A method for preparing poly-l-glutamic acid benzyl ester nanofiber bone scaffold by 3D printing one-step method

Abstract

The application discloses a method for preparing poly-L-glutamic acid benzyl ester nanofiber bone scaffolds by a 3D printing one-step method, relates to the technical field of 3D printing, and comprises the following steps: preparing poly-L-glutamic acid benzyl ester / nano-hydroxyapatite composite ink, wherein the ink has excellent formability and shear thinning characteristics, can realize the construction of a 3D printing scaffold with a spatial spiral structure, is suitable for an extrusion type 3D printing system, and can be used for the accumulation and building of a three-dimensional structure under the assistance of an ethanol bath, and the printed nanofiber bone scaffold has obvious nanofiber structure on the micro-morphology, nHA is uniformly dispersed and attached on the disordered nanofiber, has stable spatial morphology and structure, has the basic conditions for bone tissue repair and regeneration, and has potential application value.

Description

Technical Field

[0001] This invention relates to the field of 3D printing technology, specifically to a method for preparing poly-L-glutamic acid benzyl ester-based nanofiber bone scaffolds using a one-step 3D printing process. Background Technology

[0002] The bone matrix is ​​a mixture of collagen fibrils ranging from tens of nanometers to hundreds of micrometers, osteocytes, embedded minerals, etc. Nanoscale nHA crystals and collagen molecules are periodically deposited in the gaps between collagen fibers. This precise hierarchical structure gives bone excellent mechanical heterogeneity and mechanical strength.

[0003] Extrusion 3D printing technology is an advanced technique that allows for the customization of precisely shaped scaffolds to replace damaged or diseased tissues and organs, and it is widely used in the construction of bone tissue engineering scaffolds. Using polymer materials as substrates, extrusion 3D printing technology enables the construction of bone tissue engineering scaffolds with customized structural dimensions and porosity, excellent biocompatibility and biodegradability, and tissue-matched mechanical properties. Polymer nanofiber materials possess a structure highly similar to natural ECM. In vitro cell biology has shown that cells respond to their external environment, particularly exhibiting significant interactions at the nanoscale. Nanomorphology participates in regulating the initial cell adhesion process and ultimately determines cell fate through changes in cell biochemistry and cell morphology. Therefore, utilizing 3D printing technology to construct bone scaffolds with nanofiber biomimetic structures is of great significance.

[0004] Poly(L-glutamic acid benzyl ester) (PBLG) is a synthetic polypeptide with excellent biocompatibility and low immunogenicity. Its degradation product in vivo is L-glutamic acid, an essential amino acid. Simply put, PBLG can self-assemble into a nanofiber network structure through phase separation under specific conditions. This polymer nanofiber structure can mimic the microstructure of the natural ECM in bone tissue, promoting cell-cell interactions and making it a potentially ideal material for tissue engineering. Nevertheless, many challenges remain in the development of PBLG materials for 3D printing. First, the decomposition temperature of PBLG is close to its melting temperature, making it impossible to print the material in melt form, such as through traditional fused deposition modeling or casting. Second, the self-assembly of PBLG into unique nanofiber structures requires non-solvent-induced phase separation. Summary of the Invention

[0005] To address the problems of existing technologies, the present invention aims to overcome the shortcomings of existing technologies and provide a poly(L-glutamic acid benzyl ester) and its preparation method. The poly(L-glutamic acid benzyl ester) is applied to the preparation of an ink composed of poly(L-glutamic acid benzyl ester) / nanohydroxyapatite composite. The prepared ink is suitable for extrusion 3D printing systems and can be used for the deposition and construction of three-dimensional structures with the assistance of an ethanol bath.

[0006] One of the objectives of this invention is to provide a technical solution for preparing poly-L-glutamic acid benzyl ester, as detailed below: Under a protective atmosphere, 1,4-dioxane was injected into the poly-L-glutamic acid benzyl ester carboxylic anhydride monomer. After the poly-L-glutamic acid benzyl ester carboxylic anhydride monomer was completely dissolved, a first reaction solution was obtained. An initiator was injected into the first reaction solution, and after mixing evenly, the mixture was allowed to stand at room temperature until the reaction was completed, resulting in a second reaction solution. The second reaction solution was precipitated with diethyl ether, and the obtained product was filtered and vacuum dried until constant weight, yielding the polymer product poly-L-glutamic acid benzyl ester.

[0007] Preferably, the initiator is triethylamine, and the molar ratio of the initiator to the poly(L-glutamic acid benzyl ester carboxylic anhydride) monomer is 1:100.

[0008] Preferably, the protective atmosphere is nitrogen.

[0009] A second objective of this invention is to provide a poly(L-glutamic acid benzyl ester), which is prepared using the aforementioned method for preparing poly(L-glutamic acid benzyl ester), and the structural formula of the poly(L-glutamic acid benzyl ester) is as follows:

[0010] The third objective of this invention is to provide a technical solution for preparing an ink composed of poly(L-glutamic acid benzyl ester) / nano-hydroxyapatite composite, specifically as follows: Nano-hydroxyapatite was added to 1,4-dioxane to obtain a dispersion; the poly-L-glutamic acid benzyl ester was dissolved in the dispersion, and after a first stirring treatment, the poly-L-glutamic acid benzyl ester was completely dissolved to form a homogenized ink.

[0011] More preferably, the first stirring time is 24 hours.

[0012] The third objective of this invention is to provide an ink composed of poly(L-glutamic acid benzyl ester) / nanohydroxyapatite composite, prepared by the method described above, wherein the ink comprises poly(L-glutamic acid benzyl ester) and nanohydroxyapatite in a mass ratio of 1:0.4~1.

[0013] The fourth objective of this invention is to provide a technical solution for a one-step 3D printing method for preparing poly-L-glutamic acid benzyl ester-based nanofiber bone scaffolds using an ink composed of poly-L-glutamic acid benzyl ester / nano-hydroxyapatite composite, as detailed below: At room temperature, the ink composed of the poly(L-glutamic acid benzyl ester) / nanohydroxyapatite composite was transferred into a 3D printing device, and the nanofiber bone scaffold was 3D printed in the receiving phase environment of an organic solvent bath. During printing, the extrusion needle was completely immersed in the organic solvent bath. After printing, the nanofiber bone scaffold was fully soaked in an organic solvent liquid bath for 4 hours.

[0014] Preferably, the receiving phase environment of the organic solvent bath used in the printing process is an ethanol bath or an ethanol gel bath.

[0015] Preferably, the organic solvent liquid bath used for thorough soaking after printing is an ethanol bath.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention integrates 3D printing technology with nanofiber fabrication technology to prepare a biomimetic bone scaffold in one step. Using additive manufacturing technology, non-solvent-induced phase separation induces self-assembly to form a nanofiber structure. The prepared organic-inorganic composite ink is then used to construct a biomimetic bone tissue engineering scaffold with a nanofiber structure through extrusion 3D printing technology. This scaffold has good biocompatibility based on biomaterials and a three-dimensional nanofiber network similar to ECM collagen fiber bundles. The tunable mechanical properties and hydrophilic and hydrophobic properties indicate the potential application value of this scaffold in the field of bone tissue repair and regeneration.

[0017] This invention produces poly-L-glutamic acid benzyl ester by ring-opening polymerization of poly-L-glutamic acid benzyl ester carboxylic anhydride, and then prepares ink by combining poly-L-glutamic acid benzyl ester with nano-hydroxyapatite. The addition of nano-hydroxyapatite gives the ink excellent formability and shear-thinning properties, enabling the construction of 3D printing scaffolds with spatial helical structures. It is suitable for extrusion 3D printing systems and can be used for the deposition and assembly of three-dimensional structures with the assistance of an ethanol bath.

[0018] This invention uses an ethanol bath as the printing receiving phase for printing. Compared with direct printing, the printed structure has obvious interlayer stacking structure, and the structure is clear, stable, and without adhesion between structures. Using an ethanol bath to assist printing has obvious advantages, as it can help stabilize the shape of the extruder and the support.

[0019] The nanofiber bone scaffold prepared by this invention exhibits a distinct porous structure on a macroscopic scale, with the extruded strips arranged in a vertically intersecting pattern and possessing interconnected mesh-like channels. On a microscopic scale, the scaffold displays a clear nanofiber structure, and nHA is relatively uniformly dispersed and attached to the disordered nanofibers.

[0020] The nanofiber bone scaffold prepared by this invention can achieve the control of hydrophilicity / hydrophobicity and mechanical properties through process adjustment. Furthermore, the prepared nanofiber bone scaffold has a stable spatial morphology and structure. The nanofiber structure greatly increases the specific surface area, which can promote osteoblast migration and provides the basic conditions for bone tissue repair and regeneration, thus possessing potential application value. Attached Figure Description

[0021] Figure 1 The image shown is a spectral representation of the PBLG prepared in the embodiments of this invention. Figure 1 (A) is the NMR spectrum. Figure 1 (B) is the infrared spectrum; Figure 2 These are SEM and EDS images of the nanofiber bone scaffold prepared in Example 1 of this invention after drying. Figure 3 This is an image of the scaffold prepared in Example 1 of this invention after drying, wherein... Figure 3 (A) is a macroscopic photograph, Figure 3 (B) to (D) are SEM images under three different scales, corresponding to 100 μm, 10 μm and 5 μm respectively; Figure 4 This is a SEM image of the scaffold prepared in Comparative Example 2 of this invention after drying, wherein, Figure 4 (A) to (C) are SEM images under three different scales, corresponding to 100 μm, 10 μm and 5 μm respectively; Figure 5 The graphs shown are of the water contact angles tested after the scaffolds prepared in Examples 1 to 3 of this invention were dried. Figure 5 (A) is a data graph of the water contact angles corresponding to the samples of the four embodiments. Figure 5 (B) Images of the samples from the four embodiments when testing the water contact angle; Figure 6 These are data graphs obtained from compression tests performed on the stents prepared in Examples 1 to 3 of this invention after drying. Figure 6 (A) shows the compressive stress-strain diagrams of the four sample examples; Figure 6 (B) is a graph showing the elastic modulus data of the four sample examples; Figure 7The diagram shows the shear thinning properties of the PBLG / nHA inks prepared in Examples 1, 2 and Comparative Example 1 of this invention. Figure 8 These are morphological images of the PBLG / nHA inks prepared in Comparative Example 1, Example 1, and Example 2 of this invention during extrusion into the extrusion needle during 3D printing. Figure 8 (A), (F), (K)), Morphological diagrams of direct extrusion patterns of PBLG / nHA ink ( Figure 8 (B), (G), (L)), Morphological diagrams of PBLG / nHA inks extruded in an ethanol bath. Figure 8 (C), (H), (M)), Morphological images of PBLG / nHA inks printed in an ethanol bath after drying. Figure 8 (D), (I), (N)), and Figure 8 Enlarged views of the corresponding parts within the dashed boxes in (D), (I), and (K). Figure 8 (E), (J), (O)), where, Figure 8 (A) to (E) correspond to the following ratio 1. Figure 8 (F) to (J) correspond to Example 1. Figure 8 (K) to Figure 8 (O) Corresponds to Example 2; Figure 9 These are macroscopic and microscopic images of the spiral extruded strip prepared in Example 4 of the present invention, wherein... Figure 9 (A) is a macroscopic image. Figure 9 (B) is a SEM image with a scale bar of 30 μm. Figure 9 (C) is Figure 9 (B) is an enlarged image of the area within the dashed box. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] To facilitate understanding, the English terms mentioned below will be explained first: BLG-NCA: Poly-L-glutamic acid benzyl ester carboxylic anhydride; TEA: Triethylamine; nHA: Nano-hydroxyapatite.

[0024] Example 1: A method for one-step 3D printing of poly-L-glutamic acid benzyl ester-based nanofiber bone scaffolds includes the following three steps, S1 to S3: S1. Preparation of high molecular weight poly-L-glutamic acid benzyl ester; S2. Preparation of ink composed of poly(L-glutamic acid benzyl ester) / nanohydroxyapatite composite; S3. Nanofiber bone scaffolds were prepared using 3D printing.

[0025] Step S1 specifically involves: S11. Take 1 g of BLG-NCA monomer into a round-bottom flask, and under a protective atmosphere, inject 15 ml of 1,4-dioxane. After the BLG-NCA monomer is completely dissolved, the first reaction solution is obtained. S12. TEA with a molar ratio of 100:1 to BLG-NCA monomer is injected into the first reaction solution as an initiator. After mixing evenly, the mixture is allowed to stand at room temperature for 3 days. After the reaction is completed, the second reaction solution is obtained. S13. The second reaction solution was precipitated with 4 L of diethyl ether. The resulting product was filtered and vacuum dried until constant weight. The resulting polymer product, poly(L-glutamic acid benzyl ester), was labeled as PBLG.

[0026] Step S2 is as follows: S21. Take 0.3 g of nHA and add it to 10 ml of 1,4-dioxane to obtain a dispersion. Then, use stirring and sonication to make the nHA evenly dispersed in the dispersion. S22. Dissolve 0.5 g of PBLG in the dispersion. After the first stirring treatment, the PBLG is completely dissolved, forming a homogenized ink, namely PBLG / nHA ink, which appears as a viscous "plaster-like" liquid, denoted as S22. .

[0027] The S3 steps are as follows: S31. At room temperature, transfer PBLG / nHA ink into a cartridge and place the bath containing organic solvent (i.e., organic solvent bath) in the print receiving platform. S32. Select an extrusion needle with an inner diameter of 0.51 mm, set the horizontal and vertical spacing of the XY axis plane to 500 μm, and 300 μm to be the lift value on the Z axis when changing layers. Print the nanofiber bone scaffold with a pneumatic pressure of 30 kPa and a printing speed of 200 mm / min. During printing, the extrusion needle is completely immersed in the organic solvent bath, and the wiring is printed in the organic solvent bath according to the program settings. S33. After printing, the nanofiber bone scaffold is fully immersed in an organic solvent bath for 4 hours to ensure the stability of the nanofiber bone scaffold structure before the formation of the nanofiber structure.

[0028] In this embodiment, the organic solvent is ethanol, that is, an ethanol bath is used to assist in the 3D printing of PBLG / nHA nanofiber bone scaffold.

[0029] In this embodiment, nitrogen is used as the protective atmosphere in step S11.

[0030] To minimize the oxygen content in the gas above the BLG-NCA monomer, a process of repeatedly purging nitrogen through a vacuum was employed three times.

[0031] In this embodiment, the purity of nHA is greater than 97% and the particle diameter is less than 100 nm.

[0032] In this embodiment, in step S21, the mixture is stirred for 1 hour and sonicated for 15 minutes.

[0033] In this embodiment, the first stirring time is 24 hours.

[0034] In this embodiment, after the ink is prepared in step S22, in order to prevent the solvent in the ink from evaporating, the homogenized ink should be used as soon as possible after being exposed.

[0035] Example 2: The steps in this embodiment are basically the same as those in embodiment 1, with the following differences: Step S22 is replaced by: adding 0.5 g of nHA to the polymer solution, followed by a second stirring process to form a homogenized ink, namely PBLG / nHA ink, denoted as... .

[0036] Example 3: The steps in this embodiment are basically the same as those in embodiment 1, with the following differences: Step S21 is replaced by: dissolving 0.7 g of PBLG in 10 ml of 1,4-dioxane, and stirring for the first time to ensure complete dissolution of the polymer, thus obtaining a polymer solution; the PBLG / nHA ink obtained after step S22 is denoted as... .

[0037] Example 4: The steps in this embodiment are basically the same as those in embodiment 1, with the following differences: The organic solvent used was replaced with ethanol gel, that is, ethanol gel bath was used to assist in the 3D printing of PBLG / nHA nanofiber bone scaffold. In step S33, after printing, the scaffold and ethanol gel are immersed together in an ethanol bath for 4 hours to remove the ethanol gel and obtain a complete nanofiber bone scaffold.

[0038] Comparative Example 1: The steps in this comparative example are basically the same as those in Example 1, except that: Step S22 is replaced by adding nHA to the polymer solution without adding it, which means that after step S11, the PBLG / nHA ink is obtained, denoted as... .

[0039] Comparative Example 2: The steps in this comparative example are basically the same as those in Example 1, except that: The organic solvent used was replaced with diethyl ether, that is, an ether bath was used to assist in the 3D printing of PBLG / nHA nanofiber bone scaffolds.

[0040] The samples prepared in each embodiment and comparative example were tested, and the details are as follows: like Figure 1 As shown, the high molecular weight poly(L-glutamic acid benzyl ester) PBLG prepared in step S1 was subjected to NMR (nuclear magnetic resonance imaging). Figure 1 (A) and infrared spectrum ( Figure 1 The test (B) showed that the structural formula of poly(L-glutamic acid benzyl ester) PBLG is:

[0041] After testing, PBLG's weight-average molecular weight is approximately 25 × 10⁻⁶. 4 The viscosity-average molecular weight is approximately 18 × 10⁻⁶. 4 The molecular weight distribution is approximately 1.742.

[0042] The nanofiber bone scaffold (also referred to as the scaffold) printed after step S3 exhibits noticeable shrinkage after drying. This is due to the loss of a small amount of polymer PBLG as the solvent evaporates and detaches from the scaffold. Furthermore, the dried scaffold can be handled and transferred with tweezers, demonstrating good device stability. Figure 2 As shown, the SEM image of the scaffold prepared in Example 1 after drying ( Figure 2 (A)), the scaffold has a regular porous structure, and the extruded strips are vertically interlaced. The diameter after shrinkage is measured to be approximately 350~400 nm, which is 68~80% of the extrusion diameter of the extrusion pillow. The corresponding EDS image ( Figure 2 (B) and Figure 2 (C) shows that Ca / P elements are uniformly distributed on the scaffold, and nHA is uniformly dispersed and attached throughout the scaffold. Meanwhile, in Figure 2In (A), the multi-layered stacked structure can be clearly observed, indicating that the support has good support performance and will not collapse after printing; each layer of the support is a porous structure formed by perpendicular intersections in the X and Y directions, and there are also obvious pores in the Z-axis direction; there is also a certain degree of fusion between the extrusion strips, indicating that the multi-layered structure is not simply stacked, but has a certain healing and bonding effect between layers, making the whole structure more stable.

[0043] Figure 3 The images show macroscopic photographs and SEM images of the scaffold prepared in Example 1 after drying. The macroscopic photographs show that scaffolds of various shapes and specifications can be 3D printed according to actual needs, and the structure is stable. The SEM images show that Example 1 has a distinct nanofiber microstructure.

[0044] Figure 4 The SEM image of the scaffold prepared in Comparative Example 2 after drying shows that no nanofiber structure was generated in Comparative Example 2.

[0045] Figure 5 The results show the contact angle test results of the scaffolds prepared in Examples 1 to 3 after drying. The results indicate that the water contact angle of Example 1 is approximately 70.90°~72.45°, the water contact angle of Comparative Example 2 is approximately 81.14°~81.28°, the water contact angle of Example 2 is approximately 62.04°~64.38°, and the water contact angle of Example 3 is approximately 88.20°~88.26°. With higher nHA content, the water contact angle on the material surface decreases, and the improvement in the hydrophilicity of the scaffold is greater, effectively improving the hydrophobicity of the scaffold and facilitating the adhesion and growth of bone tissue cells on the scaffold material surface.

[0046] Figure 6 The graphs show the mechanical properties obtained from compression tests in Examples 1 to 3. Figure 6 (A) is the compressive stress-strain diagram. Figure 6 (B) is based on Figure 6 (A) The calculated elastic modulus diagram shows that Examples 1, 2 and 3 all have a compression modulus of more than 10 MPa, which can meet the needs of bone tissue transplantation materials in most human weight-bearing areas.

[0047] Figure 7 The graph shows the shear thinning properties of the PBLG / nHA inks prepared in Examples 1, 3 and Comparative Example 1. It can be seen that as the shear rate increases, the ink viscosity gradually decreases, which can reduce the chance of clogging during printing. From the three curves in the graph, it can be seen that the PBLG / nHA ink prepared in Example 2 has better shear thinning properties.

[0048] Figure 8This image shows a comparative test of the extrusion performance of PBLG / nHA inks. The morphology of the ink as it is extruded from the extrusion needle was observed, and the stability of the printed pattern was observed after drying. The printability and microstructure of the inks were investigated. Inks with different components can all achieve continuous and uniform extrusion in ethanol. Figure 8 As shown in (A) to (E), the P5H0 ink without added nHA in Comparative Example 1 is a viscous fluid when directly printed from the extrusion needle. It has insufficient formability and cannot form a three-dimensional structure after extrusion. With the assistance of an ethanol bath, the basic prototype of the network support set by the printing program can be obtained, but there is obvious adhesion between the meshes and no clear stacking boundary required for 3D printing between layers. Figure 8 As shown in (F) to (L), both inks containing nHA in Examples 1 and 2 were able to form shapes after being extruded from the extrusion needle. After drying, the patterns extruded in the ethanol bath showed a grid framework that was basically consistent with the program design, and the framework morphology remained intact with no adhesion film between the grids and no obvious collapse. P5H5 exhibited better formability and maintained its extruded shape more stably than P5H3. Stereo microscopy revealed a clear interlayer stacking structure, which is the basis for interlayer printing, and this effect became even more pronounced with increasing nHA content. Compared to direct printing inks, using an ethanol bath for assisted printing has significant advantages, as it helps stabilize the morphology of the extruded strip and the framework.

[0049] Figure 9 In Example 4, the extrusion needle extrudes PBLG / nHA ink into an ethanol gel bath to form an extrusion strip. After soaking in an ethanol solution to remove the ethanol gel, the extrusion strip was found to be helical, exhibiting good morphology and structure, enabling the construction of a 3D printed scaffold with a spatial helical structure. SEM images of the dried scaffold prepared in Example 4 show that the scaffold has a distinct nanofiber microstructure.

[0050] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended embodiments and their equivalents.

Claims

1. A method for preparing an ink composed of poly(L-glutamic acid benzyl ester) / nano-hydroxyapatite composite, characterized in that, Nano-hydroxyapatite was added to 1,4-dioxane to obtain a dispersion. The poly(L-glutamic acid benzyl ester) was dissolved in the dispersion, and after a first stirring treatment, the poly(L-glutamic acid benzyl ester) was completely dissolved to form a homogenized ink. The mass ratio of poly(L-glutamic acid benzyl ester) to nano-hydroxyapatite was 1:0.4~1. The structural formula of the poly(L-glutamic acid benzyl ester) is as follows: ； The ink has shear-thinning properties, making it suitable for extrusion 3D printing. It can form nanofiber structures in situ through non-solvent phase separation in an ethanol bath or ethanol gel bath.

2. The method for preparing ink according to claim 1, characterized in that, The first stirring time is 24 hours.

3. The method for preparing ink according to claim 1, characterized in that, The preparation method of the poly-L-glutamic acid benzyl ester is as follows: under a protective atmosphere, 1,4-dioxane is injected into the poly-L-glutamic acid benzyl ester carboxylic anhydride monomer. After the poly-L-glutamic acid benzyl ester carboxylic anhydride monomer is completely dissolved, a first reaction solution is obtained; an initiator is injected into the first reaction solution, and after mixing evenly, the mixture is allowed to stand at room temperature until the reaction is completed, resulting in a second reaction solution; the second reaction solution is precipitated with diethyl ether, and the obtained product is filtered and vacuum dried until constant weight, yielding the polymer product poly-L-glutamic acid benzyl ester.

4. The method for preparing ink according to claim 3, characterized in that, The initiator is triethylamine, and the molar ratio of the initiator to the poly(L-glutamic acid benzyl ester carboxylic anhydride) monomer is 1:

100.

5. The method for preparing ink according to claim 3, characterized in that, The protective atmosphere is nitrogen.

6. The ink composed of poly(L-glutamic acid benzyl ester) / nano-hydroxyapatite composite obtained by the preparation method according to any one of claims 1 to 5, characterized in that, The ink composed of the poly(L-glutamic acid benzyl ester) / nanohydroxyapatite composite comprises poly(L-glutamic acid benzyl ester) and nanohydroxyapatite in a mass ratio of 1:0.4~1.

7. A method for preparing poly-L-glutamic acid benzyl ester-based nanofiber bone scaffolds by one-step 3D printing using the ink as described in claim 6, characterized in that, At room temperature, ink composed of poly(L-glutamic acid benzyl ester) / nanohydroxyapatite composite was transferred into a 3D printing device, and 3D printing of nanofiber bone scaffolds was carried out in the receiving phase environment of an organic solvent bath. During printing, the extrusion needle was completely immersed in the organic solvent bath. After printing, the nanofiber bone scaffold was fully soaked in an organic solvent liquid bath for 4 hours.

8. The method for preparing poly-L-glutamic acid benzyl ester-based nanofiber bone scaffolds by one-step 3D printing according to claim 7, characterized in that, The receiving phase environment of the organic solvent bath used in the printing process is an ethanol bath or an ethanol gel bath.

9. The method for preparing poly-L-glutamic acid benzyl ester-based nanofiber bone scaffolds by one-step 3D printing according to claim 7, characterized in that, The organic solvent bath used for thorough soaking after printing is an ethanol bath.

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