CN116376816A - Microsphere-blended biological 3D printing gel material and preparation method and application thereof - Google Patents
Microsphere-blended biological 3D printing gel material and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedical materials, and particularly relates to a microsphere blend biological 3D printing gel material, and a preparation method and application thereof. The biological 3D printing gel material provided by the invention comprises photo-curing hydrogel and polymer porous microspheres loaded with target cells; the target cells are supported in the pore structure of the polymeric porous microsphere. The biological 3D printing gel material provided by the invention utilizes the three-dimensional pore structure of the polymer porous microsphere to load target cells to form a 'cell sphere microstructure', so that the target cells can form a microscopic living environment of the three-dimensional structure in the polymer porous microsphere, and the living environment of the target cells in vivo can be effectively simulated; meanwhile, the porous polymer microsphere is used as a carrier, and under the irradiation curing of an ultraviolet lamp, the porous structure can protect target cells in the microsphere to reduce the damage of ultraviolet rays and the cell damage caused by 3D printing, so that the target cells can maintain good cell activity.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a microsphere blend biological 3D printing gel material, and a preparation method and application thereof.
Background
Osteoarthritis generally refers to inflammatory diseases that occur in the joints of the human body and surrounding tissues and are caused by inflammation, infection, degeneration, trauma, or other factors. Clinically, the symptoms of red, swelling, heat, pain, dysfunction and joint deformity of joints are that the serious symptoms cause joint disability and influence the life quality of patients. Therefore, finding a suitable regimen for treating osteoarthritis is a focus of research in the medical arts today.
While organisms have self-healing capabilities themselves, the ability to self-repair is limited, and when body damage exceeds a certain limit, self-repair becomes difficult to achieve, with traditional medical techniques helping little to such conditions. Along with the development of medicine, regenerative medicine and tissue engineering try to implant a bracket embedded with cells into damaged tissues of a patient, so that the tissue repair capability of the lesion is improved; or in vitro culturing human tissue, and cutting the healthy artificially cultured tissue to replace the lesion part so as to achieve the treatment effect. The functional expression of cells in an in-vitro environment is often different from that in-vivo environment, and in order to realize in-vitro tissue culture and maintain the normal functional expression of cells in the in-vitro environment, it is often necessary to construct a tissue engineering scaffold simulating the in-vivo environment as a cell carrier.
In the traditional porous scaffold design, hydrogel materials such as methacryloyl gelatin and the like are generally mixed with cell slurry to be used as biological ink, and a tissue frame is obtained through 3D printing, so that a three-dimensional cell model is cultured in vitro, but the biological ink obtained by the cell slurry is printed on the microenvironment of cells through 3D, so that a two-dimensional structure is formed, and the two-dimensional cell scaffold model has the problems that the complicated spatial structure of in-vivo tissues cannot be simulated, and the expression of genes and proteins cannot be fully caused by the cells; but also severely reduces cell activity when cured by irradiation of ultraviolet light.
Disclosure of Invention
The invention aims to provide a microsphere blended biological 3D printing gel material, a preparation method and application thereof, and in-vitro bionic tissues obtained by the biological 3D printing gel material can improve the survival rate of cells and the proliferation and growth effects of the cells in vitro when in-vitro culture.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a biological 3D printing gel material, which comprises photo-curing hydrogel and polymer porous microspheres loaded with target cells; the target cells are supported in the pore structure of the polymeric porous microsphere.
Preferably, the material of the polymer porous microsphere comprises one or more of polylactic acid-glycolic acid copolymer, polylactic acid and derivatives thereof, alginic acid and derivatives thereof, chitosan and derivatives thereof, and hyaluronic acid and derivatives thereof.
Preferably, the preparation method of the polymer porous microsphere comprises the following steps:
respectively injecting the oil phase and the water phase into a microfluidic device for mixing, and obtaining polymer microspheres under the shearing action of surface tension and the water phase; the aqueous phase comprises polyvinyl alcohol and water; the oil phase comprises a degradable polymer, a pore-forming agent, water and an organic solvent;
and freeze-drying the polymer microsphere to obtain the polymer porous microsphere.
Preferably, in the water phase, the mass percentage of the polyvinyl alcohol is 0.04-2%;
in the oil phase, the pore-forming agent is gelatin, the organic solvent comprises dichloromethane and/or trifluoroethanol, the mass ratio of the polymer to the organic solvent is 1 (10-100), and the mass ratio of the pore-forming agent to the water is 0.07-0.1) (1-1.5); the mass ratio of the polymer to the pore-forming agent is 1 (1-2).
Preferably, the injection rate of the aqueous phase is 2mL/min; the injection speed of the oil phase is 0.05mL/min;
the temperature of the mixing is 0-10 ℃.
Preferably, the photo-curing hydrogel is one or more of an aqueous solution of methacryloyl gelatin, an aqueous solution of acrylated alginate and derivatives thereof, an aqueous solution of acrylated hyaluronic acid and an aqueous solution of acrylated chitosan;
the mass concentration of the photo-curing hydrogel is 50-150 mug/mL.
Preferably, the target cells include one or more of embryonic osteoblasts, embryonic chondrocytes, aortic endothelial cells, osteogenic cells, and osteoclasts.
The invention provides a preparation method of the biological 3D printing gel material, which comprises the following steps:
immersing the polymer porous microsphere in a target cell suspension to obtain a polymer porous microsphere loaded with target cells;
and mixing the photo-cured hydrogel and the polymer porous microsphere loaded with the target cells to obtain the biological 3D printing gel material.
The invention provides an application of the biological 3D printing gel material according to the technical scheme or the biological 3D printing gel material prepared by the preparation method according to the technical scheme in preparing in-vitro bionic tissues.
The invention provides an in-vitro bionic tissue, which is obtained by performing 3D printing on biological ink and then performing photo-curing under the condition of ultraviolet irradiation;
the biological ink comprises the biological 3D printing gel material according to the technical scheme or the biological 3D printing gel material prepared by the preparation method according to the technical scheme and a photoinitiator.
The invention provides a biological 3D printing gel material, which comprises photo-curing hydrogel and polymer porous microspheres loaded with target cells; the target cells are supported in the pore structure of the polymeric porous microsphere. The biological 3D printing gel material provided by the invention utilizes the three-dimensional pore structure of the polymer porous microsphere to load target cells to form a 'cell sphere microstructure', so that the target cells can form a microscopic living environment of the three-dimensional structure in the polymer porous microsphere, and the living environment of the target cells in vivo can be effectively simulated; meanwhile, the porous polymer microsphere is used as a carrier, and under the irradiation curing of an ultraviolet lamp, the porous structure can protect target cells in the microsphere to reduce the damage of ultraviolet rays, so that the target cells can maintain good cell activity. The results of the examples show that the biological 3D printing gel material provided by the invention has good proliferation and growth effects as target cells in external bionic tissues obtained by printing with biological ink.
Further, in the present invention, the preparation method of the polymer porous microsphere comprises the following steps: respectively injecting the oil phase and the water phase into a microfluidic device for mixing, and obtaining polymer microspheres through the shearing action of the water phase; the aqueous phase comprises polyvinyl alcohol and water; the oil phase comprises a degradable polymer, a pore-forming agent, water and an organic solvent; and freeze-drying the polymer microsphere to obtain the polymer porous microsphere. The invention adopts a microfluidic method to prepare the polymer porous microsphere, and the preparation method and the flow are simple, easy to operate, mild in reaction condition and short in period.
Drawings
FIG. 1 is a flow chart of the preparation of a biological 3D printing gel material and a 3D printing layered osteochondral model according to an embodiment of the present invention;
FIG. 2 is an SEM image of a layered osteochondral model prepared in example 1;
FIG. 3 is a CLSM image of the layered osteochondral model prepared in example 2, showing cell adhesion growth;
FIG. 4 is a graph showing the distribution of cells on PLGA microspheres using H & E staining of the layered osteochondral model prepared in example 3;
FIG. 5 is a graph showing the results of comparison of cell activities of a 3D tumor model and a 2D culture model prepared according to the present invention.
Detailed Description
The invention provides a biological 3D printing gel material, which comprises photo-curing hydrogel and polymer porous microspheres loaded with target cells; the target cells are supported in the pore structure of the polymeric porous microsphere.
In the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
The biological 3D printing gel material provided by the invention comprises photo-curing hydrogel.
In the present invention, the photocurable hydrogel is preferably one or more of an aqueous solution of methacryloyl gelatin, an aqueous solution of acryloylacrylate and its derivatives, an aqueous solution of acryloylahyaluronic acid and an aqueous solution of acryloylactochitosan; more preferably an aqueous solution of methacryloyl gelatin, an aqueous solution of acrylic acid alginate one or more of an aqueous solution of acrylated hyaluronic acid and an aqueous solution of acrylated chitosan; more preferably an aqueous solution of methacryloyl gelatin.
In the present invention, the mass concentration of the photocurable hydrogel is preferably 50 to 150. Mu.g/mL, particularly preferably 50. Mu.g/mL, 75. Mu.g/mL, 100. Mu.g/mL or 150. Mu.g/mL.
The biological 3D printing gel material provided by the invention comprises polymer porous microspheres loaded with target cells; the target cells are supported in the pore structure of the polymeric porous microsphere.
In the present invention, the material of the porous polymer microsphere preferably includes one or more of polylactic acid-glycolic acid copolymer, polylactic acid and its derivatives, alginic acid and its derivatives, chitosan and its derivatives, and hyaluronic acid and its derivatives; more preferably comprises one or more of polylactic acid-glycolic acid copolymer, polylactic acid, alginic acid, chitosan and hyaluronic acid; polylactic-co-glycolic acid (PLGA) is more preferred.
In the present invention, the polymer porous microspheres have a particle size of about 450 to 550 μm.
In the present invention, the specific surface area of the porous polymer microsphere is preferably 200 to 500m 2 The void porosity per gram is preferably 20 to 50% and the average pore diameter is preferably 20 to 50. Mu.m.
In the present invention, the preparation method of the polymer porous microsphere preferably comprises the following steps:
mixing the oil phase and the water phase, and obtaining polymer microspheres under the shearing action of surface tension and the water phase; the aqueous phase comprises polyvinyl alcohol and water; the oil phase comprises a polymer, a pore-forming agent, water and an organic solvent;
and freeze-drying the polymer microsphere to obtain the polymer porous microsphere.
The method comprises the steps of respectively injecting an oil phase and a water phase into a microfluidic device for mixing, and obtaining polymer microspheres under the shearing action of surface tension and the water phase; the aqueous phase comprises polyvinyl alcohol and water; the oil phase comprises a degradable polymer, a porogen, water and an organic solvent.
In the present invention, in the aqueous phase, the water is preferably ultrapure water; the polyvinyl alcohol (PVA) content is preferably 0.04 to 2% by mass, more preferably 0.05 to 1.8% by mass.
In the present invention, the preparation method of the aqueous phase preferably comprises the steps of: mixing the polyvinyl alcohol and water to obtain the water phase. The temperature of the mixing is preferably 20 to 80 ℃, more preferably 20 to 50 ℃; the mixing is performed under stirring, and the stirring time is preferably 0.2 to 1h. In the present invention, the aqueous phase preferably comprises at ambient conditions of 4 ℃.
In the present invention, in the oil phase, the water is preferably ultrapure water; the porogen is preferably Gelatin (GEL), and the organic solvent preferably comprises Dichloromethane (DCM) and/or trifluoroethanol, more preferably DMC; the degradable polymer preferably comprises one or more of polylactic acid-glycolic acid copolymer, polylactic acid and derivatives thereof, alginic acid and derivatives thereof, chitosan and derivatives thereof, and hyaluronic acid and derivatives thereof; more preferably comprises one or more of polylactic acid-glycolic acid copolymer, polylactic acid, alginic acid, chitosan and hyaluronic acid; polylactic-co-glycolic acid (PLGA) is more preferred.
In the present invention, the mass ratio of the degradable polymer to the organic solvent is preferably 1 (10 to 100), more preferably 1 (11 to 95); the mass ratio of the pore-forming agent to the water is preferably (0.07-0.1): 1-1.5; the mass ratio of the polymer to the porogen is preferably 1 (1-2).
In the present invention, the preparation method of the oil phase preferably includes the steps of: first dissolving the degradable polymer in an organic solvent to obtain a degradable polymer solution; dissolving the second pore-forming agent Kong Jidi in water to obtain a pore-forming agent solution; mixing the degradable polymer solution and the porogen solution. In the present invention, the volume ratio of the porogen solution to the degradable polymer solution is preferably 1 (1) to (5). In the present invention, the temperature of the second solvent is preferably 30 to 40 ℃, more preferably 35 ℃; the second solvent is preferably stirred for a period of time of preferably 40 to 45 minutes. In the present invention, the mixing is preferably performed under ultrasonic conditions.
In the present invention, the injection rate of the aqueous phase is preferably 2mL/min; the injection rate of the oil phase is preferably 0.05mL/min.
In the present invention, the temperature of the mixing is preferably 0 to 10 ℃, more preferably 0 to 5 ℃.
After the polymer microsphere is obtained, the polymer microsphere is freeze-dried to obtain the polymer porous microsphere.
In the present invention, the polymer microspheres are preferably washed with water before the freeze-drying, and there is no particular requirement for the specific implementation of the washing.
In the present invention, the target cells preferably include one or more of embryonic osteoblasts, embryonic chondrocytes, aortic endothelial cells, osteogenic cells, and osteoclasts, more preferably include at least two of embryonic osteoblasts, embryonic chondrocytes, aortic endothelial cells, osteogenic cells, and osteoclasts; it further preferably includes at least two of embryonic osteoblasts, embryonic chondrocytes and aortic endothelial cells.
In the present invention, the loading amount of the target cells (i.e., the mass percentage of the target cells to the target cell-loaded polymer porous microspheres) is preferably 20 to 50%.
In the present invention, the mass ratio of the target cell-supporting polymer porous microspheres to the photocurable hydrogel is preferably (1:10) to (1:100), more preferably (2:8) to (1:100).
The biological 3D printing gel material provided by the invention has the characteristics of good biocompatibility, uniform size, good biodegradability, low cytotoxicity and the like.
The invention provides a preparation method of the biological 3D printing gel material, which comprises the following steps:
immersing the polymer porous microsphere in a target cell suspension to obtain a polymer porous microsphere loaded with target cells;
and mixing the photo-cured hydrogel and the polymer porous microsphere loaded with the target cells to obtain the biological 3D printing gel material.
The invention dips the polymer porous microsphere into the target cell suspension to obtain the polymer porous microsphere loaded with the target cells.
In the present invention, the porous polymer microspheres are preferably subjected to a sterilization treatment prior to the impregnation, and the present invention is not particularly limited to the specific implementation of the sterilization treatment.
In the present invention, the concentration of cells in the target cell suspension is preferably 1.6X10 -4 ～2×10 -4 And each mL.
In the present invention, the method for preparing the target cell suspension preferably comprises the steps of: inoculating the target cells into a culture medium for culture to obtain the cell suspension. In the present invention, the medium preferably includes Fetal Bovine Serum (FBS) and penicillin-streptomycin; the volume percentage of the Fetal Bovine Serum (FBS) in the culture medium is preferably 5-10% (v/v); the penicillin-streptomycin is preferably 2% (v/v) by volume. In the present invention, the temperature of the culture is preferably 37℃and the relative humidity of the culture is preferably 90%.
In the present invention, the impregnation is preferably performed in wells of a 96-well plate, and there is no particular requirement for the impregnated body to be carried out.
After the polymer porous microsphere loaded with the target cells is obtained, the photo-curing hydrogel and the polymer porous microsphere loaded with the target cells are mixed to obtain the biological 3D printing gel material. The invention has no special requirements for the specific implementation of the mixing.
The invention provides an application of the biological 3D printing gel material according to the technical scheme or the biological 3D printing gel material prepared by the preparation method according to the technical scheme in preparing in-vitro bionic tissues.
The invention provides an in-vitro bionic tissue, which is obtained by performing 3D printing on biological ink and then performing photo-curing under the condition of ultraviolet irradiation;
the biological ink comprises the biological 3D printing gel material according to the technical scheme or the biological 3D printing gel material prepared by the preparation method according to the technical scheme and a photoinitiator.
In the present invention, the photoinitiator is particularly preferably a blue photoinitiator (LAP). The invention has no special requirement on the dosage of the photoinitiator, and ensures that the photocuring reaction is smoothly carried out.
In the present invention, the operating parameters of 3D printing preferably include: the pressure is preferably 0.3-0.4 MPa, the cooling temperature of the platform is preferably 4 ℃, and the temperature of the spray head is preferably 12 ℃.
In the present invention, the time of the photo-curing is preferably 10s.
In the invention, the initial bionic tissue is obtained after the photo-curing, and the invention preferably dries the initial bionic tissue to obtain the in-vitro bionic tissue. In the present invention, the drying is preferably freeze-drying.
In the invention, the in vitro bionic tissue is particularly preferably a layered osteochondral model.
In the present invention, the layered osteochondral model preferably includes endothelial osteoblast model units and chondrocyte model units arranged in a layered manner. In the invention, the endothelial osteoblast model unit is obtained by performing light curing on the first biological ink under the condition of ultraviolet irradiation after 3D printing. The chondrocyte model unit is obtained by performing light curing on the second biological ink under the condition of ultraviolet irradiation after 3D printing. In the invention, the first biological ink comprises a photo-cured hydrogel and first target cell-loaded polymer porous microspheres, and target cells loaded in the first target cell-loaded polymer porous microspheres are embryonic osteoblasts and aortic endothelial cells. In the invention, the second biological ink comprises a photo-cured hydrogel and second target cell-loaded polymer porous microspheres, wherein target cells loaded in the second target cell-loaded polymer porous microspheres are embryonic cartilage cells.
The layered osteochondral model provided by the invention preferably has a multi-layer structure, the number of layers and the types and proportions of the used biological ink can be adjusted according to the osteochondral model which is required to be constructed, and the multi-layer structure can protect cells in the microsphere under the irradiation curing of an ultraviolet lamp, so that the cells can keep good cell activity. Meanwhile, the layered bone cartilage model provided by the invention can protect the microspheres from being severely extruded in the pressure range of 3D printing, so that the cell viability is not affected by pressure change, and the layered bone cartilage model is beneficial to maintaining the cell activity under ultraviolet lamp curing and 3D printing.
The invention utilizes a microfluidic method to prepare the polymer porous microsphere with good biological properties such as biocompatibility, biodegradability and the like, and loads chondrocytes and endothelial-osteoblasts to form micro-tissues of the cartilage and the endothelial-osteoblasts respectively. Finally, the invention disperses the microsphere loaded with cells in gelatin to form biological ink, and performs 3D printing to generate a layered osteochondral 3D model. The in-vitro bionic tissue prepared by the invention has the advantages of good biocompatibility, uniform size, good biodegradability, low cytotoxicity and the like, and can be applied to the fields of drug screening, tissue engineering and the like.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation flow of the biological 3D printing gel material and the 3D printing layered bone cartilage model is as shown in figure 1: (1) 5 g of polyvinyl alcohol (PVA) was weighed, 500mL of ultrapure water was added, and then stirred at 80℃for 1 hour to completely dissolve, thereby obtaining a PVA solution, which was cooled to room temperature and then stored at 4 ℃. 0.07 g of Gelatin (GEL) was weighed, 1mL of ultrapure water was added, and then stirred at 35℃for 45 minutes to completely dissolve, thereby obtaining a gelatin solution. Then, 0.04 g of polylactic acid-glycolic acid copolymer (PLGA) was weighed into another beaker, and 2mL of Dichloromethane (DCM) was added to obtain a dichloromethane solution of PLGA. Adding gelatin solution into PLGA dichloromethane solution, performing ultrasonic emulsification to obtain oil phase, using PVA solution as water phase, and using microfluidic device to obtain milky microsphere with uniform size, wherein injection speed of water phase is 2mL/min and injection speed of oil phase is 0.05mL/min at 4deg.C. The microspheres are washed by water and freeze-dried to obtain porous microspheres.
(2) The method comprises the steps of co-culturing a mouse embryo osteoblast (MC 3T 3-E1) and a Mouse Aortic Endothelial Cell (MAEC) in a culture medium of 10% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin at 37 ℃ and a relative humidity of 90% to obtain a cell suspension containing the mouse embryo osteoblast (MC 3T 3-E1) and the Mouse Aortic Endothelial Cell (MAEC) at the same time, wherein the concentration of the MC3T3-E1 and the MAEC is 1.6X10 -4 mL -1 . Culturing mouse embryo chondrocyte (C518) in culture medium containing 10% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin at 37deg.C with relative humidity of 90% to obtain cell suspension containing mouse embryo chondrocyte (C518) with concentration of C518 of 1.6X10 -4 mL -1 。
(3) Disinfecting the microspheresInto wells of a 96-well plate, 300. Mu.L of a concentration of 1.6X10 were added -4 mL -1 The cell suspension of the mouse embryonic chondrocytes C518 of (C) to obtain polymer microspheres (marked as 3D-CS) loaded with chondrocytes. The microspheres were sterilized and placed in wells of a 96-well plate, and 300. Mu.L of each 1.6X10 were added -4 mL -1 Cell suspensions produced by co-culturing the mouse embryonic osteoblasts MC3T3-E1 and the mouse aortic endothelial cells MAEC, give polymer microspheres (designated EO-MT) loaded with osteoblasts and endothelial cells simultaneously.
(4) A mixture of EO-MT and GelMA hydrogels (concentration 100. Mu.g/mL) was taken out (wherein the mass ratio of EO-MT and GelMA hydrogels was 1:10), and then biological 3D printing was performed in a 6-well cell culture dish (pressure 0.3-0.4 MPa, platform cooling temperature 4 ℃, shower nozzle temperature 12 ℃), and the hydrogels were cured after irradiation with ultraviolet light for 10 seconds. The mixture of 3D-CS and GelMA hydrogel (100 mug/mL) was printed on EO-MT layer by biological 3D printing (pressure 0.3-0.4 MPa, platform cooling temperature 4 ℃ C., spray head temperature 12 ℃ C.), wherein the mass ratio of 3D-CS and GelMA hydrogel was 1:10, and the mixture was also cured by irradiation with ultraviolet light for 10 seconds. And freeze-drying the bionic osteochondral tissue model to obtain the layered osteochondral model.
SEM characterization of the layered osteochondral model prepared in this example was performed, and the results are shown in FIG. 2, in which the layered osteochondral model prepared in this example has a thickness of 15X 3mm 3 The rectangular grid structure of (c) can be used for transporting nutrients, oxygen and metabolic waste, which is necessary for the growth and proliferation of cells. To investigate the distribution of microspheres in GelMA, the structure of the freeze-dried microspheres and GelMA composite hydrogel was also observed with SEM images.
Example 2
The preparation flow of the biological 3D printing gel material and the 3D printing layered bone cartilage model is as shown in figure 1: (1) 4.5 g of polyvinyl alcohol (PVA) was weighed, 400mL of ultrapure water was added, and then stirred at 80℃for 1 hour to completely dissolve it, to obtain a PVA solution, which was cooled to room temperature and stored at 4 ℃. 0.1 g of Gelatin (GEL) was weighed, 1.5mL of ultrapure water was added, and then stirred at 35℃for 40 minutes to completely dissolve, thereby obtaining a gelatin solution. Then, 0.1 g of polylactic acid-glycolic acid copolymer (PLGA) was weighed into another beaker, and 5mL of Dichloromethane (DCM) was added to obtain a dichloromethane solution of PLGA. Adding gelatin solution into PLGA dichloromethane solution, performing ultrasonic emulsification to obtain oil phase, using PVA solution as water phase, and using microfluidic device to obtain milky microsphere with uniform size, wherein injection speed of water phase is 2mL/min and injection speed of oil phase is 0.05mL/min at 4deg.C. The microspheres are washed by water and freeze-dried to obtain porous microspheres.
(2) The method comprises the steps of co-culturing mouse embryo osteoblast (MC 3T 3-E1) and Mouse Aortic Endothelial Cell (MAEC) in a culture medium containing 5% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin at 37deg.C and relative humidity of 90% to obtain cell suspension containing both mouse embryo osteoblast (MC 3T 3-E1) and Mouse Aortic Endothelial Cell (MAEC), wherein the concentrations of MC3T3-E1 and MAEC are 2×10 -4 mL -1 . Placing mouse embryo chondrocyte (C518) in culture medium containing 10% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin, culturing at 37deg.C and relative humidity of 90% to obtain cell suspension containing mouse embryo chondrocyte (C518) with concentration of C518 of 2X10 -4 mL -1 。
(3) The microspheres were sterilized and placed in wells of a 96-well plate, 200. Mu.L of 2X 10 concentration was added -4 mL -1 The cell suspension of the mouse embryonic chondrocytes C518 of (C) to obtain polymer microspheres (marked as 3D-CS) loaded with chondrocytes. The microspheres were sterilized and placed in wells of a 96-well plate, and 300. Mu.L of each 1.6X10 were added -4 mL -1 Cell suspensions produced by co-culturing the mouse embryonic osteoblasts MC3T3-E1 and the mouse aortic endothelial cells MAEC, give polymer microspheres (designated EO-MT) loaded with osteoblasts and endothelial cells simultaneously.
(4) A mixture of EO-MT and GelMA hydrogels (50. Mu.g/mL concentration) was removed (wherein the mass ratio of EO-MT to GelMA hydrogels was 1:100), and then bio-3D printing was performed in a 6-well cell culture dish (pressure 0.3-0.4 MPa, platform cooling temperature 4 ℃, showerhead temperature 12 ℃), and the hydrogels were cured after irradiation with ultraviolet light for 10 seconds. And continuing to print a mixture of 3D-CS and acrylic acid modified hyaluronic acid (100 mug/mL) on the EO-MT layer by biological 3D printing (the pressure is 0.3-0.4 MPa, the platform cooling temperature is 4 ℃ and the spray head temperature is 12 ℃), wherein the mass ratio of the 3D-CS to the acrylic acid modified hyaluronic acid is 1:100, and carrying out curing treatment by irradiating ultraviolet light for 10 seconds. And freeze-drying the bionic osteochondral tissue model to obtain the layered osteochondral model.
The layered osteochondral model prepared in this example was used to characterize the level of cell proliferation by nuclear staining, and it was confirmed that cells adhered to and grown on microspheres. The results are shown in FIG. 3: both MC3T3-E1 and MAEC showed good proliferation within the first 5 days. Chondrocytes in 3D-CSs also exhibit good proliferation. It was demonstrated that all three types of cells were able to proliferate well in PLGA microspheres.
Example 3
The preparation flow of the biological 3D printing gel material and the 3D printing layered bone cartilage model is as shown in figure 1:
(1) The polylactic acid-glycolic acid copolymer porous microspheres prepared in reference example 2.
(2) The method comprises the steps of co-culturing mouse embryo osteoblast (MC 3T 3-E1) and Mouse Aortic Endothelial Cell (MAEC) in a culture medium containing 5% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin at 37deg.C and relative humidity of 90% to obtain cell suspension containing both mouse embryo osteoblast (MC 3T 3-E1) and Mouse Aortic Endothelial Cell (MAEC), wherein the concentrations of MC3T3-E1 and MAEC are 2×10 -4 mL -1 . Placing mouse embryo chondrocyte (C518) in culture medium containing 10% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin, culturing at 37deg.C and relative humidity of 90% to obtain cell suspension containing mouse embryo chondrocyte (C518) with concentration of C518 of 2X10 -4 mL -1 。
(3) The microspheres were sterilized and placed in wells of a 96-well plate, 200. Mu.L of 2X 10 concentration was added -4 mL -1 Is fine in embryo cartilage of miceCell C518 cell suspension, polymer microsphere loaded with chondrocytes (designated 3D-CS) was obtained. The microspheres were sterilized and placed in wells of a 96-well plate, and 300. Mu.L of each 1.6X10 were added -4 mL -1 Cell suspensions produced by co-culturing the mouse embryonic osteoblasts MC3T3-E1 and the mouse aortic endothelial cells MAEC, give polymer microspheres (designated EO-MT) loaded with osteoblasts and endothelial cells simultaneously.
(4) A mixture of EO-MT and acrylated alginic acid (concentration: 75. Mu.g/mL) was taken out (wherein the mass ratio of EO-MT to acrylated alginic acid was 1:100), and then biological 3D printing was performed in a 6-well cell culture dish (pressure 0.3-0.4 MPa, platform cooling temperature 4 ℃, shower nozzle temperature 12 ℃ C.) and the hydrogel was cured after irradiation with ultraviolet light for 10 seconds. The mixture of 3D-CS and acrylated alginic acid (with the mass ratio of 3D-CS and GelMA hydrogel of 1:100) was printed on EO-MT layer continuously by biological 3D printing (pressure of 0.3-0.4 MPa, platform cooling temperature of 4 ℃ C., spray head temperature of 12 ℃ C.), and was also cured by irradiation with ultraviolet light for 10s. And freeze-drying the bionic osteochondral tissue model to obtain the layered osteochondral model.
The layered osteochondral model prepared in the embodiment can better observe the distribution of cells on PLGA microspheres by using H & E staining. As a result, as shown in FIG. 4, tissues having a high cell density and an ordered arrangement have been formed on the microspheres.
Example 4
The preparation flow of the biological 3D printing gel material and the 3D printing layered bone cartilage model is as shown in figure 1:
(1) The polylactic acid-glycolic acid copolymer porous microspheres prepared in reference example 2.
(2) Human SV40 transfected osteoblast (hFOB) and microvascular endothelial cells (HMEC-1) were placed together in a medium of 5% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin, and cultured at 37℃under a relative humidity of 90% to give a cell suspension containing both human SV40 transfected osteoblast (hFOB) and microvascular endothelial cells (HMEC-1) at a concentration of 2X 10 -4 mL -1 . People to be treatedCulturing embryo chondrocyte in culture medium containing 10% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin at 37deg.C and relative humidity of 90% to obtain cell suspension containing human embryo chondrocyte with concentration of 2×10 -4 mL -1 。
(3) The microspheres were sterilized and placed in wells of a 96-well plate, 200. Mu.L of 2X 10 concentration was added -4 mL -1 The cell suspension containing the human embryonic chondrocytes, to obtain the polymer microsphere (marked as 3D-CS) loaded with the human embryonic chondrocytes. The microspheres were sterilized and placed in wells of a 96-well plate, and 300. Mu.L of each 1.6X10 were added -4 mL -1 Cell suspensions produced by co-culturing human SV40 transfected osteoblasts (hFOB) and microvascular endothelial cells (HMEC-1) gave polymer microspheres (designated EO-MT) loaded with both hFOB and HMEC-1.
(4) A mixture of EO-MT and GelMA hydrogel (150. Mu.g/mL) was taken out (wherein the mass ratio of EO-MT and GelMA hydrogel was 1:100), and then biological 3D printing (pressure 0.3-0.4 MPa, platform cooling temperature 4 ℃ C., shower nozzle temperature 12 ℃ C.) was performed in a 6-well cell culture dish, and the hydrogel was cured after irradiation with ultraviolet light for 10 seconds. And continuing to print by biological 3D (the pressure is 0.3-0.4 MPa, the platform cooling temperature is 4 ℃, the spray head temperature is 12 ℃) on the EO-MT layer, printing a mixture of 3D-CS and GelMA hydrogel (100 mug/mL) (the mass ratio of the 3D-CS to the GelMA hydrogel is 1:100), and irradiating the mixture for 10 seconds by ultraviolet light to carry out curing treatment. And freeze-drying the bionic osteochondral tissue model to obtain the layered osteochondral model.
Comparative example 1
(1) The method comprises the steps of co-culturing a mouse embryo osteoblast (MC 3T 3-E1) and a Mouse Aortic Endothelial Cell (MAEC) in a culture medium of 10% (v/v) Fetal Bovine Serum (FBS) and 2% (v/v) penicillin-streptomycin at 37 ℃ and a relative humidity of 90% to obtain a cell suspension containing the mouse embryo osteoblast (MC 3T 3-E1) and the Mouse Aortic Endothelial Cell (MAEC) at the same time, wherein the concentration of the MC3T3-E1 and the MAEC is 1.6X10 -4 mL -1 . Mouse embryonic chondrocytes (C518) were placed in 10% (v/v) Fetal Bovine Serum (FBS)And 2% (v/v) penicillin-streptomycin, at 37℃and 90% relative humidity to give a cell suspension containing mouse embryonic chondrocytes (C518) at a concentration of C518 of 1.6X10% -4 mL -1 。
(3) 300. Mu.L of 1.6X10 g concentration was taken -4 mL -1 300. Mu.L of the cell suspension of the mouse embryonic chondrocyte C518 at a concentration of 1.6X10, respectively -4 mL -1 The mixture of cell suspension produced by co-culturing MC3T3-E1 of mouse embryo osteoblast and MAEC of mouse aortic endothelial cells and GelMA hydrogel (with the mass ratio of the cell suspension to the GelMA hydrogel of 1:10) is subjected to biological 3D printing in a 6-hole cell culture dish (the pressure is 0.3-0.4 MPa, the platform cooling temperature is 4 ℃ and the spray head temperature is 12 ℃) and the hydrogel is solidified after being irradiated by ultraviolet light for 10 seconds. The mixture of cells and GelMA hydrogel (100. Mu.g/mL) was printed on the EO-MT layer by continuous biological 3D printing (pressure 0.3-0.4 MPa, platform cooling temperature 4 ℃ C., nozzle temperature 12 ℃ C.), wherein the total mass of the cell suspension and the mass ratio of the GelMA hydrogel were 1:10, and the mixture was also subjected to curing treatment by irradiation with ultraviolet light for 10 seconds. And freeze-drying the bionic osteochondral tissue model to obtain a 2D culture model.
Application example
The layered osteochondral model prepared by the invention is more suitable for drug screening research of osteoarthritis, anti-inflammatory drugs (such as curcumin) are added into the 3D bracket model prepared in the embodiment 1 of the invention and the 2D culture model prepared in the comparative example 1 for culturing for 24 hours, cell survival rates are detected respectively at 12 hours and 24 hours, and drug toxicity of cells in the two models is analyzed under different culture conditions and different drug concentrations. As shown in FIG. 5, after curcumin was added in 2D culture, i.e., culture dish culture, the drug concentration was 100. Mu.g mL after 12 hours and 24 hours -1 Cell viability was 21.1% and 14.0%, respectively. Under the condition of 3D culture (prepared 3D printing gel scaffold), after curcumin is added, the drug concentration is 100 mug mL after 12h and 24h -1 When the cell viability is 46.1% and 31.3% respectively, the cell tolerance to the drug is better and is closer to the focus in vivo under the 3D culture conditionBit reality.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.
Claims (10)
1. A biological 3D printing gel material, which is characterized by comprising a photo-curing hydrogel and polymer porous microspheres loaded with target cells; the target cells are supported in the pore structure of the polymeric porous microsphere.
2. The biological 3D printing gel material according to claim 1, wherein the material of the polymer porous microspheres comprises one or more of polylactic acid-glycolic acid copolymer, polylactic acid and derivatives thereof, alginic acid and derivatives thereof, chitosan and derivatives thereof, and hyaluronic acid and derivatives thereof.
3. The biological 3D printing gel material according to claim 1 or 2, wherein the preparation method of the polymer porous microspheres comprises the following steps:
respectively injecting the oil phase and the water phase into a microfluidic device for mixing, and obtaining polymer microspheres under the shearing action of surface tension and the water phase; the aqueous phase comprises polyvinyl alcohol and water; the oil phase comprises a degradable polymer, a pore-forming agent, water and an organic solvent;
and freeze-drying the polymer microsphere to obtain the polymer porous microsphere.
4. The biological 3D printing gel material according to claim 3, wherein the mass percentage of the polyvinyl alcohol in the water phase is 0.04-2%;
in the oil phase, the pore-forming agent is gelatin, the organic solvent comprises dichloromethane and/or trifluoroethanol, the mass ratio of the polymer to the organic solvent is 1 (10-100), and the mass ratio of the pore-forming agent to the water is 0.07-0.1) (1-1.5); the mass ratio of the polymer to the pore-forming agent is 1 (1-2).
5. The biological 3D printing gel material of claim 3 or 4, wherein the injection rate of the aqueous phase is 2mL/min; the injection speed of the oil phase is 0.05mL/min;
the temperature of the mixing is 0-10 ℃.
6. The biological 3D printing gel material of claim 1, wherein the photo-curable hydrogel is one or more of an aqueous solution of methacryloyl gelatin, an aqueous solution of acrylated alginate and derivatives thereof, an aqueous solution of acrylic modified hyaluronic acid, and an aqueous solution of acrylated chitosan;
the mass concentration of the photo-curing hydrogel is 50-150 mug/mL.
7. The biological 3D printing gel material of claim 1, wherein the target cells include one or more of embryonic osteoblasts, embryonic chondrocytes, aortic endothelial cells, osteogenic cells, and osteoclasts.
8. The method for preparing the biological 3D printing gel material according to any one of claims 1 to 7, comprising the steps of:
immersing the polymer porous microsphere in a target cell suspension to obtain a polymer porous microsphere loaded with target cells;
and mixing the photo-cured hydrogel and the polymer porous microsphere loaded with the target cells to obtain the biological 3D printing gel material.
9. The use of the biological 3D printing gel material according to any one of claims 1 to 7 or the biological 3D printing gel material prepared by the preparation method according to claim 8 in the preparation of in vitro bionic tissues.
10. An in-vitro bionic tissue is characterized in that the in-vitro bionic tissue is obtained by 3D printing of biological ink and photo-curing under the condition of ultraviolet irradiation;
the biological ink comprises the biological 3D printing gel material according to any one of claims 1 to 7 or the biological 3D printing gel material prepared by the preparation method according to claim 8 and a photoinitiator.
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