A biomimetic bio-ink for 3D bioprinting and a method for preparing the same

Abstract

The application discloses a kind of biomimetic biological ink for 3D biological printing and its preparation method and application;The biomimetic biological ink is composite biological ink system, at least two sub-inks are included;Each sub-ink includes hydrogel matrix and one stimulus-responsive functional material, the functional material encapsulates bioactive substance;Wherein the functional material in different sub-ink is configured to respond to different types of external non-invasive physical stimulation respectively, and structural change occurs after being stimulated to release the encapsulated bioactive substance;The application also provides the customized preparation method of the ink and the method for manufacturing gradient differentiation tissue engineering construction by multi-channel printing and programmed stimulation using the ink;The application realizes the spatiotemporal programming accurate release of bioactive substance by constructing signal release library that can respond to multiple external stimuli, effectively solves the technical problem that traditional biological ink is difficult to construct complex biomimetic gradient tissue.

Description

Technical Field

[0001] This invention relates to the field of biomaterials and tissue engineering technology, specifically to a biomimetic bio-ink for 3D bioprinting that enables spatiotemporally programmable gradient differentiation, its preparation method, and its application. Background Technology

[0002] Tissue engineering aims to repair and regenerate damaged tissues and organs. Constructing tissues with complex structures and functions, especially composite tissues containing multiple cell types in a gradient distribution, remains a major challenge. Traditional bio-inks typically have a single function and struggle to precisely control cell fate in different regions after printing, leading to a mismatch in structure and function between the regenerated and original tissues.

[0003] Existing gradient differentiation strategies largely rely on alternating printing of multiple materials and subsequent infusion of growth factors, which suffer from problems such as weak interfacial binding, uncontrollable signal release, and poor spatiotemporal precision. Therefore, developing a smart bio-ink that can pre-encapsulate multiple differentiation signals and release them through precise spatiotemporal programming via external stimuli is of vital importance for advancing the development of complex tissue engineering. Summary of the Invention

[0004] Purpose of the invention The purpose of this invention is to overcome the shortcomings of the prior art and provide a biomimetic bio-ink for 3D bioprinting. It can achieve the spatiotemporal programming and precise release of bioactive substances by constructing a signal release library that can respond to a variety of external stimuli, thereby effectively constructing complex tissues with biomimetic gradients.

[0005] Another objective of this invention is to provide a method for preparing the above-mentioned biomimetic bio-ink. This method is based on the design of the biomimetic differentiation gradient map of the target tissue. By precisely controlling the encapsulation, dispersion and multi-channel encapsulation processes of functional materials, the ink system achieves high stability, high uniformity and good biocompatibility under mild conditions. The process is stable and has good reproducibility.

[0006] Technical solution In a first aspect, the present invention provides a biomimetic bio-ink for 3D bioprinting.

[0007] The biomimetic bio-ink is a composite bio-ink system composed of at least two sub-inks with different functions. Each of the sub-inks comprises: a) A biocompatible hydrogel matrix; b) A stimulus-responsive functional material.

[0008] The stimulus-responsive functional material encapsulates at least one bioactive substance, and its shell material is designed to undergo structural changes upon receiving a preset external non-invasive physical stimulus, thereby releasing the encapsulated bioactive substance.

[0009] The key point is that the stimulus-responsive functional materials contained in the at least two sub-inks have shell materials with different physical and chemical structures, which change in structure in response to different types of external non-invasive physical stimuli.

[0010] Preferably, the biomimetic bio-ink further includes a separately configured cell-loading component. The cell-loading component is a cell suspension containing seed cells; the seed cells are preferably bone marrow-derived and adipose-derived mesenchymal stem cells with a purity of not less than 90%. In use, the seed cells and the bio-ink are simultaneously extruded and regionally co-distributed using a multi-channel bioprinter, with a preferred density of 1×10⁻⁶ cells / cm² within the final scaffold. 5 cells / mL to 1×10 7 cells / mL.

[0011] To achieve precise and specific release of bioactive substances, the shell materials of various functional materials need to be specifically designed: For photoresponsive functional materials, the shell material is a biocompatible copolymer grafted with photolytically cleavable chemical bonds, such as polyethylene glycol methacrylate-polycaprolactone copolymer. The photolytically cleavable chemical bonds are selected from o-nitrobenzyl ester groups. The grafting rate of these photolytically cleavable chemical bonds is preferably 10%-30%.

[0012] For a stimulus-responsive functional material that responds to ultrasonic stimulation, its shell material is a biocompatible polyelectrolyte cross-linked with an ultrasonic-responsive cross-linking agent. The ultrasonic-responsive cross-linking agent is selected from at least one of dithiobisacrylamide and cystamine diacrylate. The polyelectrolyte is selected from at least one of chitosan and sodium alginate. The mass percentage of the cross-linking agent is preferably 2%-8%.

[0013] For stimuli-responsive functional materials that respond to temperature changes, the shell material is a thermosensitive copolymer with a defined low critical dissolution temperature, such as poly(N-isopropylacrylamide)-polyethylene glycol copolymer, whose low critical dissolution temperature is preferably 32°C-37°C.

[0014] For a stimulus-responsive functional material that responds to magnetic field stimulation, its shell material is a thermosensitive copolymer with a defined hypercritical dissolution temperature, and dispersed with superparamagnetic Fe3O4 nanoparticles. The copolymer is poly(acrylamide-co-sodium acrylate). The average particle size of the nanoparticles is preferably 10-50 nm, with a mass percentage of 5%-20%, and the hypercritical dissolution temperature of the shell material is preferably 40℃-45℃.

[0015] The bioactive substance may be selected from at least one of vascular endothelial growth factor (VEGF), nerve growth factor (NGF), transforming growth factor (TGF-β1) and TGF-β3, and growth differentiation factor (GDF-5).

[0016] Regarding ink performance control, the mass concentration of the stimulus-responsive functional material in each sub-ink is preferably between 0.5 mg / mL and 20 mg / mL; and the mass concentration ratio of the functional material in different sub-inks is set based on the biomimetic differentiation gradient map of the target tissue, with a preferred ratio range of 1:10 to 10:1. The average particle size of the functional material is preferably between 0.2 μm and 20 μm, and the coefficient of variation of dispersion in the corresponding sub-ink is not greater than 15%.

[0017] To further ensure printing accuracy and process monitoring during application, fluorescently labeled microspheres may be added to at least one sub-ink. The fluorescently labeled microspheres preferably have an average particle size of 0.1-1 μm, a mass concentration of 0.01-0.1 mg / mL, and are made of silica.

[0018] Secondly, the present invention also provides a method for preparing the above-mentioned biomimetic ink.

[0019] The method is based on the biomimetic differentiation gradient map of the target tissue and includes the following steps: S1. Based on the biomimetic differentiation gradient map, determine the type of sub-ink required, the type of bioactive substance that the functional material in each sub-ink should be loaded with, the target mass concentration, and the combination ratio of the sub-inks.

[0020] S2. For each seed ink, based on its target response type and the characteristics of the loaded bioactive substances, select an appropriate encapsulation process to prepare stimulus-responsive functional materials loaded with the corresponding bioactive substances.

[0021] S3. Preparation of hydrogel matrix precursor solution: Dissolve commercially available biocompatible hydrogel material in a buffer solution with a pH of 7.0-7.4 and adjust its mass concentration to 5%-10% to obtain the corresponding hydrogel matrix precursor solution.

[0022] S4. Add each prepared stimulus-responsive functional material to the corresponding hydrogel matrix precursor solution prepared in S3 according to the target mass concentration determined in S1, and ultrasonically disperse it for 5-10 minutes under a power of 50-100W to obtain at least two uniformly dispersed sub-inks.

[0023] S5. The at least two sub-inks are filled and sealed in the independent channels of a multi-channel composite container according to the combination ratio determined in S1, and stored at 2℃-8℃ under light-proof conditions to form the biomimetic bio-ink.

[0024] The packaging process described in step S2 can be optimized as needed: When preparing photoresponsive materials, photolytic groups can be grafted through free radical copolymerization. The molar ratio of the photoresponsive monomer to the basic polymer monomer is preferably 1:5 to 1:20, and the amount of initiator is 0.5%-2% of the total mass of the monomer.

[0025] When preparing ultrasonically responsive materials, a crosslinking agent is added to a polyelectrolyte solution with a concentration of 0.5%-2% (w / v) for crosslinking, followed by washing and freeze-drying.

[0026] When preparing temperature-responsive materials, the thermosensitive copolymer is dissolved to form a 1%-5% solution. After adding bioactive substances, microspheres are formed by one of the emulsification-solvent evaporation method and microfluidic focusing method, and the mass ratio of hydrophilic to hydrophobic segments is controlled to be 1:9 to 3:7.

[0027] When preparing magnetic field responsive materials, Fe3O4 nanoparticles need to be ultrasonically dispersed at a power of 100W-300W for 10-30 minutes to uniformly disperse them in the shell polymer solution.

[0028] One of the beneficial effects of this invention is that by constructing a composite system containing at least two functional sub-inks that respond to different stimuli, the programmability and release specificity of the differentiation signal are guaranteed, thus solving the problem that traditional single-material inks cannot achieve complex spatiotemporal signal encoding.

[0029] The second beneficial effect of this invention is that by precisely designing and limiting the parameters of the shell materials and key components of different response units such as light, ultrasound, temperature, and magnetic field, the reliability, specificity, and biocompatibility of the stimulus response behavior of each unit are improved, demonstrating a high degree of technical controllability.

[0030] The third beneficial effect of this invention is that by specifying the monomer ratio, initiator dosage, dispersion parameters and storage conditions in the preparation process, the uniformity and stability between batches of functional units and final ink products are improved, forming a repeatable and standardized preparation process.

[0031] The fourth beneficial effect of this invention is that by quantifying the precision of the region arrangement and the spatiotemporal program parameters in the application method, it achieves precise digital control of the tissue construction process from macroscopic structure to microscopic signal release.

[0032] The fifth beneficial effect of this invention is that by integrating auxiliary units such as independent cell-loading components and highly stable fluorescently labeled microspheres, cell viability is protected, and the online calibration accuracy of the printing process and the overall success rate of the solution are improved.

[0033] Ultimately, this invention deeply integrates materials chemistry, 3D printing, and external physical field modulation technology, achieving one-stop construction from biomimetic design drawings to functionalized tissues, providing a brand-new solution for the repair of complex tissue defects in the field of regenerative medicine. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the preparation method of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions and conditions recommended by the manufacturer. Reagents and instruments used, unless otherwise specified, are all commercially available conventional products.

[0036] Example 1: A dual-response ink system for constructing bone-cartilage gradient tissue: Materials and proportions: Photoresponsive quantum ink: Matrix solution: 8% (w / v) methacrylamide gelatin (GelMA) in PBS.

[0037] Raw materials for the preparation of functional materials - photolytic microspheres: Polyethylene glycol methacrylate (PEGMA, Mw-500): 0.70 g; Polycaprolactone (PCL, Mw-50k): 0.30 g; o-Nitrobenzyl methacrylate: 0.05 g; Initiator: Azobisisobutyronitrile (AIBN): 10.5 mg; Solvent: Dichloromethane (DCM): 20 mL; Bioactive substance: Recombinant human bone morphogenetic protein-2 (rhBMP-2): 100 μg; Stabilizer: Bovine serum albumin (BSA): 1 mg; Emulsifier: Polyvinyl alcohol: 4.0 g; Aqueous phase: Deionized water: 200 mL.

[0038] Temperature-responsive sub-ink: Matrix solution: 6% (w / v) GelMA in PBS.

[0039] Raw materials for the preparation of functional materials - temperature-sensitive microspheres: Shell polymer: Poly(N-isopropylacrylamide)-polyethylene glycol copolymer: 0.30 g; Bioactive substance: Transforming growth factor-β3 (TGF-β3): 50 μg; Dispersed phase solvent: Deionized water: 10 mL; Continuous phase: Liquid paraffin: 100 mL; Crosslinking agent solution: PBS solution of N,N'-methylenebisacrylamide (BIS) (10 mM): 50 mL.

[0040] Cell-loaded components: Seeding cells: 5.0 × 10^6 third-generation human bone marrow mesenchymal stem cells (BMSCs); Culture medium: 1 mL of α-MEM medium containing 10% fetal bovine serum (FBS).

[0041] Preparation method: S1. Determine the required sub-ink types as light-responsive sub-ink and temperature-responsive sub-ink.

[0042] S2. Preparation of functional materials: Preparation of photoresponsive functional materials: A double emulsion-solvent evaporation method was employed. 0.7 g PEGMA, 0.3 g PCL, 0.05 g o-nitrobenzyl methacrylate, and 1.0% (by weight) of the initiator azobisisobutyronitrile (AIBN) were dissolved in 20 mL dichloromethane to form the oil phase. 100 μg rhBMP-2 was dissolved in 1 mL of 0.1% bovine serum albumin (BSA) in PBS to form the inner aqueous phase. Under ice bath and light-protected conditions, the inner aqueous phase was injected into the oil phase, and homogenization was carried out at 10,000 rpm for 2 minutes to form the primary emulsion. The primary emulsion was slowly poured into 200 mL of an aqueous solution containing 2% (w / v) polyvinyl alcohol, and stirred at 500 rpm to form the double emulsion. Subsequently, the system was heated to 65 °C and reacted under nitrogen protection for 6 hours to complete the free radical copolymerization of the shell polymer and graft photolytic groups. After the reaction was completed, stirring was continued for 12 hours to allow complete evaporation of the dichloromethane. The microspheres were collected by centrifugation at 8000 rpm for 10 min, washed three times with deionized water, and freeze-dried for 24 hours to obtain freeze-dried microsphere powder with an average particle size of 15 μm.

[0043] Preparation of temperature-responsive functional materials: Microfluidic focusing was employed. PNIPAAAm-PEG copolymer was dissolved in deionized water to prepare a 3% (w / v) solution as the dispersed phase, containing 50 μg of TGF-β3. Liquid paraffin was used as the continuous phase. Using a microfluidic chip, the flow rate of the dispersed phase was controlled at 10 μL / min, and the flow rate of the continuous phase at 100 μL / min, forming monodisperse droplets. The droplets flowed into a collection tank containing a crosslinking agent solution and were allowed to solidify at room temperature for 3 hours. The microspheres were collected by centrifugation, washed three times alternately with n-hexane and ethanol, and then washed twice with PBS buffer. Finally, they were dispersed in PBS for later use, with an average particle size of 12 μm.

[0044] S3. Preparation of hydrogel matrix precursor solutions: Weigh 0.8 g and 0.6 g of GelMA powder respectively, and dissolve each in 10 mL of pre-cooled PBS (pH 7.4). Stir magnetically at 300 rpm for 6 hours at 4°C in the dark until completely dissolved, obtaining 8% (w / v) and 6% (w / v) GelMA precursor solutions, respectively, and store temporarily at 4°C.

[0045] S4. Preparation and Dispersion of Sub-Ink: Accurately weigh 20 mg of lyophilized photoresponsive microsphere powder and add it to 10 mL of 8% GelMA solution. Take 1 mL of PBS dispersion containing TGF-β3 microspheres, centrifuge and discard the supernatant, then resuspend in 1 mL of 6% GelMA solution. First, magnetically stir the above two mixtures at 200 rpm for 10 minutes in an ice bath, then sonicate them in an ice bath for 5 minutes using an ultrasonic cell disruptor at 60W in pulse mode. Examine samples under a microscope, count more than 100 microspheres, and calculate the dispersion coefficient of variation to be <15%.

[0046] S5. Ink Filling and Storage: In a sterile operating room, draw the two sub-inks into two separate 5 mL sterile syringes, removing any air bubbles. Install the syringes into the Regenovo Bio-Architect... ® The printhead is stored in an independent cartridge within the dual-channel printer. The entire printhead is placed in a 4°C medical refrigerator, strictly protected from light, for later use.

[0047] Example 2 is used to construct a three-component system for the muscle-tendon-bone interface: Materials and proportions: Photoresponsive quantum ink: Matrix solution: 5% (w / v) GelMA in PBS.

[0048] Functional material: same as the photolysis microspheres prepared in Example 1.

[0049] Ultrasonic responsive sub-ink: Matrix solution: 4% (w / v) sodium alginate aqueous solution.

[0050] Raw materials for the preparation of functional materials - ultrasonically responsive microspheres: Shell polymer: Chitosan: 0.30 g; Solvent: 1% (v / v) acetic acid aqueous solution: 20 mL; Bioactive substance: Growth differentiation factor-5 (GDF-5): 200 μg; Crosslinking agent: Dithiobisacrylamide: 15 mg; Ionic crosslinking agent: Sodium tripolyphosphate (TPP): 4.0 g; Ionic crosslinking agent solvent: Deionized water: 200 mL.

[0051] Non-irritating sub-ink: Matrix solution: a mixed solution of 3% (w / v) GelMA and 1% (w / v) sodium alginate.

[0052] Cell-loaded components: Seeding cells: BMSCs: 1.5 × 10^6; C2C12 myoblasts: 1.5 × 10^6; Mixed cell suspension: The mixed cell density was adjusted to 3 × 10^6 cells / mL using DMEM medium containing 10% FBS.

[0053] Preparation method: S1: Determine the required sub-ink types as photoresponsive sub-ink, ultrasonic responsive sub-ink, and non-irritating sub-ink.

[0054] S2. Preparation of functional materials: Preparation of photoresponsive functional materials: Same as step 1 in Example 1.

[0055] Preparation of ultrasound-responsive functional materials: 0.3 g of chitosan was dissolved in 20 mL of 1% (v / v) acetic acid solution and stirred overnight. 200 μg of GDF-5 was added and mixed well. This mixture was then added dropwise at a rate of 20 mL / h to 200 mL of a stirred 2% (w / v) sodium tripolyphosphate (TPP) solution. After the addition was complete, stirring was continued for 30 minutes to induce ion pregelation. Subsequently, 0.015 g of dithiobisacrylamide was added to the system, and the mixture was placed in a 30℃ constant temperature water bath and stirred at 300 rpm for 2 hours. After the reaction was completed, microspheres were collected through a 200-mesh sieve, washed repeatedly with deionized water 5 times, and freeze-dried for 48 hours.

[0056] S3. Preparation of hydrogel matrix precursor solutions: Prepare the following three solutions respectively: Solution A: Dissolve 0.5 g GelMA in 10 mL PBS at pH 7.4 and stir at 4°C.

[0057] Solution B: Dissolve 0.4 g sodium alginate in 10 mL of deionized water and stir at room temperature until transparent.

[0058] Solution C: Mix 3% (w / v) GelMA solution and 1% (w / v) sodium alginate solution at a volume ratio of 3:1 and stir until homogeneous.

[0059] S4. Preparation and dispersion of sub-ink: Photoresponsive ink: Weigh 30 mg of lyophilized photoresponsive microsphere powder, add it to 10 mL of solution A, and ultrasonically disperse it according to the method in Example 1.

[0060] Ultrasonic responsive ink: Weigh 15 mg of lyophilized ultrasonic responsive microsphere powder and add it to 10 mL of solution B. First, pre-disperse the powder by magnetic stirring at 400 rpm for 15 minutes, and then ultrasonically disperse it in an ice bath at 70 W for 8 minutes.

[0061] Non-irritating seed ink: Solution C can be used directly as the third seed ink.

[0062] S5. Ink Filling and Storage: Fill the three types of ink into the three independent 5 mL storage tanks of the CELLINK BIO X three-channel printing system. Store the entire system in a 4℃ cold storage.

[0063] Example 3: A dual-trigger system based on magnetocaloric and thermosensitive phase transition: Materials and proportions: Magnetic field responsive sub-ink: Matrix solution: 7% (w / v) GelMA in PBS.

[0064] Raw materials for the preparation of functional materials - magnetocaloric responsive microspheres: Shell polymer: poly(acrylamide-co-sodium acrylate) (P(AAm-co-AANA), AAm: AANA = 9:1mol / mol): 0.50 g; Magnetic nanoparticles: Oleic acid-modified Fe3O4 nanoparticles: 88 mg; Solvent: Chloroform: 20 mL; Bioactive substance: Vascular endothelial growth factor (VEGF): 200 μg; Emulsifier: Span-80: 2.0 g; Aqueous phase: Deionized water: 200 mL.

[0065] Temperature-responsive sub-ink: Matrix solution: 5% (w / v) GelMA in PBS.

[0066] Functional material: The same temperature-sensitive microspheres prepared in Example 1.

[0067] Cell-loaded components: Seed cells: Human umbilical cord mesenchymal stem cells (hUMSCs): 4.0 × 10^6.

[0068] Cell suspension: The cell density was adjusted to 4 × 10^6 cells / mL using α-MEM medium containing 10% FBS.

[0069] Preparation method: S1: Determine the types of sub-inks required: magnetic field responsive sub-inks and temperature responsive sub-inks.

[0070] S2. Preparation of functional materials: Preparation of magnetic field-responsive functional materials: 0.5 g P(AAm-co-AANA) and 0.088 g Fe3O4 nanoparticles were dissolved in 20 mL chloroform and ultrasonically dispersed at 250 W for 20 min to form a homogeneous oil phase. 200 μg VEGF was dissolved in 0.5 mL PBS as the inner aqueous phase. Under ice bath conditions, the inner aqueous phase and oil phase were mixed and homogenized at 15000 rpm for 1 min to form a pre-emulsion. The pre-emulsion was poured into 200 mL of an aqueous solution containing 1% (w / v) Span-80 and stirred overnight at 400 rpm to allow complete chloroform evaporation. Microspheres were collected with the aid of a magnet and washed three times each with 0.1% Tween-80 aqueous solution, ethanol, and deionized water, and then freeze-dried.

[0071] Preparation of temperature-responsive functional materials: Same as step 1 in Example 1, except that the loading factor is replaced with NGF.

[0072] S3. Preparation of hydrogel matrix precursor solutions: Weigh 0.7 g and 0.5 g of GelMA powder respectively, dissolve them in 10 mL of PBS, stir at 4℃ to dissolve, and obtain 7% and 5% GelMA precursor solutions.

[0073] S4. Preparation and Dispersion of Sub-Ink: Magnetic field-responsive microspheres and temperature-responsive microspheres were added to the corresponding GelMA solutions at concentrations of 2.5 mg / mL and 2.0 mg / mL, respectively. All operations were performed in an ice bath. The microspheres were ultrasonically dispersed in an ice bath for 10 minutes at 80W power. Samples were taken immediately after dispersion for analysis; the coefficient of variation was <15%.

[0074] S5. Ink Filling and Storage: Quickly fill the two sub-inks into the pre-cooled dual-channel printing cartridge. Immediately transfer to a low-temperature, light-protected incubator at 2-8℃ for storage. Equilibrate on the printer's cooling stage for 30 minutes before printing.

[0075] Example 4: Highly Integrated Complex Systems and Printing Calibration Materials and proportions: Photoresponsive quantum ink: Matrix solution: 10% (w / v) GelMA in PBS.

[0076] Functional material: same as the photolysis microspheres prepared in Example 1.

[0077] Fluorescent label: Red fluorescent silica microspheres: 0.5 mg.

[0078] Ultrasonic responsive sub-ink: Matrix solution: a mixed PBS solution of 6% (w / v) GelMA and 1% (w / v) sodium alginate.

[0079] Raw materials for the preparation of functional materials - ultrasonically responsive microspheres: Chitosan (deacetamide degree 90%): 0.30 g; 1% acetic acid solution: 20 mL; Medication: Dexamethasone: 3 mg; Crosslinking agent: Cystamine diacrylate: 9 mg; 2% TPP aqueous solution: 200 mL; Fluorescent label: Green fluorescent silica microspheres: 0.5 mg.

[0080] Temperature-responsive sub-ink: Matrix solution: 5% (w / v) GelMA in PBS.

[0081] Functional material: Thermosensitive microspheres (PNIPAAm-PEG, LCST=33℃, loaded with TGF-β1), the raw materials are the same as in Example 1, except that TGF-β3 is replaced with TGF-β1.

[0082] Magnetic field responsive sub-ink: Matrix solution: 4% (w / v) hyaluronic acid methacrylamide (HAMA) in PBS.

[0083] Functional material: magnetocaloric responsive microspheres, prepared from the same raw materials as in Example 3, except that the loaded drug is dexamethasone.

[0084] Cell-loaded components: Seed cells: BMSCs: 6.0 × 10^6.

[0085] Cell suspension: The cell density was adjusted to 6 × 10^6 cells / mL using α-MEM medium containing 10% FBS.

[0086] Fluorescent labeling: 0.05 mg / mL of red (Ex / Em: 580 / 605 nm) and green (Ex / Em: 488 / 520 nm) fluorescent silica microspheres were added to the photoresponsive and ultrasonic responsive inks, respectively.

[0087] Preparation method: S1: Determine the required sub-ink types as light-responsive sub-ink, ultrasonic-responsive sub-ink, temperature-responsive sub-ink, and magnetic field-responsive sub-ink.

[0088] S2. Preparation of functional materials: Light, temperature, and magnetic field responsive functional materials: The preparation methods are the same as in Examples 1 and 3, respectively.

[0089] Ultrasonic responsive functional material: The preparation method is the same as in Example 2, except that the crosslinking agent is replaced with cystamine diacrylate, the amount of which is 3% of the mass of chitosan, and the loaded drug is dexamethasone.

[0090] S3. Preparation of hydrogel matrix precursor solution: 10% GelMA: Dissolve 1.0 g GelMA in 10 mL PBS and stir overnight at 4°C.

[0091] 6% GelMA + 1% Sodium Alginate: Mix 6% (w / v) GelMA solution with 2% (w / v) sodium alginate solution at a volume ratio of 1:1.

[0092] 5% GelMA: 0.5 g GelMA dissolved in 10 mL PBS.

[0093] 4% HAMA: Dissolve 0.4 g HAMA in 10 mL PBS and stir overnight at 4°C in the dark.

[0094] S4. Preparation and Dispersion of Sub-Inks: The four functional microspheres were added to their respective matrix solutions according to the specified ratios. Corresponding fluorescent microspheres of the corresponding colors were added to the photoresponsive and ultrasonically responsive sub-inks. Each ink was then ultrasonically dispersed in an ice bath. Light-responsive ink: 60W, 6 min; Ultrasonic-responsive ink: 70W, 8 min; Temperature-responsive ink: 60W, 5 min; Magnetic field-responsive ink: 80W, 7 min.

[0095] S5. Ink Filling and Precision Printing Preparation: Using the CELLINK BIO X6 six-channel bioprinter, select four independent channels. Precisely inject the four types of ink into four independent 3 mL printing cartridges, strictly avoiding cross-contamination. Store the loaded printhead at 4°C away from light. In the printer's software, set the imaging parameters for the red and green fluorescence channels and associate them with the corresponding printing channels for real-time extrusion path calibration during the printing process.

[0096] Example 5: Traditional control system Materials and proportions: Bio-ink: Matrix solution: 6% (w / v) GelMA in PBS.

[0097] Bioactive substances: rhBMP-2: 10 μg; TGF-β3: 5 μg.

[0098] Preparation: rhBMP-2 and TGF-β3 were directly dissolved in 10 mL of the above GelMA solution.

[0099] Cell-loaded components: Seed cells: BMSCs: 5.0 × 10^6.

[0100] Pre-mixing cell suspension: The cell density was adjusted to 1 × 10^7 cells / mL using α-MEM medium containing 10% FBS.

[0101] Preparation method: Preparation of bio-ink: Weigh 0.6 g of GelMA powder and dissolve it in 10 mL of PBS (pH 7.4). Stir at 4°C until completely dissolved. Add 10 μg rhBMP-2 and 5 μg TGF-β3 directly to the above solution and gently vortex at 4°C for 10 seconds, avoiding the formation of bubbles. Prepare the solution immediately before use.

[0102] Printing preparation: Before printing, mix the prepared growth factor-containing ink with 0.5 mL of BMSCs suspension (1×10⁻⁶). 7 Mix briefly on ice (cells / mL) to achieve a final cell density of 5 × 10⁻⁶. 6 cells / mL. Aspirate the mixture into the 3 mL cartridge of the single-channel printer and begin printing immediately.

[0103] Application effect verification The constructs from Examples 1-4 were cultured and stimulated strictly according to a pre-defined program. The results showed that the cells in Examples 1-4 had high survival rates and highly expressed osteogenic and chondrogenic markers in the stimulated regions, respectively, with a gradient transition at the interface. In contrast, the signal distribution in the control group of Example 5 was diffuse, the differentiation direction was chaotic, and an ordered gradient structure could not be formed. The construct from Example 4 successfully reproduced the specific marker distribution of the multi-layered structure of a joint under quadruple stimulation. This fully demonstrates the superior ability of this invention to guide the gradient differentiation of complex tissues through spatiotemporal programming.

[0104] Table 1. Characterization of Printed Structure Accuracy and Physical Properties ; Table 2. In vitro release kinetics of bioactive substances in response to stimulation (cumulative release rate, %) ; Table 3. Expression of differentiation-related genes after 14 days of in vitro culture (qRT-PCR, relative expression levels, with GAPDH as an internal reference). ; Table 4. Histological scores of in vivo repair efficacy (ICRS score, 12 weeks post-operation) ; in conclusion This invention constructs a highly efficient, controllable, and customizable biomimetic bio-ink system through the integration of multiple responsive functional units, a spatiotemporal programming release mechanism, and the coordinated control of multi-channel printing and external stimuli. It exhibits significant advantages in complex tissue construction, precise signal modulation, and material and process standardization, providing an innovative solution for the fields of tissue engineering and regenerative medicine, and possesses significant scientific research value and clinical application prospects.

[0105] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A biomimetic bio-ink for 3D bioprinting, characterized in that, The biomimetic bio-ink is a composite bio-ink system containing at least two sub-inks, each of which includes: a) A biocompatible hydrogel matrix; b) A stimulus-responsive functional unit; The stimulus-responsive functional material encapsulates at least one bioactive substance; The at least two types of sub-inks contain stimulus-responsive functional materials, each with a shell material having a different structure to undergo structural changes in response to different types of external non-invasive physical stimuli. For photo-responsive functional materials, the shell material is a biocompatible copolymer grafted with photolytically cleavable chemical bonds, wherein the copolymer is polyethylene glycol methacrylate-polycaprolactone copolymer; and the photolytically cleavable chemical bond is o-nitrobenzyl ester group. For a stimulus-responsive functional material that responds to ultrasonic stimulation, its shell material is a biocompatible polyelectrolyte cross-linked with an ultrasonic-responsive cross-linking agent, wherein the ultrasonic-responsive cross-linking agent is selected from at least one of dithiobisacrylamide and cystamine diacrylate, and the polyelectrolyte is selected from at least one of chitosan and sodium alginate. For a stimulus-responsive functional material that responds to temperature changes, its shell material is a thermosensitive copolymer with a defined low critical dissolution temperature, wherein the copolymer is a poly(N-isopropylacrylamide)-polyethylene glycol copolymer, and the low critical dissolution temperature is 32℃-37℃. For a stimulus-responsive functional material that responds to magnetic field stimulation, its shell material is a thermosensitive copolymer with a defined high critical dissolution temperature, wherein the copolymer is poly(acrylamide-co-sodium acrylate) and dispersed with superparamagnetic nanoparticles.

2. The biomimetic ink according to claim 1, characterized in that, It also includes an independently configured cell-loading component, which is a cell suspension containing seed cells; the seed cells are mesenchymal stem cells derived from bone marrow and adipose tissue, with a purity of not less than 90%; the seed cells and the sub-ink are respectively loaded in different channels of the printing device, and the density of the seed cells in the scaffold used for construction is 1×10⁻⁶. 5 cells / mL to 1×10 7 cells / mL.

3. The biomimetic ink according to claim 1, characterized in that, The ultrasonically responsive crosslinking agent accounts for 2%-8% of the mass of the shell material; the superparamagnetic nanoparticles are Fe3O4 with an average particle size of 10-50 nm, accounting for 5%-20% of the mass of the shell material, and the high critical dissolution temperature is 40℃-45℃.

4. The biomimetic ink according to claim 1, characterized in that, The bioactive substance is selected from at least one of vascular endothelial growth factor (VEGF), nerve growth factor (NGF), transforming growth factor (TGF-β1) and TGF-β3, and growth differentiation factor (GDF-5).

5. The biomimetic ink according to claim 1, characterized in that, In each seed ink, the mass concentration of the stimulus-responsive functional material is from 0.5 mg / mL to 20 mg / mL; in the at least two seed inks, the mass concentration ratio of the stimulus-responsive functional material is from 1:10 to 10:

1.

6. The biomimetic ink according to claim 1, characterized in that, The average particle size of the stimulus-responsive functional material is 0.2 μm to 20 μm, and the coefficient of variation of dispersion in the corresponding sub-ink is no greater than 15%.

7. The biomimetic ink according to claim 1, characterized in that, At least one sub-ink also contains fluorescently labeled microspheres having an average particle size of 0.1-1 μm and a mass concentration of 0.01-0.1 mg / mL, and is made of silica.

8. A method for preparing the biomimetic bio-ink according to any one of claims 1-7, characterized in that, Includes the following steps: S1: Based on the biomimetic differentiation gradient map of the target tissue, determine the types of sub-inks required, the types of bioactive substances that the stimulus-responsive functional materials in each sub-ink should be loaded with, the target mass concentration, and the combination ratio between each sub-ink. S2: For each seed ink, based on its target response type and the characteristics of the loaded bioactive substances, select an appropriate encapsulation process to prepare stimulus-responsive functional materials loaded with the corresponding bioactive substances. S3: Preparation of hydrogel matrix precursor solution: Dissolve commercially available biocompatible hydrogel material in a buffer solution with a pH of 7.0-7.4 and adjust its mass concentration to 5%-10% to obtain the corresponding hydrogel matrix precursor solution; S4: Add each prepared stimulus-responsive functional material to the corresponding hydrogel matrix precursor solution prepared in S3 according to the target mass concentration determined in S1, and ultrasonically disperse it for 5-10 minutes under a power of 50-100W to obtain at least two uniformly dispersed sub-inks. S5: The at least two sub-inks are filled and sealed in the independent channels of a multi-channel composite container according to the combination ratio determined in S1, and stored at 2℃-8℃ under light-proof conditions to form the biomimetic bio-ink.

9. The preparation method according to claim 8, characterized in that, In step S2: In preparing photoresponsive functional materials, photolytic chemical bonds are grafted into the shell polymer via free radical copolymerization. The molar ratio of the photoresponsive monomer to the base polymer monomer is 1:5 to 1:

20. Azobisisobutyronitrile (AIBN) is used as the initiator, with an amount of 0.5%-2% of the total monomer mass and a grafting rate of 10%-30%. When preparing ultrasonic responsive functional materials, ultrasonic responsive crosslinking agent is added to a polyelectrolyte solution with a concentration of 0.5%-2% (w / v) at a mass ratio of 2%-8%, and crosslinked at 25℃-37℃ for 1-3 hours. After crosslinking is completed, the material is washed with deionized water and freeze-dried. In preparing temperature-responsive functional materials, a thermosensitive copolymer is dissolved in an organic solvent and water to form a solution with a mass concentration of 1%-5%. The bioactive substance to be encapsulated is added, and microspheres are formed by microfluidic focusing. The mass ratio of hydrophilic segments to hydrophobic segments in the thermosensitive copolymer is 1:9 to 3:7, and the average particle size of the microspheres is 0.2 μm to 20 μm. In the preparation of magnetic field responsive functional materials, superparamagnetic Fe3O4 nanoparticles with an average particle size of 10-50 nm are uniformly dispersed in a shell polymer solution by ultrasonic dispersion.

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