CN117106175A

CN117106175A - Method for preparing poly-L-benzyl glutamate based nanofiber bone scaffold by 3D printing one-step method

Info

Publication number: CN117106175A
Application number: CN202310682006.0A
Authority: CN
Inventors: 张坤玺; 尹静波; 朱渊杰; 朱轶男; 李贵飞
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2023-08-17
Filing date: 2023-08-17
Publication date: 2023-11-24

Abstract

The invention discloses a method for preparing a poly-L-benzyl glutamate based nanofiber bone scaffold by a 3D printing one-step method, which relates to the technical field of 3D printing, and the ink is prepared by compounding poly-L-benzyl glutamate/nano hydroxyapatite, has excellent formability and shear thinning characteristics, can realize the construction of a 3D printing scaffold with a space spiral structure, is suitable for an extrusion type 3D printing system, can be used for stacking and building a three-dimensional structure under the assistance of an ethanol bath, and has obvious nanofiber structure on microscopic morphology, nHA is uniformly dispersed and attached on unordered nanofiber, has stable space morphology and structure, has basic conditions for repairing and regenerating bone tissues, and has potential application value.

Description

Method for preparing poly-L-benzyl glutamate based nanofiber bone scaffold by 3D printing one-step method

Technical Field

The invention relates to the technical field of 3D printing, in particular to a method for preparing a poly-L-benzyl glutamate nanofiber bone scaffold by a 3D printing one-step method.

Background

The bone matrix is mixed with fibril collagen, bone cells, embedded minerals and the like of tens of nanometers to hundreds of micrometers, nano-scale nHA crystals and collagen molecules are periodically deposited in gaps of the collagen fibers, and the precise hierarchical structure enables the bone to have excellent mechanical heterogeneity and mechanical strength.

Extrusion 3D printing techniques, which are advanced techniques that can customize precise geometry scaffolds and be used to replace damaged or diseased tissues and organs, are widely used in the construction of scaffolds for bone tissue engineering. The bone tissue engineering scaffold takes a polymer material as a base material, can be realized by adopting an extrusion type 3D printing technology, and has the advantages of customized structure size and pores, excellent biocompatibility and biodegradability and mechanical property matched with tissues. The polymeric nanofiber material has a structure that is highly similar to the native ECM. In vitro cell biology has shown that cells react to the external environment, particularly with obvious interactions at the nanoscale, nanotopography can be involved in regulating the initial adhesion process of cells, and ultimately cell fate is determined by changes in cell biochemistry and cell morphology. Therefore, the construction of the bone scaffold with the nanofiber bionic structure by using the 3D printing technology has important significance.

Poly-L-benzyl glutamate (PBLG), an artificially synthesized polypeptide, has excellent biocompatibility and low immunogenicity, and the degradation product in vivo is L-glutamic acid, which is an amino acid essential for human body. In short, PBLG is capable of self-assembly to form a nanofiber network structure through phase separation under specific conditions. The polymer nanofiber structure can simulate the microstructure of natural bone tissue ECM, promote cell interaction, and is a potential ideal material for tissue engineering. Nevertheless, there are still many challenges in developing PBLG materials for 3D printing. First, the PBLG has a decomposition temperature close to its melting temperature, and thus, the material cannot be printed in the form of a melt, such as in conventional melt printing, melt casting, and the like. Second, PBLG formation of unique nanofiber structures by self-assembly requires non-solvent induced phase separation to achieve.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to overcome the defects in the prior art and provide the poly-L-benzyl glutamate and the preparation method thereof, wherein the poly-L-benzyl glutamate is applied to the preparation of ink composed of poly-L-benzyl glutamate/nano hydroxyapatite, and the prepared ink is suitable for an extrusion type 3D printing system and can be used for stacking and building a three-dimensional structure under the assistance of an ethanol bath.

The invention provides a technical scheme of a preparation method of poly-L-benzyl glutamate, which comprises the following steps:

under a protective atmosphere, 1, 4-dioxane is injected into a poly-L-benzyl glutamate carboxylic anhydride monomer, and a first reaction solution is obtained after the poly-L-benzyl glutamate carboxylic anhydride monomer is completely dissolved; injecting an initiator into the first reaction liquid, uniformly mixing, and standing at room temperature until the reaction is completed to obtain a second reaction liquid; and (3) settling the second reaction solution by diethyl ether, filtering the obtained product, and drying in vacuum until the weight is constant to obtain a polymer product poly-benzyl L-glutamate.

Preferably, the initiator is triethylamine, and the molar ratio of the initiator to the poly-L-benzyl glutamate carboxylic anhydride monomer is 100:1.

Preferably, the protective atmosphere is nitrogen.

The second object of the invention is to provide poly-L-benzyl glutamate, which is prepared by adopting the preparation method of the poly-L-benzyl glutamate, wherein the poly-L-benzyl glutamate has the following structural formula:

the invention provides a technical scheme for using the poly-L-benzyl glutamate to the preparation method of the ink composed of the poly-L-benzyl glutamate/nano hydroxyapatite composite, which comprises the following steps:

adding nano hydroxyapatite into 1, 4-dioxane to obtain a dispersion liquid; and dissolving the poly-benzyl L-glutamate in the dispersion liquid, and completely dissolving the poly-benzyl L-glutamate through first stirring treatment to form homogenized ink.

Further preferably, the first stirring time is 24 hours.

The invention provides an ink composed of poly-L-benzyl glutamate/nano-hydroxyapatite, which is prepared by adopting the preparation method of the ink composed of poly-L-benzyl glutamate/nano-hydroxyapatite, and comprises the following components: poly L-benzyl glutamate and nano hydroxyapatite with the mass ratio of 1:0.4-1.

The invention provides a technical scheme of a method for preparing a poly-L-benzyl glutamate-based nanofiber bone scaffold by using an ink composed of poly-L-benzyl glutamate/nano hydroxyapatite in a 3D printing one-step method, which comprises the following steps:

transferring the ink formed by compounding the poly-L-benzyl glutamate and the nano-hydroxyapatite into 3D printing equipment in a room temperature environment, and performing 3D printing of the nano-fiber bone scaffold in a receiving phase environment of an organic solvent bath, wherein an extrusion needle head is completely immersed in the organic solvent bath during printing; after printing, the nanofiber bone scaffold is fully soaked in an organic solvent liquid bath for 4 hours.

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

Preferably, the organic solvent liquid bath for sufficient soaking after printing is an ethanol bath.

Compared with the prior art, the invention has the beneficial effects that:

the invention integrates a 3D printing technology and a nanofiber preparation technology, prepares the bionic bone scaffold in one step, takes an additive manufacturing technology as a means, and adopts nonsolvent induced phase separation to induce self-assembly to form a nanofiber structure, and the prepared organic-inorganic composite ink realizes the construction of the bionic bone tissue engineering scaffold with the nanofiber structure through an extrusion type 3D printing technology.

According to the invention, poly-L-benzyl glutamate is prepared from poly-L-benzyl glutamate carboxylic anhydride through ring-opening polymerization, and the poly-L-benzyl glutamate and nano hydroxyapatite are compounded to prepare the ink, and the ink has excellent formability and shear thinning characteristics through the addition of the nano hydroxyapatite, so that the construction of a 3D printing support with a space spiral structure can be realized, and the ink is suitable for an extrusion type 3D printing system and can be used for stacking and building a three-dimensional structure under the assistance of an ethanol bath.

Compared with direct printing, the structure printed by using the ethanol bath as a printing receiving phase has an obvious interlayer stacking structure, is clear and stable in structure, has no adhesion between the structures, has obvious superiority when being used for assisting in printing by using the ethanol bath, and can help to stabilize the shapes of extruded strips and brackets.

The nanofiber bone scaffold prepared by the invention has an obvious porous structure in a macroscopic sense, and extruded strips are vertically staggered and provided with mutually penetrated latticed channels. In microscopic morphology, the scaffold had a distinct nanofiber structure, and nHA was more uniformly dispersed and attached to the disordered nanofibers.

The nanofiber bone scaffold prepared by the invention can realize the adjustment and control of the hydrophilicity and mechanical property of the nanofiber bone scaffold through the adjustment of the process, has stable space morphology and structure, greatly improves the specific surface area of the nanofiber structure, can promote osteoblast migration, has the basic conditions for repairing and regenerating bone tissues, and has potential application value.

Drawings

FIG. 1 is a graph of a PBLG prepared in an embodiment of the present invention, wherein FIG. 1 (A) is a nuclear magnetic spectrum and FIG. 1 (B) is an infrared spectrum;

FIG. 2 is an SEM image and an EDS image of the nanofiber bone scaffold prepared in example 1 according to the present invention after drying;

FIG. 3 is a dried image of the stent prepared in example 1 of the present invention, wherein FIG. 3 (A) is a macroscopic photograph, and FIGS. 3 (B) to (D) are SEM images under three different scales, respectively, the scales corresponding to 100 μm, 10 μm and 5 μm, respectively;

FIG. 4 is a SEM image after drying of the stent prepared in comparative example 2 according to the present invention, wherein FIGS. 4 (A) to (C) are SEM images under three different scales, respectively, the scales corresponding to 100 μm, 10 μm and 5 μm, respectively;

fig. 5 is a graph showing the water contact angle measured after the scaffolds prepared in examples 1 to 3 were dried, wherein fig. 5 (a) is a data graph showing the water contact angle corresponding to the water contact angle of the samples of the four examples, and fig. 5 (B) is an image of the water contact angle measured for the samples of the four examples;

fig. 6 is a graph showing data obtained by performing compression test on the scaffolds prepared in examples 1 to 3 according to the present invention after drying, wherein fig. 6 (a) is a graph showing compressive stress-strain of four examples; FIG. 6 (B) is a graph of elastic modulus data for four example samples;

FIG. 7 is a graph showing the shear thinning performance of the PBLG/nHA inks prepared in example 1, example 2 and comparative example 1 according to the present invention;

fig. 8 is a diagram showing the morphology of the PBLG/nHA ink prepared in comparative example 1, example 1 and example 2 when extruded in an extrusion needle at the time of 3D printing (fig. 8 (a), (F), (K)), the morphology of the PBLG/nHA ink directly extruded pattern (fig. 8 (B), (G), (L)), the morphology of the PBLG/nHA ink when extruded in an ethanol bath (fig. 8 (C), (H), (M)), the morphology of the PBLG/nHA ink after dried pattern printed in an ethanol bath (fig. 8 (D), (I), (N)), and the enlarged diagram of the corresponding part in the dotted line box in fig. 8 (D), (I), (K) (fig. 8 (E), (J), (O)), wherein fig. 8 (a) to (E) correspond to comparative example 1, fig. 8 (F) to (J) correspond to example 1, fig. 8 (K) to 8 (O) correspond to example 2;

fig. 9 is a macroscopic and microscopic observation image of the spiral-shaped extruded strip prepared in example 4 of the present invention, wherein fig. 9 (a) is a macroscopic image, fig. 9 (B) is an SEM image of 30 μm in scale, and fig. 9 (C) is an enlarged image of a dotted line square portion in fig. 9 (B).

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

For ease of understanding, the english nouns mentioned below are first explained:

BLG-NCA: poly-benzyl L-glutamate carboxylic anhydride;

TEA: triethylamine;

nHA: nano hydroxyapatite.

Example 1:

a method for preparing a poly-L-benzyl glutamate nanofiber bone scaffold by a 3D printing one-step method comprises the following three steps of S1 to S3:

s1, preparing high molecular weight poly-L-benzyl glutamate;

s2, preparing ink composed of poly-L-benzyl glutamate/nano hydroxyapatite composite;

s3, preparing the nanofiber bone scaffold by adopting a 3D printing method.

Wherein the step S1 specifically comprises the following steps:

s11, taking 1g of BLG-NCA monomer, filling 15ml of 1, 4-dioxane into a round bottom flask under a protective atmosphere, and obtaining a first reaction solution after the BLG-NCA monomer is completely dissolved;

s12, injecting TEA with the molar ratio of 100:1 with BLG-NCA monomer as an initiator into the first reaction liquid, uniformly mixing, standing for 3d at room temperature, and obtaining a second reaction liquid after the reaction is completed;

s13, settling the second reaction solution by using 4L diethyl ether, filtering the obtained product, and drying in vacuum until the weight is constant to obtain a polymer product poly-benzyl L-glutamate, namely PBLG.

The step S2 specifically comprises the following steps:

s21, adding 0.3g of nHA into 10ml of 1, 4-dioxane to obtain a dispersion liquid, and uniformly dispersing the nHA in the dispersion liquid in a stirring and ultrasonic mode;

s22, 0.5g of PBLG is dissolved in the dispersion liquid, and the PBLG is completely dissolved by first stirring treatment to form homogenized ink, namely PBLG/nHA ink, which is shown as viscous 'Dan Gaozhuang' liquid and is marked as P5H3.

The step S3 specifically comprises the following steps:

s31, transferring PBLG/nHA ink into a charging barrel in a room temperature environment, and placing a bath pool (namely, an organic solvent bath) filled with an organic solvent in a printing receiving platform;

s32, selecting an extrusion needle with the inner diameter of 0.51mm, setting the transverse-longitudinal distance of an X-Y axis plane to be 500 mu m, setting 300 mu m as a lifting value on a Z axis when changing layers, printing the nanofiber bone scaffold at the printing speed of 200mm/min under 30KPa pneumatic pressure, completely immersing the extrusion needle in an organic solvent bath during printing, and setting the extrusion needle in the organic solvent bath according to a program for carrying out line printing;

and S33, after printing, fully soaking the nanofiber bone scaffold in an organic solvent bath for 4 hours to ensure the formation of a nanofiber structure after the stability of the structure of the nanofiber bone scaffold.

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

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

In order to try to maintain the oxygen content in the gas above the BLG-NCA monomer, the method of pumping vacuum and introducing nitrogen is adopted for three times.

In this example, nHA had a purity of greater than 97% and a particle diameter of less than 100nm.

In this example, in step S21, stirring is performed for 1h and ultrasound is performed for 15min.

In this example, the first stirring time was 24 hours.

In this embodiment, after the ink is prepared in step S22, the homogenized ink should be used as soon as possible after opening in order to prevent the solvent in the ink from volatilizing.

Example 2:

the procedure of this example was essentially the same as in example 1, except that:

the step S22 is replaced with: 0.5g of nHA was added to the polymer solution and subjected to a second stirring treatment to form a homogenized ink, namely PBLG/nHA ink, designated as P5H5.

Example 3:

the step S21 is replaced with: dissolving 0.7g of PBLG in 10ml of 1, 4-dioxane, and carrying out first stirring treatment to completely dissolve a polymer to obtain a polymer solution; after the step S22, the obtained PBLG/nHA ink was designated as P7H3.

Example 4:

the adopted organic solvent is replaced by ethanol gel, namely, the ethanol gel bath is adopted to assist 3D printing of the PBLG/nHA nanofiber bone scaffold;

and S33, after printing, placing the scaffold and the ethanol gel into an ethanol bath together for soaking for 4 hours to remove the ethanol gel, and obtaining the complete nanofiber bone scaffold.

Comparative example 1:

the procedure of this comparative example was substantially the same as in example 1, except that:

the step S22 is replaced with: adding no nHA into the polymer solution, namely obtaining the PBLG/nHA ink after the step S11, and marking the ink as P5H0.

Comparative example 2:

the adopted organic solvent is replaced by diethyl ether, namely diethyl ether bath is adopted to assist 3D printing of the PBLG/nHA nanofiber bone scaffold.

The samples prepared in each of the examples and comparative examples were tested as follows:

as shown in FIG. 1, the nuclear magnetism (FIG. 1 (A)) and the infrared spectrum (FIG. 1 (B)) of the high molecular weight poly-benzyl L-glutamate PBLG prepared in the step S1 are tested, which shows that the structural formula of the poly-benzyl L-glutamate PBLG is as follows:

the weight average molecular weight of PBLG was tested to be about 25X 10 ⁴ A viscosity average molecular weight of about 18X 10 ⁴ The molecular weight distribution was about 1.742.

The nanofiber bone scaffold (also simply referred to as a scaffold) obtained after the printing in the step S3 has obvious shrinkage after drying, which is caused by loss of a small amount of polymer PBLG after the polymer PBLG is separated from the scaffold along with volatilization of a solvent, and the dried scaffold can be clamped and transferred by tweezers, so that the stability of a device is good. As shown in fig. 2, SEM images (fig. 2 (a)) of the scaffolds prepared in example 1 after drying, the scaffolds had a regular porous structure, and the extruded strips were vertically staggered, and the diameters after shrinkage were measured to be about 350-400 nm, which is 68-80% of the extruded diameters of the extruded pillows, and the corresponding EDS images (fig. 2 (B) and fig. 2 (C)) showed that Ca/P elements were uniformly distributed on the scaffolds, and nHA was uniformly dispersed and adhered throughout the scaffolds. Meanwhile, in fig. 2 (a), a multi-layer stacking structure can be clearly observed, which indicates that the bracket has better supporting performance and does not collapse after printing; each layer of bracket is a porous structure formed by vertically crossing the X and Y directions, and obvious pores exist in the Z-axis direction; there is also some fusion between the extruded strips, indicating that the multi-layer structure is not simply stacked, but rather there is some healing and bonding between the layers, making the overall structure more stable.

Fig. 3 is a macroscopic photograph and SEM image of the stent prepared in example 1 after drying, and it can be seen from the macroscopic photograph that 3D printing and structural stabilization of the stent of various shape specifications can be performed according to actual needs. In SEM images, example 1 shows a pronounced nanofiber microstructure.

Fig. 4 is an SEM image of the stent prepared in comparative example 2 after drying, and it can be seen that comparative example 2 does not have the generation of a nanofiber structure.

Fig. 5 shows the contact angle test results after drying the stents prepared in examples 1 to 3, and shows that the water contact angle in example 1 is about 70.90 ° to 72.45 °, the water contact angle in comparative example 2 is about 81.14 ° to 81.28 °, the water contact angle in example 2 is about 62.04 ° to 64.38 °, and the water contact angle in example 3 is about 88.20 ° to 88.26 °. With the increase of the nHA content, the smaller the water contact angle of the surface of the material, the greater the improvement of the hydrophilicity of the scaffold, the better the hydrophobic problem of the scaffold, and the adhesion and growth of bone tissue cells on the surface of the scaffold material are facilitated.

Fig. 6 is a mechanical property diagram obtained by performing compression experiments in examples 1 to 3, fig. 6 (a) is a compressive stress-strain diagram, and fig. 6 (B) is an elastic modulus diagram deduced from fig. 6 (a), it can be seen that examples 1, 2 and 3 all have a compressive modulus of 10MPa or more, and can meet the needs of bone tissue graft materials in most of the load-bearing areas of the human body.

Fig. 7 is a graph showing the shear thinning properties of the PBLG/nHA inks prepared in examples 1 and 3 and comparative example 1, in which it can be seen that the ink viscosity gradually decreases with increasing shear rate, which reduces the chance of clogging during printing, and the PBLG/nHA ink prepared in example 2 is more excellent in the shear thinning properties as can be seen from the three curves in the graph.

Fig. 8 is a comparative graph of the extrusion performance of PBLG/nHA ink, in which the form of the ink when extruded from the extrusion needle was observed, and the same pattern was 3D printed, and the stability of the printed pattern was observed after the pattern was dried, and the printability and microstructure of the ink were studied. The inks of different compositions can achieve continuous and uniform extrusion in ethanol. As shown in fig. 8 (a) to (E), the P5H0 ink without nHA in comparative example 1 was in a viscous fluid form when extruded from an extrusion needle for direct printing, was not sufficiently formable, could not form a three-dimensional structure after extrusion, could obtain a basic prototype of the network stent set by the printing procedure with the aid of an ethanol bath, but had a distinct adhesive film between the meshes, and had no clear stacking limit required for 3D printing between the layers; as shown in fig. 8 (F) to (L), both inks of examples 1 and 2, to which nHA was added, were able to be molded after extrusion from an extrusion needle, and after drying of the extruded pattern in an ethanol bath, a grid stent substantially conforming to the programming was observed, and the morphology of the stent remained intact, no adhesive film was formed between the grids, no significant collapse was observed, and the moldability of P5H5 was better, and the morphology at extrusion was more stable than that at P5H 3; it was found by observation with a stereomicroscope that the interlayer stack structure was remarkable, which is the basis for realizing interlayer printing, and this effect was more advantageous when the content of nHA was increased. The use of an ethanol bath to assist in printing has significant advantages over direct printing inks, and can help stabilize the morphology of the extruded strip and stent.

Fig. 9 shows that the extrusion needle in example 4 extrudes PBLG/nHA ink in an ethanol gel bath to form an extrusion bar, and the extrusion bar is spiral after being immersed in ethanol solution to remove ethanol gel, so that the extrusion bar has good shape and structure, and the construction of a 3D printing stent with a space spiral structure can be realized. SEM images of the scaffolds prepared in example 4 after drying showed that the scaffolds had a distinct nanofiber microstructure.

Although embodiments of the present 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 hereto without departing from the spirit and scope of the invention as defined by the appended embodiments and equivalents thereof.

Claims

1. The preparation method of poly-L-benzyl glutamate is characterized in that 1, 4-dioxane is injected into poly-L-benzyl glutamate carboxylic anhydride monomer under protective atmosphere, and after the poly-L-benzyl glutamate carboxylic anhydride monomer is completely dissolved, a first reaction solution is obtained; injecting an initiator into the first reaction liquid, uniformly mixing, and standing at room temperature until the reaction is completed to obtain a second reaction liquid; and (3) settling the second reaction solution by diethyl ether, filtering the obtained product, and drying in vacuum until the weight is constant to obtain a polymer product poly-benzyl L-glutamate.

2. The method for preparing poly-L-benzyl glutamate according to claim 1, wherein the initiator is triethylamine, and the molar ratio of the initiator to the poly-L-benzyl glutamate carboxylic anhydride monomer is 100:1.

3. The method for preparing poly-benzyl L-glutamate according to claim 1, wherein the protective atmosphere is nitrogen.

4. The poly-benzyl L-glutamate is characterized by comprising the following structural formula:

5. the preparation method of the ink composed of the poly-L-benzyl glutamate/nano-hydroxyapatite composite is characterized by adding the nano-hydroxyapatite into 1, 4-dioxane to obtain a dispersion liquid; and dissolving the poly-benzyl L-glutamate in the dispersion liquid, and completely dissolving the poly-benzyl L-glutamate through first stirring treatment to form homogenized ink.

6. The method for preparing the ink composed of the poly-L-benzyl glutamate/nano-hydroxyapatite according to claim 5, wherein the first stirring time is 24 hours.

7. The ink composed of the poly-L-benzyl glutamate/nano-hydroxyapatite composite is characterized by comprising the following components: poly L-benzyl glutamate and nano hydroxyapatite with the mass ratio of 1:0.4-1.

8. A method for preparing a poly-L-benzyl glutamate based nanofiber bone scaffold by a 3D printing one-step method is characterized in that ink formed by compounding poly-L-benzyl glutamate/nano hydroxyapatite is transferred into 3D printing equipment in a room temperature environment, 3D printing of the nanofiber bone scaffold is carried out in a receiving phase environment of an organic solvent bath, and an extrusion needle is completely immersed in the organic solvent bath during printing; after printing, the nanofiber bone scaffold is fully soaked in an organic solvent liquid bath for 4 hours.

9. The method for preparing the poly-L-benzyl glutamate based nanofiber bone scaffold by a 3D printing one-step method according to claim 8, wherein the receiving phase environment of the organic solvent bath used in the printing process is an ethanol bath or an ethanol gel bath.

10. The method for preparing a poly-L-benzyl glutamate based nanofiber bone scaffold by a 3D printing one-step method according to claim 8, wherein the organic solvent liquid bath for full soaking after printing is an ethanol bath.