阅读说明：本技术 高强度生物医用水凝胶材料及浴支撑水凝胶3d打印方法 (High-strength biomedical hydrogel material and bath-supported hydrogel 3D printing method ) 是由 包春燕 蔡正伟 杨会婷 张洪波 胡天宙 刘多 薛源 王学斌 周耀武 项燕鑫 廖华 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种高强度生物医用水凝胶材料,是由以下重量份的组分制成：FDA15～40份,功能化生物医用大分子0～15份,引发剂0.05～0.3份,水20～200份。本发明还公开了采用所述高强度生物医用水凝胶材料进行浴支撑水凝胶3D打印方法,本发明中水凝胶组分和打印方法具有良好的生物相容性,可作为支架材料用于细胞的培养,在药物缓释、组织修复等领域具有重大潜在应用。(The invention discloses a high-strength biomedical hydrogel material which is prepared from the following components in parts by weight: 15-40 parts of FDA, 0-15 parts of functionalized biomedical macromolecules, 0.05-0.3 part of an initiator and 20-200 parts of water. The invention also discloses a 3D printing method of the bath supported hydrogel by adopting the high-strength biomedical hydrogel material, and the hydrogel component and the printing method have good biocompatibility, can be used as a scaffold material for cell culture, and have great potential application in the fields of drug sustained release, tissue repair and the like.)

1. A high-strength biomedical hydrogel material is characterized by being prepared from the following components in parts by weight:

15-40 parts of FDA, 0-15 parts of functionalized biomedical macromolecules, 0.05-0.3 part of an initiator and 20-200 parts of water.

2. The high-strength biomedical hydrogel material according to claim 1, wherein the high-strength biomedical hydrogel material is prepared from the following components in parts by weight: 15-30 parts of FDA, 1-5 parts of functionalized biomedical macromolecules, 0.1-0.2 part of an initiator and 20-200 parts of water.

3. The high strength biomedical hydrogel material of claim 1 or 2, wherein the FDA is acrylate functionalized pluronic;

the preparation method of the FDA comprises the following steps: dissolving pluronic and excessive triethylamine in anhydrous dichloromethane, cooling a reaction system to 0 ℃ under the protection of nitrogen, slowly dropwise adding excessive dichloromethane solution of acryloyl chloride, heating to normal temperature after dropwise adding, continuing reaction, washing with water after the reaction is finished, drying an organic phase, and re-precipitating in anhydrous ether to obtain the FDA.

4. The high strength biomedical hydrogel material according to claim 1 or 2, wherein the functionalized biomedical macromolecules are selected from acrylate or methacrylate functionalized biomedical macromolecules, carbohydrate macromolecules that can be complexed with ions.

5. The high strength biomedical hydrogel material according to claim 4, wherein said acrylate or methacrylate functionalized biomedical macromolecule is prepared by a process comprising the steps of: dissolving biomedical macromolecules into a solvent, adding excessive acid anhydride at the temperature of 0-80 ℃, continuing to react, dialyzing reaction liquid for 2-6 days, and freeze-drying to obtain the acrylate or methacrylate functionalized biomedical macromolecules.

6. The high strength biomedical hydrogel material according to claim 1 or 2, wherein the initiator is at least one of 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropiophenone, and lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate.

7. A3D printing method of bath supported hydrogel by using the high-strength biomedical hydrogel material of any one of claims 1 to 6, which is characterized by comprising the following steps:

firstly, establishing an STL-format three-dimensional printing model by using Solidworks software, then introducing the model to be printed into Bioplotter RP slice software for layering, finally printing by using 3D (three-dimensional) biomedical printer control software Visual machinery, placing an uncrosslinked high-strength biomedical hydrogel material into a 3D printer extrusion head, setting printing parameters, directly printing in a constant-temperature Pluronic hydrogel supporting bath, and printing by using a dispensing needle head with the inner diameter of 50-300 mu m, wherein the printing material temperature is 20-37 ℃, the extrusion pressure is 0.2-10 bar, and the printing speed is 4-35 mm/s;

secondly, after the 3D sample is printed, carrying out hydrogel crosslinking and curing by using LED light or ions in a Pluronic hydrogel supporting bath;

and thirdly, cooling the temperature of the Pluronic hydrogel supporting bath to below 4 ℃ to enable the Pluronic hydrogel supporting bath to be disassembled and assembled into a liquid state, taking out the printed sample, and washing the sample for multiple times by PBS (phosphate buffer solution) to obtain a bath supporting hydrogel model.

8. The 3D printing method of the high-strength biomedical hydrogel material for bath supported hydrogel according to claim 7, wherein the printer is an extrusion biological 3D printer;

the temperature of the 3D printing process is 20-37 ℃.

9. The 3D printing method of the bath-supported hydrogel with the high-strength biomedical hydrogel material according to claim 7, wherein the preparation method of the Pluronic hydrogel supporting bath comprises the following steps: under the ice bath condition, dissolving the pluronic in deionized water or a solution containing calcium ions, and completely dissolving the pluronic through multiple vortex oscillation to prepare a pluronic solution with the solid content of 20-40% to obtain the pluronic hydrogel supporting bath;

the LED light is ultraviolet light with the wavelength of 365nm and the light intensity of 10mW cm^-2(ii) a Or visible light with a wavelength of 395nm and a light intensity of 10mW cm^-2。

10. The 3D printing method of the high-strength biomedical hydrogel material for bath supported hydrogel according to claim 9, wherein the solution containing calcium ions is selected from the group consisting of 0.01-0.2 mM mL^-1CaCO of₃Solution, gluconolactone sustained-release solution and CaCl₂One in aqueous solution.

Technical Field

The invention belongs to the technical field of biomedical hydrogel and 3D printing, and particularly relates to a high-strength biomedical hydrogel material and a 3D printing method of bath-supported hydrogel.

Background

The hydrogel has excellent biocompatibility and processability, and has wide application prospect in the fields of biomedicine, tissue engineering and regenerative medicine because the biophysical property is close to that of soft tissues of a human body. Although related scientific research papers are increasing, the practical application of the method is only a few. The reason for this is that natural and artificial hydrogels have many unsatisfactory places in mechanical strength, biocompatibility, processing accuracy, etc. compared to human tissues, thereby greatly limiting their practical applications.

The PluonicF127 is a temperature-sensitive triblock polymer, molecules can be self-assembled to form micelles through hydrophilic-hydrophobic interaction at a certain temperature, and the reversible self-assembly performance of the PluonicF127 can provide energy dissipation for the hydrogel under the action of external force, so that the mechanical performance of the hydrogel is enhanced. Such as: okay et al proposed the addition of an interpenetrating network of acrylate monomers to a physically entangled Pluronic network to increase the toughness of the hydrogel (Polymer,2013,54(12), 2979-. It was proposed by Pnid et al to modify double bonds (F127DA) at both ends of Pluronic molecule and to use the double-bond functionalized nano-micelle as a cross-linking agent to initiate co-crosslinking of acrylate monomers to obtain hydrogels with excellent tensile and compressive properties (Chemical Communication,2015,51(40), 8512-8515). Although the mechanical properties of these hydrogels are greatly improved, the lack of biocompatibility in the hydrogel network due to the residual small molecule monomers that are incompletely polymerized limits their potential as bio-inks.

In addition to mechanical strength issues, another key scientific issue for hydrogel implementation applications is the shaping process of the hydrogel. With the continuous development of the field of tissue engineering, additive manufacturing methods typified by 3D printing have attracted a wide range of attention from clinicians and scientists. The 3D printing technology can simultaneously meet the macroscopic and microscopic structural requirements of materials by combining a computer data model, is suitable for personalized customization and has great advantages in tissue engineering. In addition to the requirement that hydrogel have appropriate mechanical properties to ensure structural stability before and after printing, the realization of 3D printing of hydrogel also puts higher demands on 3D printing technology. However, the existing printing technology has many limitations on the mechanical and chemical properties of hydrogel inks and printable structures. Recently, the Adam w.feinberg project reported a free reversible embedding printing technique (FRESH) that can directly print traditional bio-inks into a physically supported bath of gelatin microspheres to maintain the microstructure of the printed material. The method can not only realize printing of soft materials such as protein and polysaccharide hydrogel, but also be used for printing complex physiological structures. However, the preparation of gelatin microsphere support baths in this method is cumbersome and the fineness of the printed structures is limited due to the non-uniformity of the gelatin microsphere particles (Science advances.2015,1(9), e 1500758).

In conclusion, the mechanical property, the biocompatibility and the 3D printing are particularly important for designing hydrogel supports and promoting the hydrogel supports to be applied in the field of biomedicine, and the development of the biological ink and the 3D printing method which have excellent mechanical property and good biocompatibility and can realize a complex and precise structure becomes a key point for expanding the hydrogel to the field of practical biomedicine.

Disclosure of Invention

The first purpose of the invention is to provide a high-strength biomedical hydrogel material.

The second purpose of the invention is to provide a 3D printing method of the bath-supported hydrogel by using the high-strength biomedical hydrogel material.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a high-strength biomedical hydrogel material which is prepared from the following components in parts by weight:

15-40 parts of FDA, 0-15 parts of functionalized biomedical macromolecules, 0.05-0.3 part of an initiator and 20-200 parts of water.

Preferably, the high-strength biomedical hydrogel material is prepared from the following components in parts by weight:

15-30 parts of FDA, 1-5 parts of functionalized biomedical macromolecules, 0.1-0.2 part of an initiator and 20-200 parts of water.

The FDA is acrylic ester functionalized pluronic, specifically is prepared by esterification reaction of pluronic and excessive acryloyl chloride, and is preferably a product with both ends functionalized by acrylic ester; the pluronic (also known as Poloxamer, Poloxamer) is a polyoxypropylene polyoxyethylene block copolymer, specifically selected from commercial products under the designations F68 (basf), F77 (basf), F108(Sigma Aldrich), F127(Sigma Aldrich).

The preparation method of the FDA comprises the following steps: dissolving pluronic (selected from commercial products with the trade names of F68 (basf), F77 (basf), F108(Sigma Aldrich) and F127(Sigma Aldrich)) and excessive triethylamine in anhydrous dichloromethane, cooling the reaction system to 0 ℃ under the protection of nitrogen, slowly dropwise adding excessive dichloromethane solution of acryloyl chloride, heating to normal temperature to continue reaction after dropwise adding, washing with water after the reaction is finished, drying the organic phase, and re-precipitating in anhydrous ether to obtain FDA.

The functionalized biomedical macromolecules are further crosslinked by photoinitiation or ion complexation, and are selected from acrylate or methacrylate functionalized biomedical macromolecules, saccharide macromolecules capable of being complexed with ions and the like; the acrylate or methacrylate functionalized biomedical macromolecule is prepared by mixing and reacting a macromolecule and acrylic anhydride, and is preferably acrylate functionalized hyaluronic acid (HAMA), acrylate functionalized gelatin (GelMA), acrylate functionalized dextran (DexMA), acrylate functionalized chitosan (CsMA), acrylate functionalized collagen (ColMA), acrylate functionalized cellulose (CelMA), acrylate functionalized chondroitin sulfate (ChSMA) and the like; the carbohydrate macromolecules capable of being complexed with ions are sodium alginate (Alg) capable of being crosslinked with calcium ions.

The preparation method of the acrylate or methacrylate functionalized biomedical macromolecule comprises the following steps:

dissolving biomedical macromolecules into a solvent (water or Du's phosphate buffer solution (D-PBS)), adding excessive acid anhydride at the temperature of 0-80 ℃, continuing to react, dialyzing the reaction solution for 2-6 days, and freeze-drying to obtain the acrylate or methacrylate functionalized biomedical macromolecules.

After the addition of the anhydride, an excess of base, such as sodium hydroxide, is added as needed.

The biomedical macromolecules are hyaluronic acid, gelatin, dextran, chitosan, collagen, cellulose and chondroitin sulfate.

The acid anhydride is methacrylic anhydride or acrylic anhydride.

The initiator is at least one of a commercial photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methyl propiophenone (I2959, Sigma Aldrich) and lithium phenyl-2, 4, 6-trimethyl benzoyl phosphonate (LAP, Sigma Aldrich).

The second aspect of the invention provides a preparation method of the high-strength biomedical hydrogel material, which comprises the following steps: mixing FDA and water according to the proportion, adding functional biomedical macromolecules, mixing at low temperature, carrying out vortex dispersion, introducing nitrogen to remove oxygen after uniformly mixing to obtain a hydrogel precursor solution, and then adding an initiator, LED light or ions to initiate in-situ crosslinking to obtain the high-strength biomedical hydrogel material.

The invention provides a 3D printing method of bath-supported hydrogel by using the high-strength biomedical hydrogel material, which comprises the following steps:

firstly, establishing an STL-format three-dimensional printing model by using Solidworks software, then introducing the model to be printed into Bioplotter RP slice software for layering, finally printing by using 3D bio-printer control software visual machinery, placing an uncrosslinked high-strength biomedical hydrogel material into a 3D printer extrusion head, setting printing parameters, directly printing in a constant-temperature Pluronic hydrogel supporting bath, and printing by using a dispensing needle head with the inner diameter of 50-300 mu m, wherein the printing material temperature is 20-37 ℃, the extrusion pressure is 0.2-10 bar, and the printing speed is 4-35 mm/s;

secondly, after the 3D sample is printed, carrying out hydrogel crosslinking and curing by using LED light or ions in a Pluronic hydrogel supporting bath;

and thirdly, cooling the temperature of the Pluronic hydrogel supporting bath to below 4 ℃ to enable the Pluronic hydrogel supporting bath to be disassembled and assembled into a liquid state, taking out the printed sample, and washing the sample for multiple times by PBS (phosphate buffer solution) to obtain a bath supporting hydrogel model.

The printer adopts an extrusion type biological 3D printer.

The temperature of the 3D printing process is preferably 20-37 ℃.

The preparation method of the Pluronic hydrogel supporting bath comprises the following steps: under the ice bath condition, dissolving the pluronic in deionized water or a solution containing calcium ions, and completely dissolving the pluronic through multiple vortex oscillation to prepare a pluronic solution with the solid content of 20-40% to obtain the pluronic hydrogel supporting bath.

The solution containing calcium ions is selected from 0.01-0.2 mM mL^-1CaCO of₃Solution, gluconolactone sustained-release solution and CaCl₂One in aqueous solution.

The LED light is ultraviolet light with the wavelength of 365nm and the light intensity of 10mW cm^-2(ii) a Or visible light with a wavelength of 395nm and a light intensity of 10mW cm^-2。

The high-strength biomedical hydrogel material can be printed by different types of materials at the same time.

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

in order to improve the mechanical property of hydrogel and simultaneously realize 3D printing with high precision and complex structure, the high-strength biomedical hydrogel material provided by the invention takes FDA with temperature sensitivity and light sensitivity as a main component, and is blended with other functionalized biomedical macromolecules so as to prepare the biomedical hydrogel material with good mechanical property through co-crosslinking; the pluronic micelle type self-assembly mechanism can provide energy dissipation for the hydrogel under the action of external force, so that the problems of poor mechanical strength and difficult forming of the traditional hydrogel are solved. And further provides a 3D printing method, which takes the micelle type self-assembled pluronic as bath support to realize 3D printing of hydrogel with high precision and a complex structure. According to the method, the characteristic that the Pluronic can be self-assembled at a certain temperature and shows as viscous fluid under higher shear stress is utilized, the simple, stable and high-precision printing and the complex structure supporting of the ink are realized, and the Pluronic can be disassembled and the printed 3D hydrogel is released through cooling after the hydrogel is further crosslinked and cured, so that the technical problems that the low-precision and complex suspended structure in the existing hydrogel 3D printing technology are difficult to print are solved.

The invention takes biomedical macromolecules as a hydrogel framework, avoids potential toxicity caused by using micromolecular monomers in the past, improves the mechanical property of the hydrogel by combining non-covalent crosslinking and covalent crosslinking, and simultaneously realizes the 3D printing of the fine and complex hydrogel structure by applying a novel bath support printing method.

According to the 3D printing method for the bath-supported hydrogel by adopting the high-strength biomedical hydrogel material, disclosed by the invention, according to the temperature-sensitive and reversible micelle assembly of the Pluronic, the printing needle is rigid under low shear stress, but is characterized by viscous fluid under higher shear stress, so that the printing needle almost has no mechanical resistance when moving in a supporting bath, and can quickly generate self-healing, thereby realizing the complex printing with high precision and structure maintenance. The printing precision can reach 20 mu m optimally, the matching degree with a computer model is good, and the simultaneous printing of multiple materials can be realized. In addition, the bath-supported hydrogel provides a physical embedding space for the printing ink, prevents the penetration of oxygen, and avoids the problem of oxygen inhibition of the traditional photocured hydrogel.

The hydrogel component and the printing method have good biocompatibility, can be used as a scaffold material for cell culture, and have great potential application in the fields of drug release, tissue repair and the like.

According to the assembling principle of the pluronic reversible micelle, the energy dissipation can be provided for the hydrogel under the action of an external force, so that the mechanical property of the hydrogel is enhanced, the maximum compressive fracture stress can reach more than 10MPa, the maximum compressive modulus can reach 1.5MPa, and the maximum tensile fracture stress can reach 2.5 MPa.

According to the method, functionalized biomacromolecules are used as hydrogel printing materials, temperature-sensitive Pluronic is used as printing bath support, and illumination or ion complexation is used as a crosslinking curing method after printing, so that the hydrogel obtained by printing is stable in structure, simple in method and high in fineness, and can be used for continuously, stably and uniformly printing the hydrogel model with the complex three-dimensional shape.

According to the invention, the separation of the printing hydrogel model is realized by utilizing the low-temperature melting characteristic of the Pluronic support bath, a physical embedding space is provided for printing ink by utilizing the Pluronic temperature-sensitive characteristic, the permeation of oxygen is prevented, and the problem of oxygen inhibition of the traditional photocuring hydrogel is avoided. The method is characterized in that functionalized biomacromolecules are used as hydrogel printing materials, temperature-sensitive Pluronic is used as a printing support, and light or ion complexation is used as a crosslinking curing method after printing, so that the hydrogel model obtained by printing is stable in structure, simple in method and high in fineness, and can be continuously, stably and uniformly printed with the complex three-dimensional hydrogel model with the circular holes.

Drawings

FIG. 1 is a schematic diagram of the compressive (a) and tensile (b) stress-strain curves of the FDA-HAMA hydrogel prepared in example 1.

FIG. 2 is a schematic representation of 3D printing of a human ear model using FDA-HAMA hydrogel in example 2.

FIG. 3 is a schematic representation of 3D printing of a human ear model using FDA-HAMA hydrogel in example 2.

FIG. 4 is a schematic diagram of the fineness control of the FDA-HAMA 3D printed FDA-HAMA hydrogel model of example 4: a)60 μm, b)90 μm, c)210 μm needle.

Fig. 5 is a schematic diagram of a multi-ink complex 3D printed hydrogel model in example 5.

Fig. 6 is a schematic diagram of hydrogel scaffolds printed by a bath supported hydrogel 3D printing method for MC3T3 cell co-culture in example 6.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Hyaluronic acid (HA, 48kDa) in the examples of the present invention was purchased from Shandong Furrida pharmaceutical company; sodium alginate, gelatin, pluronic F127, pluronic F108, the photoinitiator 2-hydroxy-4- (2-hydroxyethoxy) -2-methylpropiophenone (I2959), the photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoylphosphonate (LAP), the duchenne phosphate buffer (D-PBS), rat tail Collagen (Collagen) and Fetal Bovine Serum (FBS) were purchased from Sigma Aldrich. Pluronic F77 was purchased from basf, and dextran, chitosan, cellulose, gelatin, chondroitin sulfate, triethylamine, sodium hydroxide, methylene chloride, acryloyl chloride, methacrylic anhydride were all purchased from alatin. The chemical reagents are analytically pure and do not need further purification. The mouse fibroblast NIH-3T3 cells used in the present invention were purchased from the cell bank of Chinese academy of sciences.