CN114796617A - Composite 3D printing ink and application thereof - Google Patents
Composite 3D printing ink and application thereof Download PDFInfo
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- CN114796617A CN114796617A CN202210574342.9A CN202210574342A CN114796617A CN 114796617 A CN114796617 A CN 114796617A CN 202210574342 A CN202210574342 A CN 202210574342A CN 114796617 A CN114796617 A CN 114796617A
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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 obtained by mixing acellular matrix gel microspheres and a gel material. According to the invention, the acellular matrix is prepared into hydrogel microspheres and then added into a gel material to form the composite 3D printing ink, so that the printability and the biological activity of the ink are effectively compatible, the speed of exerting the function of a biological active substance in the ink is improved, the negative influence of the phase separation of two hydrogels on a printing process is avoided, and the composite 3D printing ink also has the application potential of packaging or loading cells on the microspheres for modular construction, and manufacturing a multi-scale three-dimensional composite structure support to adjust the microenvironment and the macroscopic environment of the cells.
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 bio-ink is used as a joint point of equipment and cells, is required to be rapidly solidified while ensuring the printing precision, is required to protect the cells in the printing process, and provides a function of a microenvironment supporting the growth of the cells, and is the most important part in the printing process. The bio-ink has three requirements, namely printability, biocompatibility and mechanical property. Printability refers to the property of a material that can be shaped in a precisely controllable manner in the spatial and temporal dimensions, i.e. the material used for printing must be deposited precisely in the designated space in a certain time, which is directly related to whether the 3D printed product can achieve the desired structural and dimensional accuracy. Printability the formability of bio-inks was evaluated, including adjustable viscosity of the bio-material, fast transition from sol to gel state, and a wide range of printable parameters. Taking extrusion printing as an example, the printability generally comprises 3 layers, namely that the viscosity of the biological ink can be regulated, such as through temperature change, shear stress and the like. Only if the viscosity is adjustable, a proper printing mode and printing parameters can be designed. ② the bio-ink needs to be in fluid or semi-fluid state before printing to avoid nozzle clogging, and can be cured quickly after printing to maintain shape. The adhesion between the different layers is also very important during layer-by-layer printing, which determines whether the material or the printing process is a true 3D printing. And third, possessing or finding the printing window or the process parameter interval aiming at the material. Biocompatibility requires that the bio-ink be as similar as possible to the cellular microenvironment in the human body, be non-toxic, non-side-effect, and be suitable for cell adhesion, growth and proliferation, and also require appropriate porosity to facilitate 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 require in vitro culture, often accompanied by perfusion and degradation of nutrients, and require mechanical strength to resist external forces to maintain the topographical structure of the printed matter.
Acellular matrix (dECM) materials are derived from tissues/organs that are capable of removing immunogenicity by suitable acellular methods, retaining active substances such as proteins (collagen, fibronectin, laminin, etc.), polysaccharides (glycoglycan, proteoglycans, glycoproteins, etc.), and growth factors contained in the original tissues/organs. Compared with a single-component natural material, dECM can better simulate a primary tissue microenvironment, provide more attachment sites and nutrients for cells, ensure cell adhesion and growth and promote tissue regeneration. Also, studies have shown that dECM materials can be processed into various forms of hydrogels, cryoabrasive particles, microcarriers, etc., with varying structural and biomechanical properties, all of which can perform their biological functions. Therefore, bioink containing dmems hold great promise for high bioactivity and tissue-specific incorporation into custom-made complex bioscaffolds by 3D bioprinting. The dECM gel has the capability of self-assembling into gel, 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 limiting the application of the gel in biological printing. To date, many studies have been explored to improve the dmecm material for 3D printing. The method mainly comprises the following steps: chemically modifying the dECM material, such as methyl allene modification, introducing double bonds, and changing a crosslinking mode so as to enhance the mechanical property of the dECM material; compounding dECM with other high molecular materials, which is a better method for keeping biocompatibility, and considering printability and bioactivity to a certain extent for several systems commonly used at present, such as bio-ink directly mixed by methacrylated gelatin and acellular matrix (GelMA + dECM) hydrogel, but as GelMA generates temperature response sol-gel transformation at a temperature lower than body temperature and the acellular matrix hydrogel generates gelation at the body temperature, the difference of the gelation mechanism and temperature-sensitive transformation of the GelMA and the acellular matrix hydrogel increases the complexity of the process for extrusion printing, and the phase separation is easy to occur in the stretching and mixing process in the printing process, so that the non-uniformity of a printing structure and the blockage of a nozzle are caused; the addition of GelMA into powder prepared from acellular matrix greatly improves the printability, but the powder is not uniform and has poor degradability, which is not favorable for quick action.
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 purpose, the invention adopts the technical scheme that: in a first aspect, a composite 3D printing ink is provided, which is obtained by mixing acellular matrix gel microspheres and a gel material.
According to the invention, the acellular matrix is prepared into hydrogel microspheres and then added into a gel material to form the composite 3D printing ink, so that the printability and the biological activity of the ink are effectively compatible, the speed of exerting the function of a biological active substance in the ink is improved, the negative influence of the phase separation of two hydrogels on a printing process is avoided, and the composite 3D printing ink also has the application potential of packaging or loading cells on the microspheres for modular construction, and manufacturing a multi-scale three-dimensional composite structure support to adjust the microenvironment and the macroscopic environment of the cells.
The acellular matrix gel microspheres retain specific active substances such as proteins, polysaccharides, growth factors and the like in tissues, can better simulate microenvironment, are beneficial to cell adhesion, growth and reproduction, promote the regeneration of the matrix and provide excellent cell compatibility and histocompatibility. The sources of the acellular matrix in the invention can be tissues and organs such as peripheral nerves, spinal cords, small intestinal mucosa, cornea and the like.
The gel material is used as an ink matrix, has quite good temperature-sensitive performance and shear thinning performance, and provides printability for composite 3D printing ink.
Optionally, the composite 3D printing ink further comprises a photoinitiator, the photoinitiator has a mass-volume concentration of 0-10 mg/mL, and the photoinitiator can rapidly initiate a gel material to undergo radical polymerization to generate an crosslinked network after the composite 3D printing ink is printed, so that the mechanical strength and the shape fidelity of the printing structure are remarkably improved. Preferably, the photoinitiator is one of phenyl-2, 4, 6-trimethylbenzoyllithium phosphite (LAP) and 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (Irgacure 2959).
As an optional embodiment of the composite 3D printing ink of the present invention, the method for preparing the acellular matrix gel microspheres comprises the following steps:
(1) adding the acellular matrix powder into a pepsin solution for digestion to obtain an acellular matrix solution with the mass fraction of 0.5-1%, adding a NaOH solution to adjust the pH to be neutral, adding a 10 x PBS solution to ensure that the ionic strength of the acellular matrix solution is 1, and taking the acellular matrix solution as a 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 a water phase inlet, introducing the solution obtained in the step (2) into an oil phase inlet, and generating water-in-oil droplets at the junction of the chip microchannel by the oil-water phase under the action of a surfactant; the obtained liquid drops flow in the rubber tube and are gelatinized through a water bath at 37 ℃ to obtain the acellular matrix gel microspheres.
The volume fraction of the surfactant plays an important role in the formation of the microspheres, 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 microspheres is unstable and can be aggregated and bonded; when the volume fraction of the surfactant is within the range of 0.5-5%, the stability of an 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 demulsify and wash and have certain biological toxicity, so that the biological activity of the composite 3D printing ink is influenced.
The 10 x PBS 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 action enables the freely coiled polypeptide chain to be rapidly converted to a beta-folded structure, so that the acellular matrix solution can be gelatinized.
Optionally, the surfactant in step (2) above is a polyethylene glycol block copolymer, which is available under the trade name Krytox- (PEG) -Krytox block polymer, and is purchased 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 velocity ratio of the oil phase to the water phase is v Oil /v Water (W) 2.5-20, the particle size of the acellular matrix gel microspheres is gradually reduced along with the increase of the speed ratio of the oil phase to the water phase, and the invention can control the particle size of the acellular matrix gel microspheres 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% to 75%.
Optionally, the particle size of the acellular matrix gel microspheres is 60-300 μm.
Optionally, the mass-volume concentration of the gel material is 50-90 mg/mL. Gel materials provide printability to composite 3D printing inks, and gel materials below the above mass-volume concentrations provide insufficient printability.
Optionally, the gel material is gelatin or methacrylated gelatin. Methacrylated gelatin (GelMA) can be rapidly cured by ultraviolet light 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 the printing needle used in the printing process of the composite 3D printing ink is 0.08-0.41 mm.
Optionally, the diameter of the printing needle is 1-2 times of the particle size of the acellular matrix gel microspheres.
In the printing process, the printing needle head and the particle size of the microsphere have a certain relation, and the microsphere has certain elasticity, so that the microsphere with the inner diameter larger than that of the needle head can be extruded in the extrusion printing process, but the microsphere can deform and break to a certain extent. The inventor researches to find that when the diameter of a printing needle is 2 times of the particle size of the microsphere, the microsphere can be extruded smoothly and maintains the shape. Therefore, in the printing process, the proper particle size of the microspheres and the proper printing needle head can be selected according to actual conditions.
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 derived from peripheral nerves, which is adopted by the invention, can not only retain the active ingredients and tissue specificity of extracellular matrix, but also maintain the nanofiber structure similar to the extracellular matrix, can create a microenvironment beneficial to 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 microspheres, the obtained acellular matrix microspheres can be uniformly emulsified, and the acellular matrix microspheres have mild gelatinization conditions (37 ℃ and neutral pH) and good potential of encapsulating cells in the microspheres.
(3) According to the invention, the acellular matrix is prepared into hydrogel microspheres, and then the hydrogel microspheres are added into a gel material to form the composite 3D printing ink, so that the printing property and the biological activity of the ink are effectively compatible, the speed of the bioactive substances in the ink playing a role is increased, and the negative influence of the phase separation of two hydrogels on a printing process is avoided; the method also has the application potential that cells are encapsulated or loaded on microspheres for modular construction, and a multi-scale three-dimensional composite structure support is manufactured to adjust the micro environment and the macro 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 methacrylated gelatin and the results before and after modification of the gelatin 1 H-NMR chart;
FIG. 3 is a pictorial representation of methacrylated gelatin and acellular matrix gel prepared in example 1;
FIG. 4 shows the difference v Oil /v Water (W) An optical micrograph of the prepared acellular matrix gel microspheres; FIG. 4A is v Oil /v Water (I) 2.5, v in fig. 4B Oil(s) /v Water (W) V in fig. 4C is 5 Oil /v Water (W) 7.5, v in fig. 4D Oil /v Water (W) V in fig. 4E, 10 Oil /v Water (W) 15, v in fig. 4F Oil /v Water (W) ＝20；
FIG. 5 shows the particle size of acellular matrix gel microspheres as a function of v Oil(s) /v Water (W) A variation graph of (2);
FIG. 6 is a graph of viscosity as a function of shear rate for composite 3D printing ink prepared in example 5;
fig. 7 is a graph of shear modulus versus temperature for composite 3D printing inks prepared in example 5 and comparative example 1;
fig. 8 is a graph showing the change of storage modulus before and after light irradiation of the composite 3D printing inks prepared in example 5 and comparative example 1;
fig. 9 is a graph showing live and dead staining results and cell viability rates of PC12 cells cultured in the composite 3D printing ink prepared in example 5 and comparative example 1 at D1 and D4, fig. 9A, D is a graph showing live and dead staining results of PC12 cells cultured in the composite 3D printing ink prepared in comparative example 1 at D1 and D4, fig. 9B, E is a graph showing live and dead staining results of PC12 cells cultured in the composite 3D printing ink prepared in example 5 at D1 and D4, and fig. 9 shows cell viability rates of PC12 cells cultured in the composite 3D printing ink prepared in example 5 and comparative example 1 at D1 and D4;
fig. 10 is a grid structure diagram printed using the composite 3D printing ink prepared in example 5;
fig. 11 is a graph showing the microscopic observation result of the composite 3D printing ink printed mesh structure prepared in example 5 of the present invention;
FIG. 12 is a diagram showing the influence of different printing needle internal diameters on the particle size of acellular matrix gel microspheres before and after printing, FIG. 12A is a diagram showing a real object of composite 3D printing ink loaded into a syringe, FIG. 12B is a diagram showing the variation of the maximum extrusion particle size of acellular matrix gel microspheres with the printing needle internal diameter, FIGS. C (1), C (2) and C (3) are diagrams showing the variation of the particle size of acellular matrix gel microspheres before printing, with the printing needle internal diameter of 330 μm and with the printing needle internal diameter of 210 μm after printing, and FIGS. D (1), D (2) and D (3) are diagrams showing the variation of the particle size of acellular matrix gel microspheres before printing, with the printing needle internal diameter of 210 μm and with the printing needle internal diameter of 110 μm after printing, respectively;
fig. 13 is an optical micrograph of acellular matrix gel microspheres made with different volume fractions of surfactant, fig. 13A with a 0.4 volume fraction of surfactant and fig. 13B with a 6 volume fraction of surfactant.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings.
1. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
2. In the following examples, the acellular matrix powder used was prepared by the following method:
(1) pretreatment: cutting peripheral nerve tissues of the mice into tissue blocks of 5mm multiplied by 5mm, and fully washing the tissue blocks by using a PBS solution;
(2) and (3) cell removal: placing peripheral nerve tissue into 3% Triton X100 water solution, oscillating for 12 hr, taking out, rinsing in sterile distilled water for 3 times, placing into 4% sodium deoxycholate water solution, oscillating for 24 hr, and rinsing in sterile distilled water for 3 times. The above process was repeated 2 times;
(3) primary freeze-drying: placing the peripheral nerve tissue after 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 for multiple times by using sterile distilled water until no residue exists in the mixed solvent;
(6) and (3) secondary freeze-drying for later use: freeze drying the defatted peripheral nerve tissue in a freeze dryer at-80 deg.C, pulverizing, sieving with 40 mesh sieve, sealing the obtained acellular matrix powder, and storing at-40 deg.C.
3. In the following examples, the methacrylated gelatin (GelMA) used was prepared by the following method:
(1) dissolving 10g of gelatin in 100mL of PBS solution, heating the mixture to 60 ℃, and stirring until the gelatin is completely dissolved;
(2) dropwise adding 10mL of methacrylic anhydride into the gelatin solution in the step (1) by using a constant-pressure funnel at a rate of 0.25mL/min, uniformly stirring, and reacting at 60 ℃ for 3 hours;
(3) after the reaction is finished, adding a PBS solution with the pH value of 7.4 and the concentration of 0.1M to stop the reaction, wherein the adding amount of the PBS solution is 2 times of the volume of the mixture obtained in the step (2);
(4) putting the reaction solution obtained in the step (3) into a dialysis bag, putting the dialysis bag into pure water at 40 ℃, dialyzing for 3 days, and replacing the dialysis solution at 12h, 24h and 48 h; after dialysis, the solution is frozen in a refrigerator at the temperature of-40 ℃, and then the solution is placed in a freeze dryer to remove the solvent, so that white spongy solid GelMA is obtained; storing at minus 80 ℃ in dark for later use.
FIG. 2 is a schematic diagram of the synthesis of methacrylated gelatin and the results before and after modification of the gelatin 1 H-NMR graph, fig. 2H spectrum shows an increase in signal amount for methacrylate vinyl proton peaks (δ ═ 5.4 and 5.7ppm) and a decrease in signal amount for free lysine methylene proton peaks (δ ═ 2.9ppm), confirming successful modification of gelatin.
Example 1
This example is intended to illustrate the gelling properties of the acellular matrix for peripheral nervous tissue according to the present invention.
Weighing 10mg of acellular matrix powder, digesting the acellular matrix powder with 1ml of pepsin solution to obtain an acellular matrix solution with the mass fraction of 1%, adjusting the pH of the acellular matrix solution to be neutral by using NaOH solution to stop digestion, adding 10 XPBS solution to ensure that the ionic strength of the acellular matrix solution is 1, and gelling the acellular matrix solution at 37 ℃ to obtain acellular matrix gel with the mass fraction of 1%.
Fig. 3 is a schematic diagram of the gel of the acellular matrix of this example, and it can be seen from fig. 3 that the acellular matrix of peripheral nerve tissue can be transformed from a liquid state to a solid state by sol-gel transformation under the conditions of 37 ℃ and pH 7.
Example 2
This example illustrates an acellular matrix microsphere and a method for preparing 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 mother plate, wherein the cross-linking agent is sylgard 184 of Dow Corning company, and curing the mixture for 4 hours at 70 ℃;
after solidification, peeling the polydimethylsiloxane mold from the mother plate, and punching a channel inlet and a channel outlet on the mold by using a puncher;
after the punching is finished, carrying out oxygen plasma treatment on the polydimethylsiloxane mold, adhering the polydimethylsiloxane mold to a polydimethylsiloxane film, and finally curing the polydimethylsiloxane film at 70 ℃ for 4 hours to obtain the micro-channel chip;
(2) preparation of the aqueous phase: adding the acellular matrix powder into a pepsin solution for digestion to obtain an acellular matrix solution with the mass fraction of 1%, adding a NaOH solution to adjust the pH to be neutral, and adding a 10 x PBS solution to ensure that the ionic strength of the acellular matrix solution is 1;
(2) preparation of oil phase: adding the Krytox- (PEG) -Krytox block polymer into perfluorobutyl methyl ether for dissolving to obtain a Krytox- (PEG) -Krytox block polymer solution with the volume fraction of 1%;
(3) respectively horizontally placing the syringes filled with water phase and oil phase on a micro-injection pump, connecting the needle with the inlet of the chip by using a silicone 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 the speed ratio of the oil phase to the water phase is adjusted to be v within the flow rate range Oil /v Water (W) 2.5, 5, 7.5, 10, 15, 20, the aqueous and oil phases formed stable water-in-oil droplets at the inlet of the chip, the water-in-oil dropletsPassing through a silica gel tube placed in a water bath at 37 deg.C at the outlet of the chip, and allowing the water-in-oil droplets to gelatinize in the silica gel tube to obtain acellular matrix gel microspheres with diameter of 60-300 μm.
FIG. 4 shows the difference v Oil /v Water (W) An optical micrograph of the prepared acellular matrix gel microspheres; FIG. 4A is v Oil /v Water (W) 2.5, v in fig. 4B Oil /v Water (W) V in fig. 4C is 5 Oil /v Water (W) 7.5, v in fig. 4D Oil /v Water (W) V in fig. 4E, 10 Oil /v Water (W) 15, v in fig. 4F Oil /v Water (W) 20; FIG. 5 shows the particle size of acellular matrix gel microspheres as a function of v Oil /v Water (W) A variation diagram of (2). As can be seen from FIGS. 4 and 5, with v Oil /v Water (W) The particle size of the acellular matrix gel microspheres is gradually reduced. When v is Oil /v Water (W) The average particle diameters were 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, and 77.1. + -. 3.5. mu.m, respectively, for 2.5, 5, 7.5, 10, 15, and 20, respectively. When v is Oil /v Water (I) Beyond 20, increasing the flow rate of the oil phase or decreasing the flow rate of the aqueous phase does not result in a major change in the particle size of the microspheres, but only changes the rate of microsphere production. When v is Oil /v Water (W) When the amount is less than 5, the clogging of the pipe is likely to occur.
Example 3
This example is illustrative 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 microns obtained in the embodiment 2 sequentially through the steps of centrifuging and cleaning, transferring the washed acellular matrix gel microspheres into a PBS (phosphate 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 37 ℃ to obtain GelMA solution; and (3) 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 in the composite 3D printing ink, the volume fraction of the dECM-MS is 25%, the mass-volume fraction of the GelMA is 90mg/mL, and the mass-volume fraction of the LAP is 10 mg/mL.
Example 4
This example is illustrative 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 microns obtained in the embodiment 2 sequentially through the steps of centrifuging and cleaning, transferring the washed acellular matrix gel microspheres into a PBS (phosphate 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 37 deg.C to obtain GelMA solution; and (3) 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 in the composite 3D printing ink, the volume fraction of the dECM-MS is 75%, the mass-volume fraction of the GelMA is 50mg/mL, and the mass-volume fraction of the LAP is 1 mg/mL.
Example 5
This example is illustrative 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 microns obtained in the embodiment 2 sequentially through the steps of centrifuging and cleaning, transferring the washed acellular matrix gel microspheres into a PBS (phosphate 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 37 deg.C to obtain GelMA solution; and (3) isovolumetrically and 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 in the composite 3D printing ink, the volume fraction of dECM-MS is 50%, the mass-volume fraction of GelMA is 80mg/mL, the mass-volume fraction of LAP is 3mg/mL, and the composite 3D printing ink in the embodiment is marked as 8% GelMA + MS.
Comparative example 1
The present comparative example provides a method of preparing a composite 3D printing ink, comprising the steps of:
and adding GelMA into a PBS buffer solution containing LAP, and dissolving at 37 ℃ to obtain a GelMA solution, so as to obtain the composite 3D printing ink, wherein in the composite 3D printing ink, the mass-volume fraction of GelMA is 80mg/mL, the mass-volume fraction of LAP is 3mg/mL, and the composite 3D printing ink of the comparative example is marked as 8% GelMA.
Effect example 1
The rheological mechanical properties of the composite 3D printing ink prepared by the invention are tested.
The printing inks prepared in example 5 and comparative example 1 were tested for their rheological mechanical properties using a rotational rheometer, and the results are shown in fig. 6 to 8. It can be seen from fig. 6 that the composite 3D printing ink prepared in example 5 exhibits 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 interval of the composite 3D printing ink prepared in example 5 did not undergo significant shift compared to comparative example 1.
As can be seen from fig. 8, the modulus of the printed ink prepared in example 5 before light irradiation was larger than that of the printed ink prepared in comparative example 1, which corresponds to the test results 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 both cured within 60 seconds, and the modulus of the printing ink prepared in example 5 was smaller than that of the printing ink prepared in comparative example 1 after photo-crosslinking. The above results indicate that before photocuring, the GelMA cross-linked network is not formed and is in a fluid state, and the added acellular matrix gel microspheres improve the strength of GelMA to some extent, but as photocuring progresses, the size of the acellular matrix gel microspheres is larger, which hinders the formation of the GelMA cross-linked network, 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 growth and migration of cells are facilitated, which is identical with the experimental result of effect example 2.
Effect example 2
The effect example researches the bioactivity of the composite 3D printing ink, 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 microns obtained in the embodiment 2 sequentially through the steps of centrifuging and cleaning, then transferring the washed acellular matrix gel microspheres into 75% alcohol solution for soaking for 2 hours for disinfection, centrifuging after soaking is finished, transferring the obtained precipitate into PBS buffer solution, centrifuging and compacting to obtain the acellular matrix gel microspheres, and recording as dECM-MS;
(2) soaking GelMA in 75% alcohol solution for 2 hr for disinfection, and after soaking, placing the alcohol solution containing GelMA in a vacuum drying oven to remove solvent 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, and 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 in the composite 3D printing ink, the volume fraction of dECM-MS is 50%, the mass-volume fraction of GelMA is 80mg/mL, the mass-volume fraction of LAP is 3mg/mL, and the composite 3D printing ink of the embodiment is marked as 8% GelMA + microspheres;
(4) and (3) adding the GelMA obtained in the step (2) into PBS buffer solution containing LAP, and dissolving at 37 ℃ to obtain GelMA solution, namely obtaining the composite 3D printing ink, wherein in the composite 3D printing ink, the mass-volume fraction of GelMA is 80mg/mL, the mass-volume fraction of LAP is 3mg/mL, and the composite 3D printing ink of the comparative example is marked as 8% GelMA-2.
The biological activities of the 8% GelMA + microspheres and 8% GelMA-2 obtained in the effect example are tested, and the specific steps are as follows:
respectively wrapping PC12 cells with 8% GelMA + microspheres and 8% GelMA-2 for 3D printing (2 × 105 cells are wrapped in per ml of ink), placing the printed sample in a 48-pore plate, and crosslinking by ultraviolet irradiation after printing, wherein the illumination intensity is 5mw/cm 2 The time is 50s, and after crosslinking and solidification, cell viability detection is carried out on D1 and D4 respectively. The results are shown in fig. 9, and it can be seen from fig. 9 that 8% GelMA + microspheres are more favorable for the growth of PC12 cells than 8% GelMA-2, and have excellent biocompatibility, which is attributed to the composite 3D printing ink of the present inventionThe water contains acellular matrix microspheres.
Effect example 3
The present effect example investigated the printing performance and positioning performance in the printing 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 cylinder and printed at room temperature on a square grid of 20 × 20mm length × width with 1.5mm internal line spacing, 0.32mm layer height and 0.41mm printing needle internal diameter. After printing, ultraviolet irradiation is adopted for crosslinking, and the illumination intensity is 5mw/cm 2 The time was 50 s. The green fluorescently labeled acellular matrix gel microspheres were clearly visible in the clear GelMA scaffold when viewed under the fluorescence microscope, and the shapes of the acellular matrix gel microspheres were well preserved without severe deformation and rupture (fig. 10 and 11). The composite 3D printing ink can be applied to the construction of a bionic multi-scale composite scaffold.
Effect example 4
The effect example researches the matching property of the particle size of the acellular matrix microsphere and the printing needle.
(1) Acellular matrix gel microspheres with particle sizes of 89 +/-22 microns and 154 +/-22 microns respectively are prepared by the preparation method of 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.
Loading the composite 3D printing ink containing acellular matrix gel microspheres with the particle size of 89 +/-22 microns into a printing cylinder, and printing at room temperature by respectively using a printing needle with the inner diameter of 110 microns and the inner diameter of 210 microns, wherein the composite 3D printing ink has the advantages that when the inner diameter of the printing needle is 110 microns, the acellular matrix gel microspheres deform, the average particle size becomes small, the printing effect becomes poor, and when the inner diameter of the printing needle is 210 microns, the composite 3D printing ink has a better printing effect, the microspheres are not broken, and the shape is not changed (figure 12).
Loading the composite 3D printing ink containing the acellular matrix gel microspheres with the particle size of 154 +/-22 microns into a printing cylinder, and printing at room temperature by respectively using the printing needle with the inner diameter of 210 microns and the printing needle with the inner diameter of 330 microns, wherein the acellular matrix gel microspheres deform and have poor printing effect and small average particle size when the printing needle with the inner diameter of 210 microns, and the composite 3D printing ink has better printing effect, is not cracked and has no change in shape when the printing needle with the inner diameter of 330 microns (figure 12).
Comparative example 2
The comparative example provides a preparation method of 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 mother plate, wherein the cross-linking agent is sylgard 184 of Dow Corning company, and curing the mixture for 4 hours at 70 ℃;
after solidification, peeling the polydimethylsiloxane mold from the mother plate, and punching a channel inlet and a channel outlet on the mold by using a puncher;
after the punching is finished, carrying out oxygen plasma treatment on the polydimethylsiloxane mold, adhering the polydimethylsiloxane mold to a polydimethylsiloxane film, and finally curing the polydimethylsiloxane film at 70 ℃ for 4 hours to obtain the micro-channel chip;
(2) preparation of an aqueous phase: adding the acellular matrix powder into a pepsin solution for digestion to obtain an acellular matrix solution with the mass fraction of 0.8%;
(2) preparation of oil phase: adding the Krytox- (PEG) -Krytox block polymer into perfluorobutyl methyl ether for dissolving to respectively obtain Krytox- (PEG) -Krytox block polymer solution with volume fraction of 0.4 and 6 percent;
(3) and horizontally placing the injector filled with the water phase and the oil phase on a micro-injection pump respectively, connecting the needle with the inlet of the chip by using a silicone tube, injecting the water phase and the oil phase respectively, 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 droplets at the inlet of the chip, the water-in-oil droplets flow through the silicone tube placed in a water bath with the temperature of 37 ℃ at the outlet of the chip, and the water-in-oil droplets are gelatinized in the silicone tube to obtain the acellular matrix gel microspheres.
Observing the morphology of the obtained acellular matrix gel microspheres by using an optical microscope, as shown in fig. 13A, 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 and adhesion can occur; as shown in fig. 13B, when the volume fraction of the surfactant is 6%, the oil-water interface of the obtained acellular matrix gel microspheres is stable, but the subsequent demulsification and washing processes are complicated, and the acellular matrix gel microspheres have certain biological toxicity.
Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. The composite 3D printing ink is characterized by being mainly obtained by mixing acellular matrix gel microspheres and a gel material.
2. The composite 3D printing ink according to claim 1, wherein the method for preparing the acellular matrix gel microspheres comprises the following steps:
(1) adding the acellular matrix powder into a pepsin solution for digestion to obtain an acellular matrix solution with the mass fraction of 0.5-1%, adding a NaOH solution to adjust the pH to be neutral, adding a 10 x PBS solution to ensure that the ionic strength of the acellular matrix solution is 1, and taking the acellular matrix solution as a 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 a water phase inlet, introducing the solution obtained in the step (2) into an oil phase inlet, and generating water-in-oil droplets at the junction of the chip microchannel by the oil-water phase under the action of a surfactant; the obtained liquid drops flow in the rubber tube and are gelatinized through a water bath at 37 ℃ to obtain the acellular matrix gel microspheres.
3. The composite 3D printing ink according to claim 1, wherein the volume fraction of the acellular matrix gel microspheres in the composite 3D printing ink is 25% to 75%.
4. The composite 3D printing ink according to claim 1, wherein the acellular matrix gel microspheres have a particle size of 60-300 μm.
5. The composite 3D printing ink as claimed in claim 1, wherein the gel material has a mass-volume concentration of 50 to 90 mg/mL.
6. The composite 3D printing ink according to claim 1, wherein the gel material is gelatin or methacrylated gelatin.
7. Use of the composite 3D printing ink according to any one of claims 1 to 6 for bio-3D printing.
8. The use according to claim 7, wherein the composite 3D printing ink has a printing needle diameter of 0.08-0.41mm during printing.
9. The use of claim 8, wherein the printing tip has a diameter of 1 to 2 times the size of the acellular matrix gel microspheres.
10. The use according to claim 7, wherein the gel material is photocrosslinked after printing of the composite 3D printing ink is completed.
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