CN114796617B - Composite 3D printing ink and application thereof - Google Patents
Composite 3D printing ink and application thereof Download PDFInfo
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- CN114796617B CN114796617B CN202210574342.9A CN202210574342A CN114796617B CN 114796617 B CN114796617 B CN 114796617B CN 202210574342 A CN202210574342 A CN 202210574342A CN 114796617 B CN114796617 B CN 114796617B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
- B33Y70/10—Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y80/00—Products made by additive manufacturing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The invention discloses composite 3D printing ink and application thereof, and belongs to the technical field of biological ink. The composite 3D printing ink is mainly prepared by mixing acellular matrix gel microspheres and gel materials. According to the invention, the decellularized matrix is prepared into the hydrogel microsphere and then added into the gel material to form the composite 3D printing ink, so that the printing property and the bioactivity of the ink are effectively compatible, the acting speed of bioactive substances in the ink is improved, the negative influence of phase separation of two hydrogels on a printing process is avoided, and the application potential of packaging or loading cells on the microsphere for modularized construction and manufacturing a multi-scale three-dimensional composite structure bracket for regulating the microenvironment and macroscopic environment of the cells is provided.
Description
Technical Field
The invention belongs to the technical field of biological ink, and particularly relates to composite 3D printing ink and application thereof.
Background
The biological 3D printing technology is a new technological means for finally manufacturing a three-dimensional structure by taking a computer three-dimensional model as a drawing and assembling special biological ink, and has good application prospect and huge social value in the fields of tissue repair and regenerative medicine. The biological ink is used as the joint point of equipment and cells, so that the printing precision is ensured, the cells are protected in the printing process, and the effect of supporting the microenvironment for cell growth is provided, so that the biological ink is the most important part in the printing process. The bio-ink has three requirements relating to three aspects of printability, biocompatibility and mechanical properties. Printability refers to the property of a material to be precisely controllable in both the spatial and temporal dimensions, i.e. the material used for printing must be deposited precisely in a specified space in a certain time, which is directly related to whether the 3D printed product achieves the desired structural and dimensional accuracy. Printability the printability of the bio-ink was evaluated, including adjustable viscosity of the bio-material, rapid transition from sol to gel, and a wide range of printable parameters, among others. In the case of extrusion printing, printability generally includes 3 layers (1) the viscosity of the bio-ink is to be controlled, such as by temperature changes, shear stress, etc. Only the viscosity is adjustable, so that a proper printing mode and printing parameters can be designed. (2) The bio-ink needs to be in a fluid or semi-fluid state prior to printing to avoid clogging the nozzles and to be able to solidify quickly after printing to maintain its shape. Bonding between different layers is also very important during layer-by-layer printing, which determines whether the material or the printing process is truly 3D printing. (3) A print window or process parameter interval for the material is owned or can be found. Biocompatibility requires that the bio-ink be as similar as possible to the cellular microenvironment in the human body, is free of toxic and side effects, is suitable for cell adhesion, growth and proliferation, and also requires a suitable porosity for facilitating the transport of nutrients. Mechanical properties require that the gel state bio-ink be mechanically strong to support subsequent culturing and implantation processes; bioprinted structures typically need to be cultured in vitro, during which time, often accompanied by infusion and degradation of nutrients, some mechanical strength is required to resist external forces, maintaining the topographical structure of the printed matter.
The decellularized matrix (dcm) material is derived from a tissue/organ, which is capable of removing immunogenicity by a suitable decellularizing method, and retaining active substances such as proteins (collagen, fibronectin, laminin, etc.), polysaccharides (glycane, proteoglycan, glycoprotein, etc.), and growth factors contained in the original tissue/organ. Compared with the natural material with single component, dECM can better simulate the microenvironment of the original tissue, provide more attachment sites and nutrients for cells, ensure the adhesion and growth of the cells and promote the regeneration of the tissue. Further, studies have shown that dcms materials can exert their biological functions in a variety of forms including hydrogels, cryo-abrasive particles, microcarriers, and the like, which are diverse in structure and biomechanical properties. Therefore, dcms-containing bio-inks are very promising for high bioactivity and tissue-specific incorporation into custom complex biological scaffolds by 3D bioprinting. The dECM gel has the capability of self-assembling gel forming and can be directly used for extrusion printing, but the hydrogel obtained by the gel forming mode has weak mechanical strength and poor structural fidelity, and is a main defect of limiting the application of the dECM gel in biological printing. To date, many studies have been explored for improving the dcms materials for 3D printing. Mainly comprises the following steps: (1) chemical modification, such as methylpropionalization modification, is carried out on the dECM material, double bonds are introduced, and the crosslinking mode is changed so as to enhance the mechanical property of the dECM material; (2) compounding dvcm with other polymeric materials is a better method for preserving biocompatibility, and for several systems commonly used at present, such as bio-ink in which methacryloylated gelatin and acellular matrix (gelma+dcm) hydrogel are directly mixed, printability and bioactivity are considered to a certain extent, but because GelMA undergoes temperature-responsive sol-gel transition at a temperature lower than body temperature, and acellular matrix hydrogel undergoes gelation at body temperature, the difference of the two gelling mechanisms and temperature-sensitive transition increases the complexity of the process for extrusion printing, and phase separation easily occurs during the stretching and mixing process in the printing process, leading to non-uniformity of the printing structure and blockage of nozzles; the addition of acellular matrix as powder to GelMA, while providing a great improvement in printability, is detrimental to rapid onset of action due to non-uniformity and poor degradability of the powder.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides composite 3D printing ink and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: in a first aspect, a composite 3D printing ink is provided, which is mainly obtained by mixing acellular matrix gel microspheres and gel materials.
According to the invention, the decellularized matrix is prepared into the hydrogel microsphere and then added into the gel material to form the composite 3D printing ink, so that the printing property and the bioactivity of the ink are effectively compatible, the acting speed of bioactive substances in the ink is improved, the negative influence of phase separation of two hydrogels on a printing process is avoided, and the application potential of packaging or loading cells on the microsphere for modularized construction and manufacturing a multi-scale three-dimensional composite structure bracket for regulating the microenvironment and macroscopic environment of the cells is provided.
The acellular matrix gel microsphere retains specific proteins, polysaccharides, growth factors and other active substances in tissues, can better simulate microenvironment, is beneficial to cell adhesion, growth and reproduction, promotes regeneration of matrix, and provides excellent cell compatibility and tissue compatibility. The acellular matrix can be derived from peripheral nerves, spinal cords, small intestinal mucosa, cornea and other tissues and organs.
The gel material is used as an ink matrix, has quite good temperature sensitivity and shear thinning performance, and provides printability for the composite 3D printing ink.
Optionally, the composite 3D printing ink further comprises a photoinitiator, wherein the mass-volume concentration of the photoinitiator is 0-10 mg/mL, and the photoinitiator can rapidly initiate the gel material to undergo free radical polymerization to generate a crosslinked network after the composite 3D printing ink is printed, so that the mechanical strength and shape fidelity of a printing structure are remarkably improved. Preferably, the photoinitiator is one of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP), 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (Irgacure 2959).
As an alternative embodiment of the composite 3D printing ink of the present invention, the preparation method of the acellular matrix gel microspheres comprises the following steps:
(1) Adding acellular matrix powder into pepsin solution for digestion to obtain acellular matrix solution with mass fraction of 0.5% -1%, adding NaOH solution to adjust pH to neutrality, and adding 10 XPBS solution to make ionic strength of acellular matrix solution be 1, and taking the acellular matrix solution as water phase;
(2) Adding a surfactant into perfluorobutyl methyl ether to obtain a surfactant solution with the volume fraction of 0.5-5% as an oil phase;
(3) Introducing the acellular matrix solution obtained in the step (1) into an aqueous phase inlet, introducing the solution obtained in the step (2) into an oil phase inlet, and generating water-in-oil droplets at the interface of a chip microchannel under the action of a surfactant; the obtained liquid drops flow in a rubber tube and gel through water bath at 37 ℃ to obtain the acellular matrix gel microsphere.
The volume fraction of the surfactant plays an important role in the formation of the microsphere, the volume fraction of the surfactant is less than 0.5%, the obtained water-in-oil droplets cannot be stably dispersed in a continuous phase, and the oil-water interface of the obtained acellular matrix gel microsphere is unstable and aggregation adhesion can occur; when the volume fraction of the surfactant is in the range of 0.5-5%, the stability of the oil-water interface of the acellular matrix gel microspheres is increased along with the increase of the volume fraction of the surfactant, but the post-treatment process of the acellular matrix gel microspheres is more complicated along with the increase of the volume fraction of the surfactant, and when the volume fraction of the surfactant is higher than 5%, the acellular matrix gel microspheres are difficult to demulsifie and wash and have certain biotoxicity, so that the bioactivity of the composite 3D printing ink is affected.
The 10 XPBS solution belongs to a high-concentration salt solution, the high-concentration salt solution is introduced into a solution system of the acellular matrix, and the rapid salting-out effect enables the polypeptide chain which is curled freely to be rapidly converted into a beta-sheet structure, so that the acellular matrix solution can be gelled.
Alternatively, the surfactant in step (2) above is a polyethylene glycol block copolymer, commercially available under the trade name Krytox- (PEG) -Krytox block polymer, available from RAN Biotech.
Optionally, in the step (3), the flow rate of the water phase is 0.1-1mL/h, the flow rate of the oil phase is 5-20mL/h, and the speed ratio of the oil phase to the water phase is v Oil (oil) /v Water and its preparation method The particle size of the decellularized matrix gel microsphere is gradually reduced along with the increase of the speed ratio of the oil phase to the water phase, which is 2.5-20, and can be controlled by adjusting the speed ratio of the oil phase to the water phase.
Optionally, the volume fraction of the acellular matrix gel microspheres in the composite 3D printing ink is 25% -75%.
Optionally, the particle size of the acellular matrix gel microsphere is 60-300 μm.
Optionally, the mass-volume concentration of the gel material is 50-90 mg/mL. The gel material provides printability to the composite 3D printing ink, which is insufficient than the gel material of the above mass-volume concentration.
Optionally, the gel material is gelatin or methacryloylated gelatin. The methacryloylated gelatin (GelMA) can be rapidly cured by uv irradiation in the presence of a photoinitiator to form a scaffold structure.
In a second aspect, there is provided the use of a composite 3D printing ink in biological 3D printing.
Optionally, the diameter of a printing needle used in the printing process of the composite 3D printing ink is 0.08-0.41mm.
Optionally, the diameter of the printing needle head is 1-2 times of the particle size of the acellular matrix gel microsphere.
In the printing process, the particle sizes of the printing needle head and the microspheres have a certain relation, and the microspheres have certain elasticity, so that the microspheres with larger inner diameters than the needle head can be extruded in the extrusion printing process, but the microspheres can be deformed and broken to a certain extent. The inventors have found that when the diameter of the printing needle is 2 times the particle diameter of the microspheres, the microspheres can be extruded smoothly and maintain the shape. Therefore, in the printing process, the proper microsphere particle size and the proper printing needle head can be selected according to the actual situation.
Optionally, after the printing of the composite 3D printing ink is completed, the gel material is subjected to illumination crosslinking.
Compared with the prior art, the invention has the beneficial effects that:
(1) The acellular matrix hydrogel from the peripheral nerve source adopted by the invention can not only keep the active components and tissue specificity of the extracellular matrix, but also maintain the nanofiber structure similar to the extracellular matrix, can create a microenvironment favorable for cell survival, has excellent biocompatibility, and is more suitable for cell growth.
(2) Compared with other common methods, the method can control the production process of the acellular matrix microsphere, the obtained acellular matrix microsphere can be uniformly emulsified, the adhesive tape forming piece of the acellular matrix microsphere is mild (37 ℃ and pH is neutral), and the acellular matrix microsphere has good potential of encapsulating cells in the microsphere.
(3) The acellular matrix is prepared into the hydrogel microspheres and then added into the gel material to form the composite 3D printing ink, so that the printability and the bioactivity of the ink are effectively compatible, the acting speed of bioactive substances in the ink is improved, and the negative influence of the phase separation of two hydrogels on a printing process is avoided; the method also has the application potential of encapsulating or loading cells on microspheres for modularized construction and manufacturing a multi-scale three-dimensional composite structure bracket to regulate the microenvironment and macroscopic environment of the cells.
Drawings
FIG. 1 is a flow chart of the preparation and application of the composite 3D printing ink of the present invention;
FIG. 2 is a schematic diagram of the synthesis of methacryloylated gelatin, before and after modification of gelatin 1 H-NMR chart;
FIG. 3 is a physical diagram of methacryloylated gelatin and acellular matrix gel prepared in example 1;
FIG. 4 shows different v Oil (oil) /v Water and its preparation method Optical microscopy of the prepared acellular matrix gel microspheres; FIG. 4A is v Oil (oil) /v Water and its preparation method =2.5, fig. 4B is v Oil (oil) /v Water and its preparation method =5, fig. 4C is v Oil (oil) /v Water and its preparation method =7.5, fig. 4D is v Oil (oil) /v Water and its preparation method =10, fig. 4E is v Oil (oil) /v Water and its preparation method =15, fig. 4F is v Oil (oil) /v Water and its preparation method ＝20；
FIG. 5 shows the particle size of the acellular matrix gel microspheres as a function of v Oil (oil) /v Water and its preparation method Is a variation graph of (2);
FIG. 6 is a graph of viscosity versus shear rate for the composite 3D printing ink prepared in example 5;
FIG. 7 is a graph of shear modulus versus temperature for the composite 3D printing inks prepared in example 5 and comparative example 1;
FIG. 8 is a graph showing the change in storage modulus before and after irradiation of light of the composite 3D printing ink prepared in example 5 and comparative example 1;
FIG. 9 is a graph showing the results of live-dead staining of the cells of the composite 3D printing ink-cultured PC12 prepared in example 5 and comparative example 1 at D1 and D4, and a graph showing the survival rate of the cells of the composite 3D printing ink-cultured PC12 prepared in comparative example 1 at D1 and D4, FIG. 9A, D is a graph showing the results of live-dead staining of the cells of the composite 3D printing ink-cultured PC12 prepared in example 5 at D1 and D4, and FIG. 9B, E is a graph showing the survival rate of the cells of the composite 3D printing ink-cultured PC12 prepared in example 5 and comparative example 1 at D1 and D4;
FIG. 10 is a diagram of a grid structure printed with the composite 3D printing ink prepared in example 5;
FIG. 11 is a graph of microscopic observations of a composite 3D printing ink printed lattice structure prepared in example 5 of the present invention;
fig. 12 is a graph showing the effect of different inside diameters of the printing needle on the particle size of the decellularized matrix gel microspheres before and after printing, fig. 12A is a physical graph of the composite 3D printing ink after being loaded into a syringe, fig. 12B is a graph showing the variation of the maximum extrusion particle size of the decellularized matrix gel microspheres with the inside diameter of the printing needle, fig. C (1), C (2) and C (3) are respectively a graph showing the variation of the particle size of the decellularized matrix gel microspheres before printing, the inside diameter of the printing needle is 330 μm, the inside diameter of the printing needle is 210 μm after printing, and fig. D (1), D (2) and D (3) are respectively a graph showing the variation of the particle size of the decellularized matrix gel microspheres before printing, the inside diameter of the printing needle is 210 μm and the inside diameter of the printing needle is 110 μm after printing;
fig. 13 is an optical micrograph of acellular matrix gel microspheres prepared with different volume fractions of surfactant, fig. 13A with 0.4% volume fraction of surfactant and fig. 13B with 6% volume fraction of surfactant.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples and the accompanying drawings.
1. Materials, reagents and the like used in the examples described below were commercially available unless otherwise specified.
2. In the following examples, the acellular matrix powder used was prepared by the following method:
(1) Pretreatment: the peripheral nerve tissue of the mice is sheared into tissue blocks with the length of 5mm multiplied by 5mm, and the tissue blocks are sufficiently cleaned by using PBS solution;
(2) Decellularization: the peripheral nerve tissue is put into 3% Triton X100 water solution for 12h of shaking, taken out and rinsed 3 times in sterile distilled water, then put into 4% sodium deoxycholate water solution for 24h of shaking, and finally rinsed 3 times in sterile distilled water. The above process was repeated 2 times;
(3) Primary freeze-drying: placing the peripheral nerve tissue subjected to cell removal in a freeze dryer at the temperature of minus 80 ℃ for freeze drying;
(5) Degreasing: degreasing the peripheral nerve tissue subjected to primary freeze-drying by using a mixed solvent of ethanol and dichloromethane, wherein the volume ratio of the ethanol to the dichloromethane is 1:2, draining the solvent after degreasing, and washing the peripheral nerve tissue for multiple times by using sterile distilled water until the mixed solvent has no residue;
(6) Secondary freeze-drying for standby: and (3) freeze-drying the peripheral nerve tissue subjected to degreasing in a freeze dryer at-80 ℃, crushing, sieving with a 40-mesh sieve, sealing the obtained acellular matrix powder, and storing at-40 ℃ for later use.
3. In the following examples, the methacryloylated gelatin (GelMA) used was prepared by the following method:
(1) 10g of gelatin was dissolved in 100mL of PBS, the mixture was heated to 60℃and stirred until gelatin was completely dissolved;
(2) Dropwise adding 10mL of methacrylic anhydride into the gelatin solution in the step (1) at a concentration of 0.25mL/min by using a constant pressure funnel, uniformly stirring, and reacting for 3 hours at 60 ℃;
(3) After the reaction was completed, a PBS solution having a ph=7.4 and a concentration of 0.1M was added to stop the reaction, wherein the addition amount of the PBS solution was 2 times the volume of the mixture obtained in the step (2);
(4) Putting the reaction liquid obtained in the step (3) into a dialysis bag, putting the dialysis bag into pure water at 40 ℃, dialyzing for 3 days, and changing the dialysis liquid at 12 hours, 24 hours and 48 hours; freezing the solution in a refrigerator at-40 ℃ after dialysis is finished, and then removing the solvent in a freeze dryer to obtain white spongy solid GelMA; and (5) keeping the materials away from light at the temperature of minus 80 ℃ for standby.
FIG. 2 is a schematic diagram of the synthesis of methacryloylated gelatin, before and after modification of gelatin 1 H-NMR chart, H-spectrum of fig. 2 shows an increase in the signal of methacrylate vinyl proton peaks (δ=5.4 and 5.7 ppm) and a decrease in the signal of free lysine methylene proton peaks (δ=2.9 ppm), confirming successful modification of gelatin.
Example 1
This example is intended to illustrate the gelation properties of the decellularized matrix of peripheral nerve tissue of the present invention.
10mg of acellular matrix powder is weighed, 1ml of pepsin solution is used for digestion to obtain acellular matrix solution with the mass fraction of 1%, the pH of the acellular matrix solution is regulated to be neutral by NaOH solution to terminate digestion, 10 XPBS solution is added to enable the ionic strength of the acellular matrix solution to be 1, and the acellular matrix gel with the mass fraction of 1% is obtained through gelation at 37 ℃.
Fig. 3 is a physical diagram of the decellularized matrix gel of the present embodiment, and it can be seen from fig. 3 that the decellularized matrix of the peripheral nerve tissue can be transformed from a liquid state to a solid state by sol-gel transformation at 37 ℃ and ph=7.
Example 2
This example illustrates a decellularized matrix microsphere and a method of making the same, comprising the steps of:
(1) Preparing a micro-channel chip: mixing polydimethylsiloxane and a cross-linking agent in a mass ratio of 10:1, pouring the mixture on a master, wherein the cross-linking agent is sylgard 184 of the Dow Corning company, and then curing the mixture for 4 hours at 70 ℃;
after curing, peeling the polydimethylsiloxane mould from the mother plate, and then punching a channel inlet and a channel outlet on the mould by using a puncher;
after the punching is finished, bonding the polydimethylsiloxane mould to a polydimethylsiloxane film after oxygen plasma treatment, and finally curing for 4 hours at 70 ℃ to obtain the micro-channel chip;
(2) Preparation of an aqueous phase: adding acellular matrix powder into pepsin solution for digestion to obtain acellular matrix solution with mass fraction of 1%, adding NaOH solution to adjust pH to neutrality, and adding 10×PBS solution to make ionic strength of acellular matrix solution 1;
(2) Preparation of an oil phase: adding the Krytox- (PEG) -Krytox block polymer into perfluorobutyl methyl ether for dissolution to obtain a Krytox- (PEG) -Krytox block polymer solution with the volume fraction of 1%;
(3) Respectively placing the syringes filled with water phase and oil phase on a micro-injection pump horizontally, connecting the needle with the inlet of the chip by using a silica gel tube, respectively injecting the water phase and the oil phase, wherein the flow rate of the water phase is 0.1-1mL/h, the flow rate of the oil phase is 5-20mL/h, and adjusting the speed ratio of the oil phase and the water phase to v in the flow rate range Oil (oil) /v Water and its preparation method =2.5, 5,7.5, 10, 15, 20, the aqueous and oily phases formed stable water-in-oil droplets at the inlet of the chip, the water-in-oil droplets flowed through a silicone tube placed in a water bath at 37 ℃ at the outlet of the chip, and the water-in-oil droplets gelled at the silicone tube, yielding acellular matrix gel microspheres with a diameter of 60-300 μm.
FIG. 4 shows different v Oil (oil) /v Water and its preparation method Optical microscopy of the prepared acellular matrix gel microspheres; FIG. 4A is v Oil (oil) /v Water and its preparation method =2.5, fig. 4B is v Oil (oil) /v Water and its preparation method =5, fig. 4C is v Oil (oil) /v Water and its preparation method =7.5, fig. 4D is v Oil (oil) /v Water and its preparation method =10, fig. 4E is v Oil (oil) /v Water and its preparation method =15, fig. 4F is v Oil (oil) /v Water and its preparation method =20; FIG. 5 shows the particle size of the acellular matrix gel microspheres as a function of v Oil (oil) /v Water and its preparation method Is a variation graph of (a). As can be seen from fig. 4 and 5, as v Oil (oil) /v Water and its preparation method The particle size of the acellular matrix gel microspheres gradually decreases. When v Oil (oil) /v Water and its preparation method 2.5,5,7.5, 10, 15 and 20, the corresponding average particle diameters are 128.4.+ -. 7.6. Mu.m, 113.8.+ -. 7.9. Mu.m, 106.8.+ -. 7.9. Mu.m, 100.2.+ -. 4.8. Mu.m, 81.8.+ -. 5.1. Mu.m, 77.1.+ -. 3.5. Mu.m. When v Oil (oil) /v Water and its preparation method After exceeding 20, the oil phase is improvedThe flow rate of the aqueous phase is not changed greatly by the particle size of the microspheres, but only by the rate of the microsphere output. When v Oil (oil) /v Water and its preparation method When the number is less than 5, clogging of the pipe tends to occur.
Example 3
This example is for illustration of a composite 3D printing ink and method of making the same
(1) Removing the oil phase in the acellular matrix gel microspheres with the particle size of 100.2+/-4.8 mu m obtained in the embodiment 2 sequentially through the steps of centrifugation and cleaning, transferring the washed acellular matrix gel microspheres into PBS buffer solution, centrifuging and compacting for later use, and marking as dECM-MS;
(2) Adding GelMA into PBS buffer solution containing LAP, and dissolving at 37deg.C to obtain GelMA solution; uniformly mixing the dECM-MS obtained in the step (1) and the GelMA solution obtained in the step (2) to obtain the composite 3D printing ink, wherein the volume fraction of the dECM-MS in the composite 3D printing ink is 25%, the mass-volume fraction of the GelMA is 90mg/mL, and the mass-volume fraction of the LAP is 10mg/mL.
Example 4
This example is for illustration of a composite 3D printing ink and method of making the same
(1) Removing the oil phase in the acellular matrix gel microspheres with the particle size of 100.2+/-4.8 mu m obtained in the embodiment 2 sequentially through the steps of centrifugation and cleaning, transferring the washed acellular matrix gel microspheres into PBS buffer solution, centrifuging and compacting for later use, and marking as dECM-MS;
(2) Adding GelMA into PBS buffer solution containing LAP, and dissolving at 37deg.C to obtain GelMA solution; uniformly mixing the dECM-MS obtained in the step (1) and the GelMA solution obtained in the step (2) to obtain the composite 3D printing ink, wherein the volume fraction of the dECM-MS in the composite 3D printing ink is 75%, the mass-volume fraction of the GelMA is 50mg/mL, and the mass-volume fraction of the LAP is 1mg/mL.
Example 5
This example is for illustration of a composite 3D printing ink and method of making the same
(1) Removing the oil phase in the acellular matrix gel microspheres with the particle size of 100.2+/-4.8 mu m obtained in the embodiment 2 sequentially through the steps of centrifugation and cleaning, transferring the washed acellular matrix gel microspheres into PBS buffer solution, centrifuging and compacting for later use, and marking as dECM-MS;
(2) Adding GelMA into PBS buffer solution containing LAP, and dissolving at 37deg.C to obtain GelMA solution; uniformly mixing the dECM-MS obtained in the step (1) and the GelMA solution obtained in the step (2) in equal volume to obtain the composite 3D printing ink, wherein in the composite 3D printing ink, the volume fraction of the dECM-MS is 50%, the mass-volume fraction of the GelMA is 80mg/mL, the mass-volume fraction of the LAP is 3mg/mL, and the composite 3D printing ink in the embodiment is recorded as 8% GelMA+MS.
Comparative example 1
The comparative example provides a preparation method of a composite 3D printing ink, which comprises the following steps:
adding GelMA into PBS buffer solution containing LAP, dissolving at 37deg.C to obtain GelMA solution, and recording the composite 3D printing ink with GelMA mass-volume fraction of 80mg/mL and LAP mass-volume fraction of 3mg/mL as 8% of GelMA.
Effect example 1
The rheological mechanical properties of the composite 3D printing ink prepared by the invention are tested.
The rheological mechanical properties of the printing inks prepared in example 5 and comparative example 1 were measured using a rotary rheometer, and the results are shown in fig. 6 to 8. From fig. 6, it can be seen that the composite 3D printing ink prepared in example 5 shows shear thinning properties, indicating that the composite 3D printing ink prepared in example 5 has good printing properties.
As can be seen from fig. 7, the sol-gel transition zone of the composite 3D printing ink prepared in example 5 did not significantly shift as compared to comparative example 1.
As can be seen from fig. 8, the modulus of the printing ink prepared in example 5 was greater than that of the printing ink prepared in comparative example 1 before irradiation with light, which corresponds to the test result of fig. 7. The printing inks prepared in example 5 and comparative example 1 were photo-crosslinked, respectively, and as can be seen from fig. 8, the printing inks prepared in example 5 and comparative example 1 were cured within 60 seconds, and after the photo irradiation, the modulus of the printing ink prepared in example 5 was smaller than that of the printing ink prepared in comparative example 1. The above results show that before photocuring, the GelMA crosslinked network is not formed yet, and is in a fluid state, and the added acellular matrix gel microspheres have a certain improvement on the strength of GelMA, but as photocuring is carried out, the formation of the GelMA crosslinked network is blocked due to the larger volume of the acellular matrix gel microspheres, so that the modulus of the printing ink prepared in example 5 is reduced compared with that of the printing ink prepared in comparative example 1, and the printing ink is more beneficial to the growth and migration of cells, which is consistent with the experimental result of effect example 2.
Effect example 2
The biological activity of the composite 3D printing ink is researched in the effect example, and the composite 3D printing ink of the effect example is prepared by the following specific steps:
(1) Removing the oil phase in the acellular matrix gel microspheres with the particle size of 100.2+/-4.8 mu m obtained in the embodiment 2 sequentially through the steps of centrifugation and cleaning, transferring the washed acellular matrix gel microspheres into 75% alcohol solution for soaking for 2 hours for disinfection, centrifuging after the soaking is finished, transferring the obtained precipitate into PBS buffer solution, centrifuging and compacting to obtain the acellular matrix gel microspheres, and marking as dECM-MS;
(2) Soaking GelMA in 75% alcohol solution for 2 hr for disinfection, and vacuum drying to obtain sterile GelMA;
(3) Adding the GelMA obtained in the step (2) into PBS buffer solution containing LAP, dissolving at 37 ℃ to obtain GelMA solution, uniformly mixing the dECM-MS obtained in the step (1) and the GelMA solution obtained in the step (2) in equal volume to obtain composite 3D printing ink, wherein the volume fraction of the dECM-MS in the composite 3D printing ink is 50%, the mass-volume fraction of the GelMA is 80mg/mL, the mass-volume fraction of the LAP is 3mg/mL, and the composite 3D printing ink of the embodiment is marked as 8% GelMA+ microsphere;
(4) Adding the GelMA obtained in the step (2) into PBS buffer solution containing LAP, dissolving at 37 ℃ to obtain the GelMA solution, and obtaining the composite 3D printing ink, wherein the mass-volume fraction of the GelMA in the composite 3D printing ink is 80mg/mL, the mass-volume fraction of the LAP is 3mg/mL, and the composite 3D printing ink of the comparative example is recorded as 8% GelMA-2.
The biological activity of the 8% GelMA+ microsphere and the 8% GelMA-2 obtained in the effect example is tested, and the specific steps are as follows:
respectively wrapping 8% GelMA+ microsphere and 8% GelMA-2 in PC12 cells for 3D printing (2×105 cells are wrapped in per milliliter of ink), placing the printed sample in 48 pore plate, and crosslinking with ultraviolet irradiation at illumination intensity of 5mw/cm 2 After crosslinking and curing, cell viability was detected at D1 and D4, respectively, for 50s. As shown in fig. 9, it can be seen from fig. 9 that the 8% gelma+ microspheres are more favorable for the growth of PC12 cells than the 8% gelma-2, and have excellent biocompatibility due to the acellular matrix microspheres contained in the composite 3D printing ink of the invention.
Effect example 3
This effect example investigated the printing performance and positioning performance in the printed structure of the composite 3D printing ink prepared in example 5.
The composite 3D printing ink prepared in example 5 was loaded into a printing cartridge, and a square grid of 20 x 20mm length x width was printed at room temperature, the internal line spacing of the square grid was 1.5mm, the layer height was 0.32mm, and the inside diameter of the printing stylus was 0.41mm. After printing, crosslinking by ultraviolet irradiation, wherein the illumination intensity is 5mw/cm 2 The time was 50s. The green fluorescent-labeled decellularized matrix gel microspheres were clearly observed in the transparent GelMA scaffold when viewed under a fluorescent microscope, and the shapes of the decellularized matrix gel microspheres were well preserved without severe deformation and rupture (fig. 10 and 11). The composite 3D printing ink disclosed by the invention can be applied to the construction of a bionic multi-scale composite bracket.
Effect example 4
The effect example is to study the matching of the particle size of the acellular matrix microsphere and the printing needle.
(1) Acellular matrix gel microspheres with particle diameters of 89+/-22 microns and 154+/-22 microns are prepared by the preparation method of the example 2.
(2) Composite 3D printing inks containing acellular matrix gel microspheres with particle sizes of 89±22 μm and 154±22 μm, respectively, were prepared using the preparation method of example 5.
The composite 3D printing ink containing the decellularized matrix gel microspheres with the particle diameter of 89±22 μm was loaded into a printing cartridge, and printing was performed at room temperature using a printing head with an inner diameter of 110 μm and 210 μm, respectively, and it was found that the decellularized matrix gel microspheres were deformed when the inner diameter of the printing head was 110 μm, the average particle diameter was reduced, the printing effect was deteriorated, and the printing effect of the composite 3D printing ink was better when the inner diameter of the printing head was 210 μm, the microspheres were not broken, and the shape was not changed (fig. 12).
The composite 3D printing ink containing the decellularized matrix gel microspheres with the particle diameters of 154±22 μm was loaded into a printing cartridge, and printing was performed at room temperature using the printing head having the inner diameters of 210 μm and 330 μm, respectively, and it was found that the decellularized matrix gel microspheres were deformed when the printing head had the inner diameter of 210 μm, the printing effect was deteriorated, the average particle diameter was small, and the printing effect of the composite 3D printing ink was better when the printing head had the inner diameter of 330 μm, the decellularized matrix gel microspheres were not broken, and the shape was not changed (fig. 12).
Comparative example 2
The comparative example provides a method for preparing acellular matrix microspheres, which comprises the following steps:
(1) Preparing a micro-channel chip: mixing polydimethylsiloxane and a cross-linking agent in a mass ratio of 10:1, pouring the mixture on a master, wherein the cross-linking agent is sylgard 184 of the Dow Corning company, and then curing the mixture for 4 hours at 70 ℃;
after curing, peeling the polydimethylsiloxane mould from the mother plate, and then punching a channel inlet and a channel outlet on the mould by using a puncher;
after the punching is finished, bonding the polydimethylsiloxane mould to a polydimethylsiloxane film after oxygen plasma treatment, and finally curing for 4 hours at 70 ℃ to obtain the micro-channel chip;
(2) Preparation of an aqueous phase: adding the acellular matrix powder into pepsin solution for digestion to obtain acellular matrix solution with the mass fraction of 0.8%;
(2) Preparation of an oil phase: adding the Krytox- (PEG) -Krytox block polymer into perfluorobutyl methyl ether for dissolution to obtain a Krytox- (PEG) -Krytox block polymer solution with the volume fraction of 0.4 and 6 percent respectively;
(3) And respectively horizontally placing an injector filled with a water phase and an oil phase on a micro-injection pump, connecting a needle head with an inlet of a chip by using a silica gel tube, respectively injecting the water phase and the oil phase, wherein the flow rate of the water phase is 0.5mL/h, the flow rate of the oil phase is 10mL/h, the water phase and the oil phase form stable water-in-oil liquid drops at the inlet of the chip, the water-in-oil liquid drops flow through the silica gel tube placed in a water bath with the temperature of 37 ℃ at the outlet of the chip, and the water-in-oil liquid drops are gelled in the silica gel tube to obtain the acellular matrix gel microspheres.
Observing the morphology of the obtained acellular matrix gel microspheres by using an optical microscope, wherein when the volume fraction of the surfactant is 0.4%, the obtained acellular matrix gel microspheres cannot be stably dispersed in a continuous phase, and the oil-water interface of the acellular matrix gel microspheres is unstable and aggregation adhesion can occur as shown in fig. 13A; as shown in fig. 13B, when the volume fraction of the surfactant is 6%, the oil-water interface of the obtained acellular matrix gel microsphere is stable, but the subsequent demulsification and washing processes are complicated, and meanwhile, the acellular matrix gel microsphere has certain biotoxicity.
Finally, it should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, and that those skilled in the art will understand that the technical scheme of the invention may be modified or equally substituted without departing from the spirit and scope of the technical scheme of the invention.
Claims (4)
1. The application of the composite 3D printing ink in biological 3D printing is characterized in that the composite 3D printing ink is mainly obtained by mixing acellular matrix gel microspheres and gel materials, and the preparation method of the composite 3D printing ink comprises the following steps:
(1) Adding acellular matrix powder into pepsin solution for digestion to obtain acellular matrix solution with mass fraction of 0.5% -1%, adding NaOH solution to adjust pH to neutrality, and adding 10 XPBS solution to make ionic strength of acellular matrix solution be 1, and taking the acellular matrix solution as water phase;
(2) Adding a surfactant into perfluorobutyl methyl ether to obtain a surfactant solution with the volume fraction of 0.5-5%, wherein the surfactant is a polyethylene glycol block copolymer and is used as an oil phase;
(3) Introducing the acellular matrix solution obtained in the step (1) into an aqueous phase inlet, introducing the solution obtained in the step (2) into an oil phase inlet, and generating water-in-oil droplets at the interface of a chip microchannel under the action of a surfactant; the obtained liquid drops flow in a rubber tube and gel through water bath at 37 ℃ to obtain acellular matrix gel microspheres, wherein the particle size of the acellular matrix gel microspheres is 60-300 mu m;
(4) Dissolving a gel material at 37 ℃ to obtain a gel solution, wherein the gel material is gelatin or methacryloylated gelatin;
(5) Uniformly mixing the acellular matrix gel microspheres with a gel solution to obtain composite 3D printing ink; the diameter of a printing needle used in the printing process of the composite 3D printing ink is 0.08-0.41mm, and the diameter of the printing needle is 1-2 times of the particle size of the acellular matrix gel microsphere.
2. The use of the composite 3D printing ink according to claim 1 in biological 3D printing, wherein the volume fraction of the acellular matrix gel microspheres in the composite 3D printing ink is 25% -75%.
3. Use of the composite 3D printing ink according to claim 1 in biological 3D printing, wherein the mass-volume concentration of the gel material is 50-90 mg/mL.
4. Use of the composite 3D printing ink according to claim 1 in biological 3D printing, wherein the gel material is photo-crosslinked after the composite 3D printing ink is printed.
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