3D printing composite bio-ink, biological scaffold and preparation method thereof

Abstract

The invention discloses a 3D printing composite bio-ink, a biological scaffold and a preparation method thereof, the advantages of tussah silk fibroin (TSF), methylacryloylated gelatin (GelMA) and nano-hydroxyapatite (nHA) are systematically and innovatively integrated, and the 3D printing composite bio-ink is prepared through material ratio optimization, multi-mode cross-linking strategy innovation and 3D printing process fine adjustment. The 3D printing TSF / GelMA / nHA bone repair scaffold integrating mechanical adaptability, biological functionality and structural accuracy is constructed in a customizable mode, and the key problems that in the prior art, mechanical performance is insufficient, printing performance is good, and a multifunctional integrated structure is difficult to manufacture are effectively solved.

Description

A 3D-printed composite bio-ink, a bio-scaffold, and its preparation method Technical Field

[0001] This invention relates to the fields of biomaterials and tissue engineering, and in particular to a 3D printing composite bio-ink, a bio-scaffold, and a method for preparing the same. Background Technology

[0002] 3D bioprinting technology, as a disruptive innovation in the field of biomedical engineering, has great advantages in the construction of complex tissues and organs, and is especially widely used in the field of bone tissue engineering.

[0003] Currently, few studies systematically investigate and optimize the proportions, interactions, cross-linking synergies, and effects on the overall properties (such as rheology / printability, mechanics, etc.) of specific combinations of tussah silk fibroin (TSF), methacrylamide gelatin (GelMA), and nano-hydroxyapatite (nHA). In particular, achieving the optimal balance between the toughness of TSF and the rigidity of nHA, and the bioactivity of GelMA, remains a technical challenge for this multi-component bio-ink in extrusion 3D printing, particularly in maintaining high precision (micrometer-level features), complex porous structures (such as gradient pores and biomimetic structures), and good interlayer bonding. Existing literature reports that bio-inks have resulted in relatively simple structures or structures with limited precision. Summary of the Invention

[0004] The purpose of this invention is to provide a 3D-printed composite bio-ink, a bio-scaffold, and its preparation method. The core innovation lies in the systematic and innovative integration of the advantages of three materials: tussah silk fibroin (TSF), methacrylamide gelatin (GelMA), and nano-hydroxyapatite (nHA). Through material ratio optimization, innovative multi-mode cross-linking strategies, and fine-tuning of the 3D printing process, a 3D-printed TSF / GelMA / nHA bone repair scaffold with "mechanical adaptability, biofunctionality, and structural precision" can be customarily constructed. This effectively solves the key problems commonly found in existing technologies, such as insufficient mechanical properties, poor printability, and difficulties in manufacturing multifunctional integrated structures.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a 3D printing composite bio-ink, which is composed of the following components in weight percentage: 5%~8% tussah silk fibroin, 15%~25% methacrylamide gelatin, 10%~20% nano hydroxyapatite, 0.25%~0.5% photoinitiator, and the balance being deionized water.

[0007] As a preferred formulation, the 3D printing composite bio-ink consists of the following components by weight percentage: 5% tussah silk fibroin, 20% methacrylamide gelatin, 15% nano-hydroxyapatite, 0.5% photoinitiator, and the balance deionized water. This results in superior mechanical and printing properties.

[0008] In some specific technical solutions of the present invention, the mass ratio of the tussah silk fibroin to the methacrylamide gelatin is 1:(3~5).

[0009] As an example, the mass ratio of tussah silk fibroin to methacrylamide gelatin can be set to any of the following ratios: 1:3, 1:4, or 1:5.

[0010] In some specific technical solutions of the present invention, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP).

[0011] Secondly, the present invention provides a method for preparing the above-mentioned 3D printing composite bio-ink, comprising the following steps:

[0012] S1. Preparation of TSF solution: Dissolve tussah silk fibroin in a portion of deionized water to obtain TSF solution;

[0013] S2. Preparation of GelMA solution: Dissolve methacrylamide gelatin in the remaining deionized water, remove bubbles, and obtain GelMA solution;

[0014] S3. GelMA solution and nHA powder are mixed: Nano-hydroxyapatite is added to the GelMA solution prepared in step S2 and ultrasonically dispersed to form a uniform slurry.

[0015] S4. Add the TSF solution obtained in step S1 to the slurry in step S3 and stir to mix evenly.

[0016] S5. Add photoinitiator to step S4, stir and mix in the dark to obtain 3D printing composite bio-ink.

[0017] In some specific technical solutions of the present invention, in step S3, the ultrasonic dispersion conditions are: ultrasonic power of 200W~400W and ultrasonic dispersion time of 20min~50min.

[0018] For example, the ultrasonic power can be set to 200W, 250W, 300W, 400W, etc., and the ultrasonic dispersion time can be set to 20min, 25min, 30min, 50min, etc.

[0019] In some specific technical solutions of the present invention, in step S4, the stirring and mixing conditions are: stirring speed of 200 rpm to 400 rpm and stirring time of 20 min to 40 min.

[0020] For example, the stirring speed can be set to 200 rpm, 250 rpm, 300 rpm, 400 rpm, etc., and the stirring time can be set to 20 min, 30 min, 35 min, 40 min, etc.

[0021] In some specific technical solutions of the present invention, in step S5, the light-protected stirring and mixing conditions are: stirring speed of 100 rpm to 200 rpm and stirring time of 15 min to 45 min.

[0022] For example, the stirring speed can be set to 100 rpm, 120 rpm, 140 rpm, 150 rpm, 180 rpm, 200 rpm, etc., and the stirring time can be set to 15 min, 20 min, 25 min, 30 min, 40 min, 45 min, etc.

[0023] Thirdly, the present invention provides a biological scaffold, which is prepared by 3D printing of the above-mentioned 3D printing composite biological ink.

[0024] Fourthly, the present invention provides a method for preparing the above-mentioned biological scaffold, comprising the following steps:

[0025] F1, Primary Photocrosslinking: Using the above-mentioned 3D printing composite bio-ink, 3D printing is performed according to a preset three-dimensional model, with a strength of 10mW / cm². 2 ~15mW / cm 2 Irradiate with 405nm blue light for 90s~120s to obtain a shaped scaffold model;

[0026] F2. Secondary physical crosslinking pretreatment: The scaffold model obtained in step F1 is immersed in 75% ethanol for toughening treatment for 10 min to 30 min;

[0027] F3. Post-treatment of tertiary ion bonding: Immerse the scaffold model treated in step F2 in a 10% CaCl2 solution for 1 min to 10 min for strengthening treatment;

[0028] F4. Freeze-drying: The scaffold model processed in step F3 is freeze-dried to obtain a biological scaffold.

[0029] In some specific technical solutions of this invention, in step F1, the 3D printing process conditions are set as follows: printing temperature is set to 18℃~25℃, layer height is set to 0.2mm±0.05mm, filament diameter is set to 0.3mm±0.05mm, spacing is set to 0.4mm±0.05mm, printing speed is set to 5mm / s~15mm / s, and extrusion speed is set to 0.5mm. 3 / s~3.0mm 3 / s.

[0030] Preferably, in step F1, the 3D printing process conditions are set as follows: printing temperature is set to 23℃, layer height is set to 0.2mm, filament diameter is set to 0.3mm, spacing is set to 0.4mm, printing speed is set to 10mm / s, and extrusion speed is set to 1.0mm. 3 / s.

[0031] In some specific technical solutions of the present invention, in step F4, the freeze-drying process includes the following operations: first, the scaffold model sample is quenched in liquid nitrogen for 30s to 60s, and then transferred to -80°C to completely freeze the scaffold model sample into a hard block; then, the scaffold model sample is placed in the sample chamber of a freeze dryer with a cold trap temperature below -80°C for freeze-drying.

[0032] Specifically, the freeze-drying time is 12h to 60h.

[0033] Compared with existing technologies, this invention provides a 3D printing composite bio-ink, a bio-scaffold, and a method for preparing the same, which has the following beneficial effects:

[0034] (1) In terms of material design, a TSF / GelMA dual-network interpenetrating structure is adopted to achieve structural innovation by encapsulating nHA: On the one hand, a dual-network interpenetrating structure is constructed, with TSF forming a physically cross-linked tough network (β-sheet crystalline domain) and GelMA forming a photocross-linked active network (covalent bond). nHA is synergistically encapsulated by the dual networks to avoid stress concentration; on the other hand, an interface strengthening mechanism is formed by using the carboxyl groups of surface-modified nHA to form ionic bonds with the amino groups of GelMA, and then using CaCl2 solution treatment to utilize Ca 2+ It bridges the phosphate group of nHA with the carboxyl group of GelMA, thereby improving the interfacial binding force.

[0035] (2) In terms of process design, a multi-stage time-sequential crosslinking method is adopted to achieve innovation: In the printing process, primary photocrosslinking is first used to irradiate the nozzle with 405nm blue light in real time to extrude the filament and make it instantly shaped (<0.5s) to prevent interlayer collapse and achieve immediate structural shaping; after printing, secondary physical crosslinking pretreatment is used to induce TSF to form β-sheet crystals (crystallinity of 40%~50%) by soaking in 75% ethanol to achieve toughened scaffold; then, tertiary ion bonding posttreatment is used to promote the bonding of nHA-GelMA interface by soaking in 10% CaCl2 solution to improve the interlayer bonding strength to 1.8MPa (the traditional process is 0.6~0.8MPa) and achieve enhanced mechanical properties.

[0036] (3) This invention optimizes the ratio of TSF, GelMA, and nHA to construct a ternary synergistic system. The system achieves complementary performance through "TSF (tough scaffold) + GelMA (bioactive) + nHA (osteoconductive)". The dual-network interpenetrating structure resolves the contradiction that "high nHA content inevitably leads to brittleness". Specific innovative 3D printing parameters (pressure, speed, temperature) and post-processing techniques (gradient crosslinking, freeze-drying) are formulated to construct a biomimetic porous structure. Multi-level temporal crosslinking overcomes the technical barrier of "difficulty in printing high nHA content" and solves the problem of "incompatibility between printing accuracy and mechanical properties". The bio-scaffold prepared by this invention can be applied to the repair of tissue-engineered bone defects such as trabeculae and skull defects. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below through detailed embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0039] Example 1

[0040] This embodiment provides a 3D printing composite bio-ink composed of the following components by weight percentage: 5% tussah silk fibroin, 20% methacrylamide gelatin, 15% nano-hydroxyapatite, 0.5% photoinitiator, and the balance being deionized water. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP).

[0041] The preparation method of this 3D printing composite bio-ink includes the following steps:

[0042] S1. Preparation of TSF solution: Dissolve tussah silk fibroin in a portion of deionized water to obtain TSF solution;

[0043] S2. Preparation of GelMA solution: Dissolve the lyophilized methacrylamide gelatin powder in the remaining deionized water, remove air bubbles, and obtain GelMA solution;

[0044] S3. GelMA solution and nHA powder are mixed: Nano hydroxyapatite powder is added to the GelMA solution prepared in step S2 and ultrasonically dispersed for 30 minutes with an ultrasonic power of 300W to form a uniform slurry.

[0045] S4. Add the TSF solution obtained in step S1 dropwise to the slurry in step S3, and stir at 300 rpm for 30 minutes until the mixture is homogeneous.

[0046] S5. Add the photoinitiator to step S4 and stir and mix at 150 rpm for 20 minutes under light-protected conditions to obtain 3D printing composite bio-ink.

[0047] Example 2

[0048] This embodiment provides a 3D printing composite bio-ink, composed of the following components by weight percentage: 5% tussah silk fibroin, 20% methacrylamide gelatin, 10% nano-hydroxyapatite, 0.5% photoinitiator, and the balance being deionized water. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP).

[0049] The preparation method of this 3D printing composite bio-ink is the same as that in Example 1, so it will not be described again.

[0050] Example 3

[0051] This embodiment provides a 3D printing composite bio-ink, composed of the following components by weight percentage: 5% tussah silk fibroin, 20% methacrylamide gelatin, 20% nano-hydroxyapatite, 0.5% photoinitiator, and the balance being deionized water. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP).

[0052] The preparation method of this 3D printing composite bio-ink is the same as that in Example 1, so it will not be described again.

[0053] Example 4

[0054] This embodiment provides a biological scaffold, the preparation method of which includes the following steps:

[0055] F1. Primary photocrosslinking: The 3D printing composite bio-ink prepared in Example 1 was used for 3D printing according to a preset three-dimensional model, with a strength of 15 mW / cm². 2 The 405nm blue light was applied for 100 seconds to obtain a shaped support model. The 3D printing process conditions were set as follows: printing temperature 23℃, layer height 0.2mm, filament diameter 0.3mm, spacing 0.4mm, printing speed 10mm / s, and extrusion speed 1.0mm. 3 / s.

[0056] F2, Secondary physical crosslinking pretreatment: The scaffold model obtained in step F1 is immersed in 75% ethanol for toughening treatment for 20 min.

[0057] F3. Post-treatment of tertiary ion bonding: Immerse the scaffold model treated in step F2 in a 10% CaCl2 solution for 5 min for strengthening treatment.

[0058] F4. Freeze-drying: First, quench the scaffold model treated in step F3 in liquid nitrogen (-196℃) for 60s, and then quickly transfer it to -80℃ (such as an ultra-low temperature freezer) to completely freeze the scaffold model sample into a hard block; then quickly remove the scaffold model sample from the ultra-low temperature freezer and immediately place it in the sample chamber of a freeze dryer with a cold trap temperature below -80℃ for freeze-drying for 24h to obtain a biological scaffold.

[0059] Example 5

[0060] This embodiment provides a biological scaffold, which is 3D printed using the 3D printing composite biological ink prepared in Example 2 according to a preset three-dimensional model. The preparation method of the biological scaffold is the same as that in Example 4, so it will not be described again.

[0061] Example 6

[0062] This embodiment provides a biological scaffold, which is 3D printed using the 3D printing composite biological ink prepared in Example 3 according to a preset three-dimensional model. The preparation method of the biological scaffold is the same as that in Example 4, so it will not be described again.

[0063] Example 7

[0064] This embodiment provides a biological scaffold, which is based on Example 4 with an adjustment to the ethanol treatment time. Specifically, in step F2, the scaffold model is immersed in 75% ethanol for toughening treatment for 10 minutes. The remaining steps and processes are the same as in Example 4, and therefore will not be repeated.

[0065] Example 8

[0066] This embodiment provides a biological scaffold, which is based on Example 4 with an adjustment to the ethanol treatment time. Specifically, in step F2, the scaffold model is immersed in 75% ethanol for toughening treatment for 30 minutes. The remaining steps and processes are the same as in Example 4, and therefore will not be repeated.

[0067] Comparative Example 1

[0068] This comparative example provides a 3D printing composite bio-ink composed of the following components by weight percentage: 20% methacrylamide gelatin, 15% nano-hydroxyapatite, 0.5% photoinitiator, and the balance being deionized water. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP).

[0069] The preparation method of this 3D printing composite bio-ink includes the following steps:

[0070] S1. Preparation of GelMA solution: Dissolve lyophilized methacrylamide gelatin powder in deionized water, remove bubbles, and obtain GelMA solution;

[0071] S2. GelMA solution and nHA powder are mixed: Nano hydroxyapatite powder is added to the GelMA solution prepared in step S1 and ultrasonically dispersed for 30 minutes with an ultrasonic power of 300W to form a uniform slurry.

[0072] S3. Add the photoinitiator to step S2 and stir and mix at 150 rpm for 20 minutes under light-protected conditions to obtain 3D printing composite bio-ink.

[0073] Comparative Example 2

[0074] This comparative example provides a biological scaffold, which is 3D printed using the 3D printing composite bio-ink prepared in Comparative Example 1 according to a preset three-dimensional model. The preparation method is the same as that in Example 4, so it will not be described again.

[0075] Comparative Example 3

[0076] This comparative example provides a biological scaffold, which is based on Example 4 but without the real-time blue light irradiation curing process. The remaining steps and processes are the same as in Example 4, so they will not be described again.

[0077] Experiment 1

[0078] The biological scaffolds prepared in Example 4 and Comparative Example 2 were subjected to compressive strength, elastic modulus and 2000 cycles of compression tests to verify the basic composition and mechanical properties. The results are shown in Table 1 below.

[0079] Table 1. Test results of Example 4 and Comparative Example 2

[0080] As shown in Table 1, this invention optimizes the ratio of TSF, GelMA and nHA to construct a ternary synergistic system, which significantly improves toughness and strength, meeting the requirements for load-bearing bone repair (>10 MPa).

[0081] Experiment 2

[0082] The bio-scaffolds prepared in Examples 4-6 were subjected to compressive strength tests and printing evaluations (continuous printing for 1 hour and recording the number of blockages) to optimize and verify the nHA content. The results are shown in Table 2 below.

[0083] Table 2. Test results of Examples 4-6

[0084] As shown in Table 2, the printability and strength are optimal when the nHA content is 15%, with a clogging rate of 0%. This invention, through optimized material proportioning, achieves both good mechanical and printability properties.

[0085] Experiment 3

[0086] The biological scaffolds prepared in Examples 4, 7 and 8 were subjected to crystallinity (XRD) and interlayer bonding strength (universal testing machine) tests, respectively. The multi-level crosslinking sequence was optimized and verified by adjusting the ethanol treatment time. The results are shown in Table 3 below.

[0087] Table 3 shows the experimental results of Examples 4, 7, and 8.

[0088] As shown in Table 3, when the ethanol treatment time is adjusted to 20 min, the interlayer strength is significantly improved.

[0089] Experiment 4

[0090] The bio-scaffolds prepared in Example 4 and Comparative Example 3 were subjected to complex structure printing accuracy evaluation and verification. The bio-scaffolds used a biomimetic trabecular bone structure model (pore size gradient of 50~500μm), and the printing equipment was a SunP BioMaker2i. Accuracy evaluation was performed by analyzing the matching degree between CT scans and the design model to determine structural fidelity (%), and by measuring the minimum pore size and interlayer misalignment. The results are shown in Table 4 below.

[0091] Table 4, Test Results of Example 4 and Comparative Example 3

[0092] As shown in Table 4, the present invention achieves the printing of microstructures at the 50μm level with a fidelity of >96%, which far exceeds that of traditional solutions.

[0093] The above embodiments are merely illustrative of the concept and technical solution of the present invention and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

[0094] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A 3D printing composite bio-ink, characterized in that, It consists of the following components by weight percentage: 5%~8% tussah silk fibroin, 15%~25% methacrylamide gelatin, 10%~20% nano hydroxyapatite, 0.25%~0.5% photoinitiator, and the balance deionized water.

2. The 3D printing composite bio-ink according to claim 1, characterized in that, The mass ratio of the tussah silk fibroin to the methacrylamide gelatin is 1:(3~5).

3. The 3D printing composite bio-ink according to claim 1, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

4. A method for preparing a 3D printing composite bio-ink as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Prepare TSF solution: Dissolve tussah silk fibroin in a portion of deionized water to obtain TSF solution; S2. Prepare GelMA solution: Dissolve methacrylamide gelatin in the remaining deionized water, remove air bubbles, and obtain GelMA solution; S3. Mix GelMA solution with nHA powder: Add nano-hydroxyapatite to the GelMA solution prepared in step S2 and ultrasonically disperse to form a uniform slurry; S4. Add the TSF solution prepared in step S1 to the slurry in step S3 and stir to mix evenly; S5. Add photoinitiator to step S4, stir and mix in the dark to obtain 3D printing composite bio-ink.

5. The method for preparing 3D printing composite bio-ink according to claim 4, characterized in that, In step S3, the ultrasonic dispersion conditions are: ultrasonic power of 200W~400W and ultrasonic dispersion time of 20min~50min.

6. The method for preparing 3D printing composite bio-ink according to claim 4, characterized in that, In step S4, the stirring and mixing conditions are: stirring speed of 200 rpm to 400 rpm and stirring time of 20 min to 40 min; in step S5, the stirring and mixing conditions in the dark are: stirring speed of 100 rpm to 200 rpm and stirring time of 15 min to 45 min.

7. A biological scaffold, characterized in that, The 3D printing composite bio-ink according to any one of claims 1 to 3 is prepared by 3D printing.

8. A method for preparing a biological scaffold as described in claim 7, characterized in that, The process includes the following steps: F1, Primary photocrosslinking: Using the above-mentioned 3D printing composite bio-ink, 3D printing is performed according to a preset three-dimensional model, with a strength of 10mW / cm. 2 ~15mW / cm 2 The scaffold model was irradiated with 405nm blue light for 90s~120s to obtain a shaped scaffold model; F2, secondary physical cross-linking pretreatment: the scaffold model obtained in step F1 was immersed in 75% ethanol for toughening treatment for 10min~30min; F3, tertiary ion bonding posttreatment: the scaffold model treated in step F2 was immersed in 10% CaCl2 solution for strengthening treatment for 1min~10min; F4, freeze drying: the scaffold model treated in step F3 was freeze dried to obtain a biological scaffold.

9. The method for preparing a biological scaffold according to claim 8, characterized in that, In step F1, the 3D printing process conditions are set as follows: printing temperature is set to 18℃~25℃, layer height is set to 0.2mm±0.05mm, filament diameter is set to 0.3mm±0.05mm, spacing is set to 0.4mm±0.05mm, printing speed is set to 5mm / s~15mm / s, and extrusion speed is set to 0.5mm. 3 / s~3.0mm 3 / s.

10. The method for preparing a biological scaffold according to claim 8, characterized in that, In step F4, the freeze-drying process includes the following operations: first, the scaffold model sample is quenched in liquid nitrogen for 30s~60s, and then transferred to -80℃ to completely freeze the scaffold model sample into a hard block; then, the scaffold model sample is placed in the sample chamber of a freeze dryer with a cold trap temperature below -80℃ for freeze-drying.

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