time is 4 hours. In step 4), 0.002% of the total mass of the raw materials, i.e., 0.00086 g of LL-37 peptide (human antimicrobial peptide) is added. The remaining operations not described in detail are the same as those in Example 1.

[0060] 2. Printing process: The printing material used for printing is: adding 50wt.% solvent to the biomaterial obtained by the reaction, stirring and mixing for 1h to obtain the recombinant wnt3a protein modified bone repair low-temperature melt printing biomaterial, and the obtained biomaterial ink is in an oligomer state. Low-temperature melt extrusion printing: Step 1) The biomaterial ink is not heated, the print head is cooled to 0-5°C, and the printing platform is cooled to -20-0°C; Step 5) The printing speed is 10~30mm / s; Step 6) Use the model slicing software Slic3r to slice and generate a printing path, and print the resulting bracket to form and maintain a stable fine structure. The remaining operations not described in detail are the same as those in Example 1.

[0061] Example 6 High temperature-low temperature combined printing method The difference from Example 1 is: 1. The preparation method of the biomaterial ink is the same as that in Example 1; 2. Printing process: When the modified polycitric acid material is extremely viscous, the biomaterial ink is difficult to extrude. When a large amount of solvent is added, the prepared biomaterial ink cannot be extruded at room temperature and needs to be printed under high temperature conditions. After plasticization, it falls on a low-temperature platform to retain its structure. At this time, it is necessary to use the shaping advantages of high-temperature printing and the structural maintenance advantages of low-temperature printing at the same time. In the above method, the high-temperature printing-low-temperature platform combined printing method is selected to obtain the possibility of material preparation.

[0062] Step 1) Since the biomaterial ink obtained in Example 1 has a high viscosity and cannot be extruded at room temperature, 1,4-dioxane (melting point of 9-11° C.) and the biomaterial ink obtained in Example 1 are mixed at a volume ratio of 1:1 and stirred evenly to be used as the biomaterial ink; the biomaterial ink is loaded into a printer barrel and passed into a printing gun 1 heated to 60-80° C., and the printing platform 2 arranged below the print head is frozen at a temperature of -20-0° C.; The 3D printing device used is Fig.14 The figure includes: a frame 2, a printing gun 1, a printing platform 3, and a movable bracket 4; the printing platform 3 and the printing gun 1 are installed on the movable bracket 4 and accommodated in the frame 2; the printing gun 1 is connected to the printer barrel (not shown) by a pipeline; the printing gun 1 and the printing platform 3 control relative movement according to a preset printing program to print. During printing, the ink ejected from the printing gun 1 falls on the printing platform 3 to form an image.

[0063] The 3D printing head and the printing platform are accommodated and arranged in the frame; 1) Soften the biomaterial ink at 60-80°C; 2) The pressure of the pneumatic extrusion nozzle is set to 30-600k / Pa; 3) Set the model height to 10-15mm; 4) Model printing layer height is 0.30-0.40mm; 5) Printing speed is 10-30mm / s; 6) According to Figure 1~2 The digital model structure and slice generation path of the target object shown are sliced ​​and printed using the model slicing software Slic3r. Using the above model size and layer height, the generated printing path has 25-50 layers. By adjusting the extrusion pressure, the material is stacked to obtain the target structure.

[0064] 3. Post-treatment method for thermal crosslinking: The obtained scaffold was dried at 45°C for 1 day, 65°C for 1 day, 85°C for 1 day, and 110°C for 2 days to perform thermal crosslinking. The remaining operations not described in detail were the same as those in Example 1.

[0065] Example 7 Printing of materials for repairing cartilage tissue damage 1. Preparation of biomaterial ink Raw materials: In step 3), the stirring time is 4 h, the proportion of hydroxyapatite is reduced to 5%, and the remaining operations not described in detail are the same as those in Example 1.

[0066] 2. Printing process: During printing, the solvent isopropanol was mixed with the biomaterial ink, and the added mass of the solvent was 50 wt.% of the mass of the biomaterial ink.

[0067] 1) Soften the biomaterial ink at 60-80°C; 2) The pressure of the pneumatic extrusion nozzle is set to 30-600k / Pa; 3) Set the model height to 10-15mm; 4) Model printing layer height is 0.30-0.40mm; 5) Printing speed is 10-30mm / s; 6) Use the model slicing software Slic3r to slice and generate the printing path, stack the materials in layers, and obtain the target structure.

[0068] 3. Post-treatment method of thermal cross-linking: Figure 8The scaffold shown was sequentially dried at 45°C for 1 day, 65°C for 1 day, 85°C for 1 day, and 110°C for 2 days for thermal crosslinking, and the remaining operations not described in detail were the same as those in Example 1.

[0069] Example 8 The difference from Example 1 is: 1. Preparation of biomaterial ink: in step 2), the solvent ratio in the polycitrate composite material is 50%, and the preparation method is the same as that in Example 1; in step 3), the stirring time is 4 h, and the amount of hydroxyapatite added is 5% of the total mass of the obtained biomaterial ink, i.e., 0.9 g; in step 4), the amount of gastrodin added is 12% of the total mass of the obtained biomaterial ink, i.e., 2.17 g; 2. Printing process: During printing, the solvent 1,4-dioxane was mixed with the biomaterial ink, and the added mass of the solvent was 10 wt.% of the mass of the biomaterial ink.

[0070] 1) Place the biomaterial ink into the printer barrel, cool the print head to -20-5°C, and cool the printing platform to -20-0°C; 2) The pressure of the pneumatic extrusion nozzle is set to 30-600k / Pa; 3) Set the model height to 10-15mm; 4) Model printing layer height is 0.30-0.40mm; 5) Printing speed is 10-30mm / s; It can be understood that the medical biomaterial ink with polycitrate material as the main body provided by the present invention has easy-to-obtain raw materials, low cost, simple synthesis method, and diverse additional functions; the biomaterial ink prepared using it uses polycitrate material in 3D printing for the first time, has good biocompatibility, and provides a new choice for digital bone tissue engineering.

[0071] The present invention provides a 3D printing method for medical biomaterial ink, which perfectly solves the defects of high viscosity and difficult printing of citrate-based materials. Through various technical means, it realizes the fine structure preparation of bioactive inks with various properties, and can adjust the pore structure, pore size and porosity of the scaffold through simple methods to meet the customization needs of bone tissue engineering implants for different parts.

[0072] Comparative Example 1 The difference from Example 2 is that the printing material used is PCL6500 material, and the obtained physical structure is as follows: Fig.11 As shown in the figure, it can be seen that the nose structure printed by this material is fuzzy, and there are a lot of dot-like protrusions on the surface, which affects the normal use of the stent in the human body.

[0073] Comparative Example 2 The difference from Example 2 is that the printing material used is PCL6500 material, and the obtained physical structure is as follows: Fig.12 As shown in the figure, compared with the structure obtained by the materials and methods provided by the present application, the meniscus cannot be printed to obtain local details, which affects the reliability of the combination of the bracket and the human body.

[0074] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A 3D printing biomaterial ink based on drug sustained-release polycitrate material, characterized in that: include: Medical polycitrate polymer, nano-hydroxyapatite, active pharmaceutical ingredient; the amount of nano-hydroxyapatite added is 5-65% of the total mass of the obtained biomaterial ink; The active pharmaceutical ingredient is any one of gastrodin, notoginseng saponin, recombinant wnt3a protein or LL-37 peptide; When the active drug used is gastrodin or notoginseng saponin, the added amount is 4-12% of the total mass of the obtained biomaterial ink; When the active drug used is recombinant wnt3a protein or LL-37 peptide, the added amount is 0-0.002% of the total mass of the obtained biomaterial ink; The particle size of nanohydroxyapatite is 60~80nm.

2. The 3D printing biomaterial ink according to claim 1, characterized in that: include: A solvent for adjusting the printing properties of biomaterial ink; the solvent is at least one of 1,4-dioxane, N,N-dimethylformamide, dimethylacetamide, isopropanol, and dimethyl sulfoxide; The amount of solvent added is 10~50wt.% of the total mass of the biomaterial ink.

3. A method for preparing a 3D printing biomaterial ink based on a drug sustained-release polycitrate material as claimed in claim 1 or 2, characterized in that: The following steps are involved: After the medical polycitrate polymer is obtained by reaction, the medical polycitrate polymer and nano-hydroxyapatite are mixed and stirred for 1 to 2 hours to obtain a medical polycitrate polymer-nano-hydroxyapatite composite; After the medical polycitrate polymer-nano hydroxyapatite composite and the active drug component are mixed or chemically grafted, the mixture is reacted for 2 hours under the condition that the added drug is active, thereby obtaining the biomaterial ink.

4. A 3D printing method for 3D printing biomaterial ink based on drug sustained-release polycitrate material as claimed in claim 1 or 2, characterized in that: The following steps are involved: The biomaterial ink is 3D printed at 0~140℃ to obtain the scaffold.

5. The 3D printing method according to claim 4, characterized in that: The biomaterial ink is softened at 60-140°C for melt extrusion printing, and the resulting scaffold is subjected to thermal cross-linking treatment; The thermal cross-linking parameters are drying at 35-40°C for 1 day, drying at 55-60°C for 1 day, drying at 70-80°C for 1 day, and drying at 100-120°C for 2 days; Melt extrusion printing includes the following steps: After softening the biomaterial ink, a pneumatic extrusion nozzle with a pressure of 30~600k / Pa was used for printing. The printing parameters were set as follows: model height 10~15mm; printing layer height 0.30~0.40mm; printing speed 1~10mm / s; and the stent was printed according to the model slicing software Slic3r to generate the printing path.

6. The 3D printing method according to claim 4, characterized in that: The method comprises the following steps: after 1,4-dioxane and biomaterial ink are uniformly mixed in a volume ratio of 1:1, the biomaterial ink is printed using a pneumatic extrusion nozzle with a pressure of 30-600 k / Pa without heating, and the printing parameters are set as follows: model height is 10-15 mm; printing layer height is 0.30-0.40 mm; printing speed is 1-30 mm / s; and a printing path is generated by slicing the model using Slic3r software to print the stent.

7. The 3D printing method according to claim 4, characterized in that: The method comprises the following steps: after dimethylacetamide and biomaterial ink are uniformly mixed at a volume ratio of 1 to 2:1, a pneumatic extrusion nozzle with a pressure of 30 to 600 k / Pa is used, and the nozzle temperature is set to 0 to 5°C, and the printing platform temperature is set to -20 to 0°C for printing, and the printing parameters are set as follows: model height is 10 to 15 mm; printing layer height is 0.30 to 0.40 mm; printing speed is 10 to 30 mm / s; a printing path is generated by slicing the model using Slic3r software to print a stent; and the obtained stent is subjected to thermal cross-linking treatment; The thermal cross-linking parameters are drying at 35~40℃ for 1d, drying at 55~60℃ for 1d, drying at 70~80℃ for 1d, and drying at 100~120℃ for 2d.

8. The 3D printing method according to claim 4, characterized in that: The method comprises the following steps: 1,4-dioxane and biomaterial ink are uniformly mixed in a volume ratio of 1-2:1, and then softened at 60-80°C, and then printed using a pneumatic extrusion nozzle with a pressure of 30-600 k / Pa, and the mixture is placed on a printing platform at -20-0°C, and the printing parameters are set as follows: a printing layer height of 0.30-0.40 mm; a printing speed of 10-30 mm / s; and a printing path generated by slicing the model using Slic3r software to print the stent.

9. The 3D printing method according to claim 4, characterized in that: The temperature of softening biomaterial ink is 40~140℃; The thermal cross-linking parameters are drying at 40°C for 1 day, drying at 60°C for 1 day, drying at 80°C for 1 day, and drying at 120°C for 2 days.

10. The 3D printing method according to claim 4, characterized in that: The obtained scaffold is used as a bone repair scaffold; the temperature of the softened biomaterial ink is 60-80°C.

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