Bio-ink and preparation method and application thereof

Abstract

The invention provides bio-ink as well as a preparation method and application thereof. The method comprises the following steps: (1) mixing nano-clay with water to obtain a nano-clay dispersion liquid; (2) adding elastin-like polypeptide into the nano-clay dispersion liquid, stirring until the elastin-like polypeptide is completely dissolved, and precipitating to obtain bio-ink; the amino acid sequence of the elastin-like polypeptide is as shown in SEQ ID NO: 2. The method is simple in process, mild in process condition and suitable for large-area popularization, the bio-ink prepared through the method has good shear thinning, printability, biocompatibility and stability, pre-crosslinking and secondary crosslinking are not needed, and the bio-ink has good application prospects in many fields.

Description

Technical Field

[0001] The present invention relates to the technical field of material engineering, and in particular to a biological ink and a preparation method and application thereof. Background Art

[0002] Bio-3D printing is a cutting-edge discipline that uses biomaterials and cells as basic units to manufacture personalized in vitro biological structures using "three-dimensional printing" technology in accordance with the requirements of biomimetic morphology and cell-specific microenvironment. It is an emerging field born from the intersection of many disciplines such as engineering, materials and life sciences. At present, the lack of printable bio-ink with good biocompatibility is considered to be one of the main obstacles to the development of bio-3D printing. Extrusion 3D printing technology is one of the most popular printing technologies at present. It can meet the printing and molding of bio-inks with different rheological properties and cross-linking mechanisms. However, in order to meet the molding and structural stacking requirements of ink microfilaments, it is usually necessary to pre-cross-link the ink material in advance to increase the viscosity, and then perform secondary cross-linking after printing to enhance the integrity and stability of the structure, which seriously limits the application of bio-ink. Therefore, it is of great significance to develop a bio-ink that does not require pre-cross-linking treatment and can stably exist in the printed structure. Summary of the invention

[0003] The present invention aims to solve one of the technical problems in the prior art at least to a certain extent. To this end, one object of the present invention is to provide a biological ink and a preparation method and application thereof.

[0004] In a first aspect of the present invention, the present invention provides a method for preparing a biological ink. According to an embodiment of the present invention, the method comprises: (1) mixing nanoclay and water to obtain a nanoclay dispersion; (2) adding the elastin-like polypeptide to the nanoclay dispersion and stirring until the elastin-like polypeptide is completely dissolved, and obtaining the bio-ink after precipitation; The amino acid sequence of the elastin-like polypeptide is shown in SEQ ID NO:2.

[0005] According to the biological ink of the above embodiment of the present invention, nanoclay as a rheological additive can give the biological ink excellent extrusion molding effect, and the elastin-like polypeptide has good biocompatibility and mechanical properties, which can give the biological ink excellent biocompatibility and stability. At the same time, the elastin-like polypeptide sequence contains rich glutamic acid groups, so a large number of charges are distributed on it, which can produce intermolecular interactions with nanoclay to form a shear-thinning, self-healing dynamic coacervate network. The formed physical cross-linked coagulation system effectively solves the cumbersome process problem of the existing biological ink requiring pre-crosslinking and secondary crosslinking. Therefore, the method is simple in process and mild in process conditions, which is suitable for large-scale promotion. The biological ink prepared by this method has good shear thinning, printability, biocompatibility and stability, and does not require pre-crosslinking and secondary crosslinking, and has good application prospects in many fields.

[0006] In addition, the method for preparing the bio-ink according to the above embodiment of the present invention also has the following technical features: In some embodiments of the present invention, in step (1), the particle size of the nanoclay is 10 nm to 100 nm.

[0007] In some embodiments of the present invention, in step (1), the nanoclay is a gel type.

[0008] In some embodiments of the present invention, in step (1), the concentration of the nanoclay in the nanoclay dispersion is no more than 18% w / v.

[0009] In some embodiments of the present invention, in step (2), the mass ratio of the nanoclay to the elastin-like polypeptide is 1:(0.3-8).

[0010] In some embodiments of the present invention, the mass ratio of the nanoclay to the elastin-like polypeptide is 1:(1.5-3).

[0011] In some embodiments of the present invention, step (2) comprises: adding the elastin-like polypeptide to the nanoclay dispersion, stirring until the elastin-like polypeptide is completely dissolved, centrifuging, and removing the gel layer to obtain the bio-ink.

[0012] In some embodiments of the present invention, the centrifugal speed is 500g~5000g, and the centrifugal time is 5min~30min.

[0013] In a second aspect of the present invention, the present invention provides a bio-ink. According to an embodiment of the present invention, the bio-ink is prepared by the above method.

[0014] In a third aspect of the present invention, the present invention proposes the application of the above-mentioned bio-ink in the field of biomaterial engineering, tissue engineering, medicine, environment or 3D printing materials.

[0015] The present invention has at least the following technical effects: (1) Nanoclay, as a rheological additive, can give bio-ink excellent extrusion molding effect. Elastin-like peptides have good biocompatibility and mechanical properties, which can give bio-ink excellent biocompatibility and stability, making bio-ink suitable for a wider range of applications.

[0016] (2) The elastin-like polypeptide sequence contains abundant glutamic acid groups, which carry a large amount of electric charges. This can generate intermolecular interactions with nanoclays to form a shear-thinning, self-healing dynamic coacervate network.

[0017] (3) Elastin-like peptides are rich in glutamic acid groups, which can generate intermolecular forces with nanoclay to form a physically cross-linked cohesive system. The above special structure enables bio-ink to be directly used in 3D printing without the need for pre-cross-linking or secondary cross-linking after printing, greatly reducing the difficulty of operation.

[0018] (4) This method has a simple preparation process and mild conditions, and the prepared bio-ink has good application prospects in many fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The appearance and molecular weight characterization results of the elastin-like polypeptides of the embodiments of the present invention; Figure 2 is a schematic diagram of the preparation process of the bio-ink of Example 1 of the present invention; Figure 3 is a shear thinning rheology result diagram of the bio-ink of Example 1 of the present invention; Figure 4 is a digital photo of the printed structure of the bio-ink of Example 1 of the present invention; Figure 5 is a microscope photograph of fibroblasts cultured in the bio-ink of Example 1 of the present invention; Figure 6 This is a diagram showing the live and dead results of fibroblasts cultured with the bio-ink in Example 1 of the present invention; Figure 7This is a graph showing the stability test results of the bio-ink of Example 1 of the present invention. DETAILED DESCRIPTION

[0021] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0022] In a first aspect of the present invention, the present invention provides a method for preparing a biological ink. According to an embodiment of the present invention, the method comprises: S100: Mixing nanoclay and water

[0023] In this step, nanoclay and water are mixed to obtain a nanoclay dispersion. Nanoclay, as a rheological additive, can give the bio-ink an excellent extrusion molding effect. Further, the nanoclay is a gel type.

[0024] According to an embodiment of the present invention, the particle size of the nanoclay is 10nm~100nm. For example, the particle size is 10nm, 20nm, 25nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, etc., or within the range of any two of the above data points. The inventors found that the particle size of the nanoclay within the above range is conducive to the cross-linking of the nanoclay and the elastin-like polypeptide, so that the bio-ink presents an excellent extrusion molding effect.

[0025] According to an embodiment of the present invention, the concentration of nanoclay in the nanoclay dispersion is not greater than 18% w / v. For example, the concentration is 1% w / v, 2% w / v, 5% w / v, 10% w / v, 14% w / v, 18% w / v, etc., or within the range of any two of the above data points.

[0026] S200: Preparation of bio-ink

[0027] In this step, elastin-like polypeptide is added to the nanoclay dispersion and stirred until the elastin-like polypeptide is completely dissolved, and bio-ink is obtained after precipitation. Elastin-like polypeptide has good biocompatibility and mechanical properties, and can give the bio-ink excellent biocompatibility and stability. At the same time, the elastin-like polypeptide sequence contains abundant glutamic acid groups, so a large number of charges are distributed on it, which can produce intermolecular interactions with nanoclay to form a shear-thinning and self-healing dynamic coacervate network. The formed physically cross-linked coacervate system effectively solves the cumbersome process problem of the existing bio-ink requiring pre-cross-linking and secondary cross-linking. Further, the appearance and molecular weight characterization results of the elastin-like polypeptide are shown in Figure 1 .

[0028] The amino acid sequence of the elastin-like polypeptide is [(VPGVG)2VPGEG(VPGVG)2]25 (SEQ ID NO:2).

[0029] (SEQ ID NO:2).

[0030] According to an embodiment of the present invention, the mass ratio of nanoclay to elastin-like polypeptide is 1: (0.3-8), preferably 1: (1.5-3). For example, the mass ratio is 1: 0.3, 1: 0.5, 1: 0.7, 1: 1, 1: 1.5, 1: 2, 1: 2.5, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, etc. The inventors found that when the mass ratio of nanoclay to elastin-like polypeptide is controlled within the above range, nanoclay and elastin-like polypeptide can be cross-linked to form a stable gel morphology, giving the bio-ink gel properties and realizing 3D printing.

[0031] According to an embodiment of the present invention, S200 further includes: adding elastin-like polypeptide to the nanoclay dispersion, stirring until the elastin-like polypeptide is completely dissolved, centrifuging, and removing the gel layer to obtain biological ink. By centrifugation, the polymerization rate of the gel is increased, and the biological ink for direct use is quickly obtained. Furthermore, the centrifugal speed is 500g~5000g, and the centrifugal time is 5min~30min.

[0032] In a second aspect of the present invention, a bio-ink is provided. According to an embodiment of the present invention, the bio-ink is prepared by the above method. As a result, the bio-ink has good shear thinning, printability, biocompatibility and stability, and does not require pre-crosslinking and secondary crosslinking, and has good application prospects in many fields.

[0033] In a third aspect of the present invention, the present invention proposes the application of the above-mentioned bio-ink in the field of biomaterial engineering, tissue engineering, medicine, environment or 3D printing materials.

[0034] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0035] Example 1

[0036] (1) 5 g of nanoclay was added to 100 mL of aqueous solution and stirred until completely dissolved to obtain a uniform nanoclay dispersion. The particle size of the nanoclay was 25 nm and the concentration of the nanoclay dispersion was 5% w / v. The nanoclay used was purchased from BYK Chemicals of Germany and was the same as in Example 2.

[0037] (2) Add 10 g of elastin-like polypeptide (SEQ ID NO: 2) to the prepared nanoclay dispersion and stir thoroughly until completely dissolved.

[0038] (3) The system of step (2) is centrifuged at a speed of 1000 g for 10 min to obtain the precipitate, which is the elastin-like polypeptide coacervate bio-ink. The specific process is as follows: Figure 2 As shown in the figure, the bio-ink can print and shape complex structures without pre-crosslinking or secondary crosslinking.

[0039] The properties of the bio-ink prepared in Example 1 were tested as follows: (1) Shear-thinning rheological investigation of 3D printed elastin-like polypeptide coacervate bio-ink

[0040] The shear thinning of the bio-ink in Example 1 was verified using a rheometer (MCR302, Anton Paar, Austria). A flat plate mold (15 mm in diameter) was used for rheological measurements. The bio-ink sample was first placed between the flat plates, and then the sample was shear tested to record the change process of viscosity.

[0041] The experimental results are shown in Figure 3 ,from Figure 3 It can be seen that with the increase of shear rate, the dynamic network breaks and the viscosity decreases significantly, indicating that the bio-ink can be extruded smoothly in extrusion 3D printing.

[0042] Figure 4 This is a digital photo of the printed structure of the biological ink of Example 1 of the present invention.

[0043] (2) 3D printed elastin polypeptide coacervate bio-ink for culturing fibroblasts

[0044] Fibroblasts (mouse origin, model: L929) were cultured in a system of RPMI 1640 basal medium + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin. The bio-ink containing fibroblasts was printed with a microfilament scaffold using an extrusion 3D printer, and then cultured with the medium. The cells were observed and photographed using a fluorescence microscope, and tested using a live-death test kit to obtain the cell survival rate.

[0045] Microscope photos of fibroblasts cultured in 3D printed elastin polypeptide coacervate bio-ink. Figure 5 ,from Figure 5 It can be seen that fibroblasts were successfully contained in the microfilament scaffold.

[0046] The live-death results of fibroblasts cultured with 3D printed elastin polypeptide coacervate bio-ink are shown in Figure 6 ,from Figure 6 It can be seen that the fibroblasts maintained a survival rate of more than 85% throughout the culture process, which shows the good cell compatibility of the bio-ink.

[0047] (3) Stability test of 3D printed elastin polypeptide coacervate bio-ink

[0048] Weigh a certain weight of dry sample (weight: W1) and put it in a weighing bottle, add 5ml phosphate buffer; put the above weighing bottle into a shaker (60r / min) and change the solution once a day; take out the sample at different time periods, such as 1 day, 3 days, and 7 days, dry it and weigh it (W2), and finally calculate the remaining mass according to the following formula. Remaining mass (%) = (W1-W2) / W1x100%

[0049] The stability test results are shown in Figure 7 ,from Figure 7 It can be seen that the mass loss of the coacervate bio-ink prepared in Example 1 was less than 10% during the 7-day incubation process, and the overall shape of the bio-ink remained intact, while the nanoclay sample had poor stability in the buffer and was completely dissolved in 3 days. The above results show that the elastin-like polypeptide coacervate bio-ink of the present application has good stability in solution.

[0050] Example 2

[0051] (1) 3 g of nanoclay was added to 100 mL of aqueous solution and stirred thoroughly until completely dissolved to obtain a uniform nanoclay dispersion. The concentration of the nanoclay dispersion was 3% w / v.

[0052] (2) Add 20 g of elastin-like polypeptide (SEQ ID NO: 2) to the prepared nanoclay dispersion and stir thoroughly until completely dissolved.

[0053] (3) The system of step (2) is centrifuged at a speed of 3000 g for 5 min, and the precipitate is the elastin-like polypeptide coacervate bio-ink. The bio-ink can print and shape complex structures without pre-crosslinking or secondary crosslinking.

[0054] As the shear rate increases, the dynamic network of the bio-ink prepared in Example 2 also breaks and the viscosity decreases, indicating that the bio-ink in Example 2 can be smoothly extruded in extrusion 3D printing. In the biocompatibility test, the fibroblasts in Example 2 maintained a survival rate of more than 84% throughout the culture process, indicating that the bio-ink in Example 2 has good cell compatibility. In the stability test, the mass loss of the bio-ink in Example 2 after 7 days was within 10%, indicating that the bio-ink in Example 2 has good stability.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a biological ink, characterized in that: include: (1) mixing nanoclay and water to obtain a nanoclay dispersion; (2) adding the elastin-like polypeptide to the nanoclay dispersion and stirring until the elastin-like polypeptide is completely dissolved, and obtaining the bio-ink after precipitation; The amino acid sequence of the elastin-like polypeptide is shown in SEQ ID NO:

2.

2. The method according to claim 1, characterized in that In step (1), the particle size of the nanoclay is 10 nm to 100 nm.

3. The method according to claim 1 or 2, characterized in that: In step (1), the nanoclay is a gel type.

4. The method according to claim 1 or 2, characterized in that: In step (1), the concentration of the nanoclay in the nanoclay dispersion is not greater than 18% w / v.

5. The method according to claim 1 or 2, characterized in that: In step (2), the mass ratio of the nanoclay to the elastin-like polypeptide is 1:(0.3-8).

6. The method according to claim 5, characterized in that The mass ratio of the nanoclay to the elastin-like polypeptide is 1:(1.5-3).

7. The method according to claim 1, characterized in that Step (2) comprises: adding the elastin-like polypeptide to the nanoclay dispersion, stirring until the elastin-like polypeptide is completely dissolved, centrifuging, and removing the gel layer to obtain the bio-ink.

8. The method according to claim 7, characterized in that The centrifugal speed is 500g~5000g, and the centrifugal time is 5min~30min.

9. A biological ink, characterized in that: Prepared by the method according to any one of claims 1 to 8.

10. Application of the bio-ink according to claim 9 in the fields of biomaterial engineering, tissue engineering, medicine, environment or 3D printing materials.

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