CN115894964A - Photo-curing porous hydrogel cell preparation and preparation method thereof - Google Patents
Photo-curing porous hydrogel cell preparation and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a light-cured porous hydrogel cell preparation and a preparation method thereof, belonging to the technical field of biological medicines. The invention aims to provide a photocuring porous hydrogel cell preparation with a dynamic self-adaptive porous structure. The cell preparation comprises a porous hydrogel material and cells, wherein the porous hydrogel material is formed by stacking hydrogel spheres and has a three-dimensional porous structure; the pore diameter of the three-dimensional porous structure is micron-sized; and the pores are adjustable under the action of cell extrusion; the particle size of the hydrogel spheres is micron-sized or nano-sized; the cells are located within the pores of the porous hydrogel material and interact with the hydrogel spheres. The cell preparation has a unique microstructure, has dynamic self-adaptive characteristics, and has important application values in the fields of biological stent materials, tissue engineering, drug delivery, living tissue/organ construction and the like.
Description
Technical Field
The invention relates to a photocuring porous hydrogel cell preparation and a preparation method thereof, belonging to the technical field of biological medicines.
Background
The cell therapy provides a new means for treating major diseases, has wide application prospect in the fields of tissue and organ restoration and reconstruction, malignant tumors, infectious diseases, aging resistance and the like, is expected to lead the treatment of related diseases to make a new breakthrough, and is a hotspot of current scientific research and industrial development. However, the activity of therapeutic cells in vivo is often low, and the therapeutic effect needs to be further improved, so that an advanced cell preparation technology is urgently needed to support the development of a new generation of cell therapy products, and the cell preparation technology is also one of the neck clamp technologies in the current cell therapy field.
The hydrogel has wide application scenes in the biomedical field. Among them, the method has great potential in the fields of cell delivery, living tissues, organ construction and the like. However, most of the main materials of the existing common photo-curing cell preparations and in vivo tissue and organ scaffolds are dense non-porous hydrogels or porous hydrogels with closed pore structures, which can form a certain barrier effect on the growth, proliferation and migration of cells, and the growth conditions of the cells in a complex microenvironment in vivo are difficult to reproduce. Therefore, there is a need to develop new bio-photocuring material systems and technical paths, and to develop new cell preparations with dynamically adaptive microstructures to meet the requirements of biomedical applications.
Disclosure of Invention
In view of the above defects, the technical problem to be solved by the present invention is to provide a photo-cured porous hydrogel cell preparation with a dynamic adaptive porous structure. The invention relates to a photocuring porous hydrogel cell preparation, which comprises a porous hydrogel material and cells; the porous hydrogel material is formed by stacking hydrogel balls and has a three-dimensional porous structure; the pore diameter of the three-dimensional porous structure is micron-sized; and the pores are adjustable under the action of cell extrusion; the particle size of the hydrogel spheres is micron-sized or nano-sized; the cells are located within the pores of the porous hydrogel material and interact with the hydrogel spheres.
In one embodiment of the present invention, the hydrogel spheres have a particle size of 50 to 50000nm; the aperture of the three-dimensional porous structure is 1-2000 mu m; the granularity of the hydrogel spheres is preferably 50-10000 nm; the aperture of the three-dimensional porous structure is 1-200 mu m.
In one embodiment of the invention, the cell comprises a somatic cell or a stem cell,
in one embodiment of the present invention, preferably the cell comprises a mesenchymal stem cell, an adipose stem cell or a chondrocyte.
The invention also provides a preparation method of the photocuring porous hydrogel cell preparation.
The preparation method of the photocuring porous hydrogel cell preparation comprises the following steps:
a. co-dissolving a photo-curing biological material monomer, a photoinitiator and a polyoxyethylene-polyoxypropylene-polyoxyethylene segmented copolymer in a solvent, and uniformly mixing to obtain biological ink;
b. uniformly mixing the biological ink in the step a with cells to obtain bioactive ink;
c. curing the bioactive ink in the step b by DLP-3D light;
d. followed by elution with a solvent to remove the non-crosslinked photocurable biomaterial monomer, residual photoinitiator and free polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer to give a photocurable porous hydrogel cell preparation.
In one embodiment of the present invention, the photocurable biomaterial monomer comprises a photocrosslinkable protein or polypeptide, and the photoinitiator comprises at least one of lithium phenyl-2, 4, 6-trimethylbenzenephosphinate, 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone, and 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2,4,6 (trimethylbenzoyl) diphenylphosphine oxide; the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer includes at least one of pluronic F127 and pluronic F68. Preferably, the photo-curable biomaterial monomer comprises at least one of methacrylated gelatin and methacrylic anhydride modified tussah fibroin.
In one embodiment of the present invention, in the bioactive ink of step b, the concentration of the photo-curable biomaterial monomer is 0.01 to 0.5g/mL, the concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer is 0.01 to 0.6g/mL, the concentration of the photoinitiator is 0.001 to 0.1g/mL, and the cell concentration is preferably 1X 10 6 ～1×10 8 One per mL.
In one embodiment of the present invention, the solvent of step a is any one of deionized water, phosphate buffer, glucose solution, physiological saline solution and cell culture solution.
In one embodiment of the invention, in the step c, the curing is to directly irradiate the bioactive ink with blue light in the range of 400-480nm to cure and shape the bioactive ink; or adding the bioactive ink into a mold, and then irradiating the mold by adopting blue light within the range of 400-480nm to solidify and mold the ink; or adopting a photocuring 3D printing technology for curing and forming; preferably, the following steps are also carried out after step c: and mechanically cutting the formed light-cured porous hydrogel material to obtain a product with a specific shape and size.
In one embodiment of the present invention, in the step c, the curing time of the bioactive ink is 8 seconds to 20 seconds.
In one embodiment of the present invention, in step d, the elution is performed by using deionized water, phosphate buffer, glucose solution, physiological saline solution or cell culture solution, and the elution time is 30 minutes to 2 hours.
The invention combines the clinical and market demands of cell therapy, develops a photocuring porous hydrogel microsphere material with a microstructure aiming at the common technical problem of cell preparations at the present stage, and can support the development of various novel cell preparations.
Compared with the prior art, the invention has the following beneficial effects:
(1) The light-cured porous hydrogel cell preparation prepared by the invention has a unique microstructure, high pore connectivity and dynamic self-adaptive characteristic, and can be loaded with bioactive components to be applied to the fields of biological scaffold materials, tissue engineering, drug delivery, living tissue/organ construction and the like.
(2) The cell preparation is prepared by taking a photo-curing biomaterial monomer and a polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer as basic raw materials through a self-assembly-3D printing-elution method, and has the advantages of simple process, mild conditions, short period, low cost, environmental friendliness, large-scale production and the like.
(3) The light-cured porous hydrogel cell preparation prepared by the invention has good processing performance, can be processed into products with different shapes, structures and sizes by utilizing a die, a light-cured 3D printing technology and mechanical cutting, can realize personalized customization so as to support the development of various innovative products, and has good application prospect.
Drawings
FIG. 1 is a graph showing the survival of fibroblasts, human Umbilical Vein Endothelial Cells (HUVEC) and chondrocytes in a non-porous hydrogel and a porous hydrogel, respectively, in example 1 of the present invention.
FIG. 2 is a comparison of migration of adipose stem cells in the cell preparation of the present invention in example 2 with GelMA cell preparation.
Fig. 3 shows the shape of the concha-shaped chondrocyte scaffold customized by the 3D printing technology in example 3 before and after implantation in vivo.
FIG. 4 is a comparison of histological evaluation of chondrocyte scaffolds of the present invention in example 4 with a GelMA cell preparation in vivo.
FIG. 5 is a scanning electron micrograph of the porous hydrogel prepared in example 5 of the present invention.
FIG. 6 is a scanning electron microscope image of a conventional hydrogel prepared in comparative example 1 of the present invention.
Detailed Description
The light-cured porous hydrogel cell preparation comprises a porous hydrogel material and cells;
the porous hydrogel material is formed by stacking hydrogel balls and has a three-dimensional porous structure;
the pore diameter of the three-dimensional porous structure is micron-sized; and the pores are adjustable under the action of cell extrusion;
the particle size of the hydrogel spheres is micron-sized or nano-sized;
the cells are located within the pores of the porous hydrogel material and interact with the hydrogel spheres.
Wherein, the porous hydrogel material has dynamic self-adaptive characteristic, and the porous structure can be self-regulated under the extrusion action of other substances (such as cells); when the porous structure is squeezed by cells, the porous structure self-regulates, so that the porous structure is beneficial to relatively free spreading and migration of the cells in the pore channels.
The porous hydrogel material has a three-dimensional porous structure formed by stacking micron-sized or nano-sized hydrogel spheres, certain spatial connection is kept among the hydrogel spheres to maintain the shape of the hydrogel, the hydrogel material has larger spatial displacement capacity to endow the hydrogel with better deformation capacity, a good physical environment is provided for cell growth and proliferation, and the biological function of cells can be regulated and controlled through a special micro-nano structure. The hydrogel with the special porous structure can wrap cells and provide a good microenvironment to improve the survival and the efficacy of the cells in vivo, provides a new treatment means for wounds, rare diseases, tumors and the like, promotes the development of the cell therapy industry, and has wide application prospect and great economic value.
The pore diameter of the three-dimensional porous structure is micron-sized, which means that the porous structure is provided with a plurality of pores, the pore diameter of each pore is not completely the same, and the pore diameter of each pore is micron-sized. The photocuring porous hydrogel has dynamic self-adaptive characteristics, and the porous structure of the photocuring porous hydrogel can be self-regulated under the extrusion action of other substances (such as cells); when the porous structure is extruded by cells, the self-regulation of the porous structure is beneficial to relatively freely spreading and migrating the cells in the pore channels, thereby providing a good physical environment for cell growth and proliferation.
In one embodiment of the present invention, the hydrogel spheres have a particle size of 50 to 50000nm; the aperture of the three-dimensional porous structure is 1-2000 mu m; preferably, the granularity of the hydrogel spheres is 50-10000 nm; the aperture of the three-dimensional porous structure is 1-200 mu m.
In one embodiment of the present invention, the porous hydrogel material is prepared from a photocurable biomaterial monomer, a photoinitiator, and a polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer as raw materials.
In one embodiment of the invention, the cell comprises a somatic cell or a stem cell,
in one embodiment of the present invention, preferably the cell comprises a mesenchymal stem cell, an adipose stem cell or a chondrocyte.
The invention also provides a preparation method of the photocuring porous hydrogel cell preparation.
The preparation method of the light-cured porous hydrogel cell preparation comprises the following steps:
a. co-dissolving a photo-curing biomaterial monomer, a photoinitiator and a polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer in a solvent, and uniformly mixing to obtain biological ink;
b. uniformly mixing the biological ink in the step a with cells to obtain bioactive ink;
c. c, performing DLP-3D photocuring molding on the bioactive ink obtained in the step b;
d. followed by elution with a solvent to remove the non-crosslinked photocurable biomaterial monomer, residual photoinitiator and free polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer to give a photocurable porous hydrogel cell preparation.
The preparation method of the photocuring porous hydrogel cell preparation takes a photocuring biomaterial monomer, a photoinitiator and a polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer as basic raw materials, is prepared by a self-assembly-3D printing-elution method, and simultaneously loads corresponding cells. The method comprises the steps of mixing polyoxyethylene-polyoxypropylene-polyoxyethylene amphiphilic block copolymer with photo-curing biological material monomer, carrying out phase separation, driving the photo-curing biological material monomer to self-assemble micro-nano liquid drops, carrying out photo-curing to form micro-nano particles, and finally eluting to obtain the stable photo-curing porous hydrogel cell preparation with the microstructure.
The photo-curing biomaterial monomer is a photo-crosslinkable protein or polypeptide commonly used in the field, and the porous structure of the invention cannot be well obtained by adopting other photo-curing material monomers, such as methacryloyl hyaluronic acid. In an embodiment of the present invention, the photo-curable biomaterial monomer is at least one of methacrylated gelatin (GelMA) and methacrylic anhydride modified tussah silk protein (ASF-MA).
The photoinitiator used in the present invention can be any photoinitiator containing free radicals in the art, including but not limited to phenyl-2, 4, 6-trimethylbenzene acyl lithium phosphinate, 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone, 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2,4,6 (trimethylbenzoyl) diphenyl phosphine oxide, and the like.
Polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymers, which are a novel class of polymeric nonionic surfactants, are at least one of F68 and F127 in a preferred embodiment of the present invention.
In one embodiment of the inventionIn the embodiment, in the bioactive ink of step b, the concentration of the photocurable biomaterial monomer is 0.01 to 0.5g/mL, the concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer is 0.01 to 0.6g/mL, the concentration of the photoinitiator is 0.001 to 0.1g/mL, and the cell concentration is 1X 10 6 ～1×10 8 One per mL.
The concentration of polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer affects the formation of the photocurable porous hydrogel material and its pore size. If the concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer is too low, the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer cannot self-assemble with the biological ink of the photo-cured biological material monomer, and the photo-cured porous hydrogel material cannot be obtained. As the concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer increases, the contour of the nanoparticles in the obtained photo-cured hydrogel is more remarkably formed, and pores formed by stacking the nanoparticles are gradually increased. When the concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer is too high, the concentration of a local polymer in a biological ink system is too high, and finally a heterostructure is formed, namely the photocuring porous hydrogel containing a part of hydrogel spheres with micron-scale dimensions is formed. On the basis of fixing the initial concentrations of the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer and the monomer of the photocuring biomaterial to enable the initial concentrations to be self-assembled to form the photocuring porous hydrogel, when the volume of the block copolymer solution is continuously increased, the connection among hydrogel spheres is gradually weakened, and finally, single dispersed hydrogel spheres are formed.
In one embodiment of the present invention, the solvent of step a is any one of deionized water, phosphate buffer, glucose solution, physiological saline solution and cell culture solution.
In one embodiment of the invention, in the step a and the step b, the blending is at least one of blowing and beating, vortex blending and stirring blending by using a pipette.
In one embodiment of the present invention, the bio-ink comprises micro-nano droplets.
The usual techniques for shaping materials by photocuring are suitable for use in the present invention.
In one embodiment of the invention, the bioactive ink is directly irradiated by blue light in the range of 400-480nm to be cured and molded.
In another embodiment of the present invention, the bioactive ink is added to a mold and then cured to shape with blue light irradiation in the range of 400-480 nm.
In another embodiment of the invention, the molding is cured using a photo-curing 3D printing technique.
Preferably, step c is followed by the steps of: and mechanically cutting the formed light-cured porous hydrogel material to obtain a product with a specific shape and size.
In one embodiment of the present invention, in the step c, the curing time of the bioactive ink is 8 seconds to 20 seconds.
The elution in step d can be performed by conventional elution methods in the art, and in one embodiment of the present invention, in step d, the elution is performed by deionized water, phosphate buffer, glucose solution, physiological saline solution or cell culture solution, and the elution time is 30 minutes to 2 hours.
The invention combines the clinical and market demands of cell therapy, develops a photocuring porous hydrogel microsphere material with a microstructure aiming at the common technical problem of cell preparations at the present stage, and can support the development of various novel cell preparations.
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
Example 1
(1) A certain amount of photoinitiator (LAP) and 1 XPBS buffer solution are added in sequence into a sample bottle to prepare a LAP solution with the mass volume percentage concentration of 0.7% (0.007 g/mL). Adding a certain amount of methacrylated gelatin (GelMA) into the LAP solution, and then placing a sample bottle at 37 ℃ until the GelMA is completely dissolved to obtain a GelMA solution with the mass volume percentage concentration of 0.15 g/mL; then, the obtained solution is filtered and sterilized by a 0.22 mu m filter membrane;
(2) Adding 1.0g of F127 and 5.0mL of 1 XPBS buffer solution into a sample bottle in sequence, and placing the sample bottle at 4 ℃ until F68 is completely dissolved to obtain an F127 solution with the mass volume percentage concentration of 0.2 g/mL; then filtering and sterilizing the obtained solution by using a 0.22 mu m filter membrane;
(3) Adding fibroblast, HUVEC and chondrocyte suspension into a 1.5mL EP tube, centrifuging (800rmp, 3 min), discarding the supernatant to obtain 3 corresponding cell precipitates;
(4) Taking 600 mu L of GelMA solution in the step 1, placing the GelMA solution in each cell sediment in the step 3, adding 300 mu L of F127 solution in the step 2, and mechanically stirring for 1 minute to obtain 3 kinds of bioactive ink;
(5) Placing 0.5mL of the bioactive ink of step 4 on a glass slide covered with a silica gel membrane, subsequently irradiating with blue light with a wavelength in the range of 400-480nm for 8 seconds to obtain a cell-encapsulating light-cured porous hydrogel in the form of hydrogel having a microstructure, and washing the light-cured porous hydrogel with 1 XPBS buffer solution for 3 times, 50 minutes each time, to remove non-crosslinked GelMA, photoinitiator and free F127;
(6) Respectively taking 600 mu L of GelMA solution in the step 1, placing the GelMA solution in the EP tube containing various cell precipitates in the step 3, adding 300 mu L of 1 XPBS buffer solution, and mechanically stirring for 1 minute to obtain 3 control group bioactive inks;
(7) Placing 0.5ml of the bioactive ink of step 6 on a slide covered with a silicone membrane, followed by irradiation with blue light having a wavelength in the range of 400-480nm for 8 seconds to obtain a cell-encapsulating, micro-structured, photo-curable, non-porous hydrogel in the form of a hydrogel, and washing the photo-curable, non-porous hydrogel with 1 x PBS buffer 3 times, 50 minutes each time, to remove non-crosslinked GelMA, photoinitiator and free F127;
(8) Subjecting the non-porous and porous hydrogels encapsulating various cells described in steps 5 and 7 to 5% CO at 37 ℃% 2 Cultured in DMEM medium containing 10% fetal bovine serum, and the live/dead cell staining test was performed on the 3 rd day after the culture.
Fig. 1 shows the proliferation of different cells in two materials, and the results show that the cell survival in the porous cell hydrogel of the present invention is significantly higher than that in the GelMA cell preparation.
Example 2
(1) A certain amount of photoinitiator (LAP) and 1 XPBS buffer solution are added in sequence into a sample bottle to prepare a LAP solution with the mass volume percentage concentration of 0.7% (0.007 g/mL). Adding a certain amount of methacrylated gelatin (GelMA) into the LAP solution, and then placing a sample bottle at 37 ℃ until the GelMA is completely dissolved to obtain a GelMA solution with the mass volume percentage concentration of 0.15 g/mL; then filtering and sterilizing the obtained solution by using a 0.22 mu m filter membrane;
(2) Adding 1.5g of F68 and 5.0mL of 1 XPBS buffer solution into a sample bottle in sequence, and placing the sample bottle at 4 ℃ until the F68 is completely dissolved to obtain an F68 solution with the mass volume percentage concentration of 0.3 g/mL; then, the obtained solution is filtered and sterilized by a 0.22 mu m filter membrane;
(3) The suspension of adipose stem cells (cell number 2X 10) was added to a 1.5mL EP tube 4 ) Centrifuging (800rmp, 3 min), and discarding the supernatant to obtain a cell pellet;
(4) Taking 600 mu L of GelMA solution in the step 1, placing the GelMA solution in the cell sediment in the step 3, adding 300 mu L of F68 solution in the step 2, and mechanically stirring for 1 minute to obtain bioactive ink;
(5) Placing 0.5mL of the bioactive ink of step 4 on a slide covered with a silica gel membrane, then irradiating for 15 seconds under blue light in the range of 400-480nm by adopting a photo-curing 3D printing technology based on a digital light processing technology to prepare cylindrical photo-curing porous cell-containing hydrogel (the height is 1mm, and the diameter of a bottom circle is 0.5 cm), and then washing the cylindrical photo-curing porous cell-containing hydrogel for 3 times with 1 XPBS buffer solution for 50 minutes each time to remove a photoinitiator and free F68;
(6) Placing 600 mu L of GelMA solution in the EP tube containing the cell sediment in the step 3, adding 300 mu L of 1 XPBS buffer solution, and mechanically stirring for 1 minute to obtain control group bioactive ink;
(7) Placing 0.5mL of the bioactive ink described in step 6 on a slide glass covered with a silicone membrane, followed by irradiating with blue light in the range of 400-480nm for 15 seconds using a digital light processing technology-based photocuring 3D printing technique to prepare a cylindrical photocuring non-porous cell-containing hydrogel (height of 1mm, bottom circle diameter of 0.5 cm), followed by washing the cylindrical photocuring non-porous cell-containing hydrogel 3 times with 1 XPBS buffer for 50 minutes each time to remove non-crosslinked GelMA, photoinitiator, and free F68;
(8) Subjecting the adipose stem cell-encapsulated non-porous and porous hydrogels described in steps 5 and 7 to 5% CO at 37 deg.C 2 Cultured in DMEM medium containing 10% fetal bovine serum, and the live/dead cell staining test was performed on the 6 th day after the culture.
Fig. 2 shows the migration of adipose-derived stem cells in the porous hydrogel, and the results show that the migration of adipose-derived stem cells in the porous hydrogel material of the present invention is better than that of GelMA cell preparations, the cells can freely migrate without being constrained, and the material can dynamically adapt to cell biomechanics to generate micro-deformation, which is beneficial to cell migration.
Example 3
(1) A certain amount of photoinitiator (LAP) and 1 XPBS buffer solution are added in sequence to a sample bottle to prepare a LAP solution with the mass volume percentage concentration of 0.7% (0.007 g/mL). Adding a certain amount of methacrylated gelatin (GelMA) into the LAP solution, and then placing a sample bottle at 37 ℃ until the GelMA is completely dissolved to obtain a GelMA solution with the mass volume percentage concentration of 0.15 g/mL; then filtering and sterilizing the obtained solution by using a 0.22 mu m filter membrane;
(2) Adding 1.25g of F68 and 5.0mL of 1 XPBS buffer solution into a sample bottle in sequence, and placing the sample bottle at 4 ℃ until the F68 is completely dissolved to obtain an F68 solution with the mass volume percentage concentration of 0.25 g/mL; then filtering and sterilizing the obtained solution by using a 0.22 mu m filter membrane;
(3) Chondrocyte cell suspension (cell number 1X 10) was added to a 1.5mL EP tube 7 ) Centrifuging (800rmp, 3 min), and discarding the supernatant to obtain a cell pellet;
(4) Taking 600 mu L of GelMA solution in the step 1, placing the GelMA solution in the cell sediment in the step 3, adding 300 mu L of F68 solution in the step 2, and mechanically stirring for 1 minute to obtain bioactive ink;
(5) Placing 0.5mL of the bioactive ink of step 4 on a glass slide covered with a silicone membrane, then preparing an auricle-shaped photocured porous cell-containing hydrogel by adopting a photocuring 3D printing technology of blue light with the wavelength ranging from 400 nm to 480nm, wherein the exposure time is 12 seconds, and then washing the auricle-shaped photocured porous cell-containing hydrogel for 3 times with 1 XPBS buffer solution for 50 minutes each time to remove a photoinitiator and free F68;
(6) Placing 600 mu L of GelMA solution in the EP tube containing the cell sediment in the step 3, adding 300 mu L of 1 XPBS buffer solution, and mechanically stirring for 1 minute to obtain control group bioactive ink;
(7) Placing 0.5mL of the bioactive ink of step 4 on a glass slide covered with a silicone membrane, then preparing an outer ear-shaped photo-cured non-porous cell-containing hydrogel by adopting a photo-curing 3D printing technology of blue light with the wavelength ranging from 400 nm to 480nm, wherein the exposure time is 12 seconds, and then washing the outer ear-shaped photo-cured non-porous cell-containing hydrogel for 3 times with 1 XPBS buffer solution for 50 minutes each time to remove non-crosslinked GelMA, photoinitiator and free F68;
(8) The chondrocyte-encapsulated, non-porous and porous hydrogels described in steps 5 and 7 were 5% CO at 37 deg.C 2 Culturing with DMEM medium containing 10% fetal calf serum, implanting under the skin of nude mice on the 7 th day after culturing, and observing morphology.
Fig. 3 shows a general view of a 3D printed outer ear, and the results show that the chondrocyte scaffold of the present invention can maintain a good biological morphology after being implanted in vivo.
Example 4
(1) A certain amount of photoinitiator (LAP) and 1 XPBS buffer solution are added in sequence into a sample bottle to prepare a LAP solution with the mass volume percentage concentration of 0.7% (0.007 g/mL). Adding a certain amount of methacrylated gelatin (GelMA) into the LAP solution, and then placing a sample bottle at 37 ℃ until the GelMA is completely dissolved to obtain a GelMA solution with the mass volume percentage concentration of 0.15 g/mL; then filtering and sterilizing the obtained solution by using a 0.22 mu m filter membrane;
(2) Adding 1.25g of F68 and 5.0mL of 1 XPBS buffer solution into a sample bottle in sequence, and placing the sample bottle at 4 ℃ until the F68 is completely dissolved to obtain an F68 solution with the mass volume percentage concentration of 0.25 g/mL; then filtering and sterilizing the obtained solution by using a 0.22 mu m filter membrane;
(3) Chondrocyte cell suspension (cell number 1X 10) was added to a 1.5mL EP tube 7 ) Centrifuging (800rmp, 3 minutes), and discarding the supernatant to obtain a cell pellet;
(4) Taking 600 mu L of GelMA solution in the step 1, placing the GelMA solution in the cell sediment in the step 3, adding 300 mu L of F68 solution in the step 2, and mechanically stirring for 1 minute to obtain bioactive ink;
(5) Through computer aided design and creation of articular cartilage patch 3D models of different shapes, introducing articular cartilage patch model data into an operating system of the photocuring 3D printing equipment;
(6) Placing 0.5mL of the bioactive ink of step 4 on a glass slide covered with a silica gel film, preparing an articular cartilage patch by adopting a photocuring 3D printing technology of blue light with the wavelength ranging from 400 nm to 480nm, wherein the exposure time is 12 seconds, and then washing the articular cartilage patch for 3 times with 1 XPBS (phosphate buffered saline) buffer solution for 50 minutes each time to remove a photoinitiator and free F68;
(7) Anesthetizing a rat by chloral hydrate, debriding joint tissues, and constructing a full-layer articular cartilage injury model of the rat;
(8) Implanting the articular cartilage patch obtained in the step 6 into an articular cartilage defect part, enabling the articular cartilage patch to be attached to the defect part, and then suturing the incision layer by layer;
(9) After three months, the joint at the repaired part is taken down for decalcification, slicing and staining treatment, and the repairing effect of the cell preparation is evaluated.
Fig. 4 shows the evaluation effect of the cell preparation in the rat cartilage defect model, and the result shows that the defect cartilage tissue treated by the cell preparation has more obvious cartilage regeneration and tissue integration effects. The injectable cartilage structure can be implanted into the articular cartilage defect for treatment by a minimally invasive surgery mode in combination with an arthroscope.
Example 5
(1) Adding a certain amount of photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphate (LAP) and deionized water in a sample bottle in sequence to prepare a LAP solution with the mass volume percentage concentration of 0.007 g/mL. Adding a certain amount of GelMA into the LAP solution, and then placing a sample bottle at 37 ℃ until the GelMA is completely dissolved to obtain a GelMA solution with the mass volume percentage concentration of 0.15 g/mL;
(2) Adding 1.0g of F127 and 5.0mL of deionized water into a sample bottle in sequence, and placing the sample bottle at 4 ℃ until the F127 is completely dissolved to obtain an F127 solution with the mass volume percentage concentration of 0.2 g/mL;
(3) Adding 0.9g of F68 and 5.0mL of deionized water into a sample bottle in sequence, and placing the sample bottle at 4 ℃ until the F68 is completely dissolved to obtain an F68 solution with the mass volume percentage concentration of 0.18 g/mL;
(4) Placing 2mL of GelMA solution in the step (1) into a sample bottle, adding 1mL of F127 solution in the step (2), and mechanically stirring for 1 minute to obtain biological ink with the initial mass volume percentage concentration of F127 of 20%;
(5) Placing 2mL of GelMA solution in the step (1) into a sample bottle, adding 1mL of F68 solution in the step (3), and mechanically stirring for 1 minute to obtain biological ink with the initial mass volume percentage concentration of F68 of 18%;
(6) Placing 2mL of the bio-ink obtained in the step (4) in a silica gel mold, irradiating for 1 minute under ultraviolet light with the wavelength of 405nm to obtain photocured porous hydrogel, washing the photocured hydrogel with deionized water for 3 times and 30 minutes/time, and removing a photoinitiator and free F127;
(7) And (3) placing 2mL of the biological ink obtained in the step (5) in a silica gel mold, irradiating for 1 minute under ultraviolet light with the wavelength of 405nm to obtain the photocuring porous hydrogel, washing the photocuring porous hydrogel with deionized water for 3 times and 30 minutes/time, and removing the photoinitiator and free F68.
Fig. 5 shows the morphology of the photo-cured hydrogel prepared from the bio-ink with the initial mass volume percentage concentration of F127 of 20% and the bio-ink with the initial mass volume percentage concentration of F68 of 18%, and the photo-cured hydrogel has a unique microstructure according to the following contents: the particle structure comprises a plurality of micro-nano particles, the micro-nano particles are piled up to form a three-dimensional porous structure, the pore diameter of the porous structure is in the micron order, and the pore connectivity is high.
Comparative example 1
(1) And (3) adding a certain amount of photoinitiator LAP and deionized water into a sample bottle in sequence to prepare a LAP solution with the mass volume percentage concentration of 0.007 g/mL. Adding a certain amount of GelMA into the LAP solution, and then placing a sample bottle at 37 ℃ until the GelMA is completely dissolved to obtain a GelMA solution with the mass volume percentage concentration of 0.15 g/mL;
(2) Taking 2mL of GelMA solution in the step (1), placing the GelMA solution in a sample bottle, adding 1mL of PBS solution, and mechanically stirring for 1 minute to obtain biological ink;
(3) Placing 2mL of the bio-ink obtained in the step (2) in a silica gel mold, irradiating for 1 minute under ultraviolet light with the wavelength of 405nm to obtain a photo-cured hydrogel, washing the photo-cured hydrogel with deionized water for 3 times and 30 minutes/time, and removing the photoinitiator.
Fig. 6 shows the morphology of a conventional photo-cured hydrogel prepared from GelMA and PBS, and it can be known from the description that the conventional photo-cured hydrogel is not formed by stacking a plurality of micro-nano particles.
Claims (10)
1. A photocurable porous hydrogel cell formulation characterized by: including porous hydrogel materials and cells;
the porous hydrogel material is formed by stacking hydrogel balls and has a three-dimensional porous structure;
the pore diameter of the three-dimensional porous structure is micron-sized; and the pores are adjustable under the action of cell extrusion;
the particle size of the hydrogel spheres is micron-sized or nano-sized;
the cells are located within the pores of the porous hydrogel material.
2. The photo-curable cellular hydrogel cell preparation of claim 1, wherein: the particle size of the hydrogel spheres is 50-50000 nm; the aperture of the three-dimensional porous structure is 1-2000 mu m; the granularity of the hydrogel spheres is preferably 50-10000 nm; the aperture of the three-dimensional porous structure is 1-200 mu m.
3. The light-curable porous hydrogel cell preparation of claim 1, wherein: the cell comprises a somatic cell or a stem cell, preferably the cell comprises a mesenchymal stem cell, an adipose stem cell or a chondrocyte.
4. The method for producing a photocurable porous hydrogel cell preparation according to any one of claims 1 to 3, comprising the steps of:
a. co-dissolving a photo-curing biological material monomer, a photoinitiator and a polyoxyethylene-polyoxypropylene-polyoxyethylene segmented copolymer in a solvent, and uniformly mixing to obtain biological ink;
b. uniformly mixing the biological ink in the step a with cells to obtain bioactive ink;
c. c, performing DLP-3D photocuring molding on the bioactive ink in the step b;
d. followed by elution with a solvent to remove the non-crosslinked photocurable biomaterial monomer, residual photoinitiator and free polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer to give a photocurable porous hydrogel cell preparation.
5. The method for preparing a photo-curable porous hydrogel cell preparation according to claim 4, wherein: the photo-curable biomaterial monomer comprises a photo-crosslinkable protein or polypeptide, and the photo-initiator comprises at least one of phenyl-2, 4, 6-trimethylbenzene acyl lithium phosphinate, 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone and 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2,4,6 (trimethylbenzoyl) diphenyl phosphine oxide; the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer comprises at least one of pluronic F127 and pluronic F68; preferably, the photo-curable biomaterial monomer comprises at least one of methacrylated gelatin and methacrylic anhydride modified tussah silk protein.
6. The method for preparing a photo-curable porous hydrogel cell preparation according to claim 4, wherein: in the bioactive ink of step b, the concentration of the photo-curable biomaterial monomer is 0.01 to 0.5g/mL, the concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer is 0.01 to 0.6g/mL, the concentration of the photoinitiator is 0.001 to 0.1g/mL, and the cell concentration is preferably 1X 10 6 ～1×10 8 one/mL.
7. The method for preparing a photo-curable porous hydrogel cell preparation according to claim 4, wherein: the solvent in the step a is any one of deionized water, phosphate buffer solution, glucose solution, normal saline solution and cell culture solution.
8. The method for preparing a photo-curable cellular hydrogel cell preparation according to claim 4, wherein: in the step c, the curing is to directly irradiate the bioactive ink by adopting blue light within the range of 400-480nm to cure and form the bioactive ink; or adding the bioactive ink into a mould, and then irradiating the bioactive ink by adopting blue light within the range of 400-480nm to solidify and mold the bioactive ink; or adopting a photocuring 3D printing technology for curing and forming; preferably, the following steps are also carried out after step c: and mechanically cutting the formed light-cured porous hydrogel material to obtain a product with a specific shape and size.
9. The method for preparing a photo-curable porous hydrogel cell preparation according to claim 4, wherein: in the step c, the curing time of the bioactive ink is 8-20 seconds.
10. The method for preparing a photo-curable cellular hydrogel cell preparation according to claim 4, wherein: and d, eluting by using deionized water, phosphate buffer solution, glucose solution, physiological saline solution or cell culture solution for 30 minutes to 2 hours.
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Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5944754A (en) * | 1995-11-09 | 1999-08-31 | University Of Massachusetts | Tissue re-surfacing with hydrogel-cell compositions |
| US6027744A (en) * | 1998-04-24 | 2000-02-22 | University Of Massachusetts Medical Center | Guided development and support of hydrogel-cell compositions |
| WO2017223529A1 (en) * | 2016-06-24 | 2017-12-28 | Washington State University | Three-dimensional tissue matrix scaffold system |
| CN109553783A (en) * | 2017-09-26 | 2019-04-02 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of photocuring hydrogel and the preparation method and application thereof |
| WO2019210209A1 (en) * | 2018-04-27 | 2019-10-31 | Regents Of The University Of Michigan | Aligned hydrogel tubes for tissue repair and regeneration |
| CN111040199A (en) * | 2019-12-31 | 2020-04-21 | 华南理工大学 | Photo-crosslinking porous hydrogel based on two water-phase immiscible emulsions and preparation method and application thereof |
| EP3666294A1 (en) * | 2018-12-13 | 2020-06-17 | Valeopharm GmbH | Porous object based on biopolymers having a flexible pore size, its preparation and use thereof |
| CN112409553A (en) * | 2020-11-25 | 2021-02-26 | 杭州术道生物科技有限公司 | Method for preparing injectable porous hydrogel microspheres by micro-fluidic ice crystal method and application thereof |
| CN112813016A (en) * | 2021-01-20 | 2021-05-18 | 温州医科大学附属第一医院 | Porous hydrogel array for three-dimensional cell sphere generation and preparation method thereof |
| CN113041215A (en) * | 2021-03-17 | 2021-06-29 | 清华大学深圳国际研究生院 | Eye surface in-situ medicine and preparation method thereof |
| CN113274554A (en) * | 2021-05-14 | 2021-08-20 | 清华大学 | Gel microsphere-based 3D printing biological ink and application thereof |
| CN113398330A (en) * | 2021-05-17 | 2021-09-17 | 四川大学 | 3D printing biological ink capable of constructing multi-level bionic pore structure and preparation method and printing method thereof |
Family Cites Families (35)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030086975A1 (en) * | 2001-11-08 | 2003-05-08 | Timothy Ringeisen | Method for making a porous Polymeric material |
| US7485670B2 (en) * | 2002-08-02 | 2009-02-03 | Cambridge Polymer Group, Inc. | Systems and methods for controlling and forming polymer gels |
| CN100546662C (en) * | 2006-09-15 | 2009-10-07 | 武汉理工大学 | Polyurethane material and preparation and purposes with anticoagulation and thrombus dissolving functions |
| US20110256628A1 (en) * | 2010-04-20 | 2011-10-20 | The University Of Washington Through Its Center For Commercialization | Adaptive tissue engineering scaffold |
| CN103131625B (en) * | 2011-11-23 | 2015-08-05 | 清华大学 | For building method and the device of three-dimensional microenvironment |
| WO2015024133A1 (en) * | 2013-08-22 | 2015-02-26 | Polyvalor Limited Partnership | Porous gels and methods for their preparation |
| CN103816573A (en) * | 2014-03-01 | 2014-05-28 | 湖南科技大学 | Preparation method of porous gelatin/hyaluronic acid composite microspheres |
| KR102404093B1 (en) * | 2015-10-14 | 2022-06-02 | 주식회사 파마리서치 | Injectable composition for regenerating or enhancing volume of skin tissues comprising hollow porous microspheres |
| EP3383447B1 (en) * | 2015-12-02 | 2021-06-23 | Otago Innovation Limited | Light-activated preparation of hydrogels |
| US20170173209A1 (en) * | 2015-12-18 | 2017-06-22 | Rousseau Research, Inc. | Wound closure compositions and method |
| CN105561375B (en) * | 2016-01-05 | 2018-11-30 | 山东省药学科学院 | A kind of gelatin liquid-absorbent hemostatic sponge and preparation method thereof of dopamine crosslinking |
| US20180371117A1 (en) * | 2017-06-15 | 2018-12-27 | The Regents Of The University Of Colorado, A Body Corporate | Synthesis and assembly of clickable microgels into cell-laden porous scaffolds |
| KR20190118694A (en) * | 2018-04-10 | 2019-10-21 | 서울대학교산학협력단 | Porous microspheres composite and a preparation method thereof |
| CN108744060B (en) * | 2018-05-29 | 2021-05-07 | 四川大学 | Bone repair material capable of being injected with multiple pore structures and preparation method thereof |
| CN109400818B (en) * | 2018-09-18 | 2020-11-24 | 中国科学院宁波材料技术与工程研究所 | Preparation method of polyacrylamide hydrogel |
| KR102251384B1 (en) * | 2018-10-02 | 2021-05-13 | 한림대학교 산학협력단 | Rapid photocuring bio-glue with adhesion, heamostatic and wound healing efficacy |
| WO2020106703A1 (en) * | 2018-11-19 | 2020-05-28 | Endomedix, Inc. | Methods and compositions for achieving hemostasis and stable blood clot formation |
| CN109529110A (en) * | 2018-12-10 | 2019-03-29 | 上海摩漾生物科技有限公司 | Porous nano hydroxyapatite sustained-release gel preparation method |
| CN109897387A (en) * | 2019-01-25 | 2019-06-18 | 华南理工大学 | Application, porous gel and its preparation of a kind of modified gelatin in water packet air lotion |
| WO2020156358A1 (en) * | 2019-01-30 | 2020-08-06 | 高雄医学大学 | Slurry for photocuring 3d printing, preparation method therefor, and method of use thereof |
| CN109880151A (en) * | 2019-02-21 | 2019-06-14 | 上海市伤骨科研究所 | A kind of preparation method and porous support materials of hydrogel porous microsphere |
| CN110169959B (en) * | 2019-05-20 | 2022-11-15 | 中国人民解放军总医院 | Growth factor slow-release microsphere, tissue engineering cartilage composite scaffold and preparation method |
| US11903815B2 (en) * | 2019-05-31 | 2024-02-20 | Tepha, Inc. | Implants assembled from skeletal polyhedron unit cells, coiled unit cells or mesh unit cells |
| CN111184909B (en) * | 2019-10-21 | 2022-08-09 | 湖北翎美生物科技有限公司 | Hyaluronic acid sustained-release filler and preparation method thereof |
| CN112917891B (en) * | 2019-12-06 | 2022-08-02 | 苏州永沁泉智能设备有限公司 | Biological 3D printing method for massive tissues |
| CN112917899B (en) * | 2019-12-06 | 2023-01-06 | 苏州永沁泉智能设备有限公司 | Biological 3D printing method based on porous structure |
| WO2021114260A1 (en) * | 2019-12-13 | 2021-06-17 | 中国科学院深圳先进技术研究院 | Coaxial electrostatic spinning gelma/plga-lysogm1, preparation method therefor and application thereof |
| CN111110922B (en) * | 2019-12-25 | 2020-10-27 | 四川大学 | Periodontal biological module for 3D biological printing and construction method and application thereof |
| US11918703B2 (en) * | 2020-08-13 | 2024-03-05 | Universidad De Los Andes | Extrudable photocrosslinkable hydrogel and method for its preparation |
| CN112245658A (en) * | 2020-10-09 | 2021-01-22 | 北京大学 | Injectable crystal gel microsphere cell amplification carrier and preparation method thereof |
| CN112999428B (en) * | 2020-11-19 | 2023-02-21 | 中国医学科学院整形外科医院 | Cell-loaded polymer porous microsphere three-dimensional biological printing ink for auricle reconstruction and manufacturing method and application thereof |
| CN113024879A (en) * | 2020-12-22 | 2021-06-25 | 苏州大学附属第一医院 | Gel microsphere and preparation method and application thereof |
| CN113278168B (en) * | 2021-01-29 | 2023-03-21 | 大连理工大学 | Two-field coupling cross-linked injectable plastic printable particle hydrogel material and preparation method and application thereof |
| CN113024847B (en) * | 2021-03-19 | 2022-09-13 | 中国科学院宁波材料技术与工程研究所 | Application of natural polysaccharide hydrogel in hemostasis field |
| CN113290844B (en) * | 2021-05-14 | 2022-10-21 | 清华大学 | Multilevel suspension printing method for constructing complex heterogeneous tissues/organs |
-
2022
- 2022-08-26 CN CN202211030925.1A patent/CN115894964A/en active Pending
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Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5944754A (en) * | 1995-11-09 | 1999-08-31 | University Of Massachusetts | Tissue re-surfacing with hydrogel-cell compositions |
| US6027744A (en) * | 1998-04-24 | 2000-02-22 | University Of Massachusetts Medical Center | Guided development and support of hydrogel-cell compositions |
| WO2017223529A1 (en) * | 2016-06-24 | 2017-12-28 | Washington State University | Three-dimensional tissue matrix scaffold system |
| CN109553783A (en) * | 2017-09-26 | 2019-04-02 | 中国科学院苏州纳米技术与纳米仿生研究所 | A kind of photocuring hydrogel and the preparation method and application thereof |
| WO2019210209A1 (en) * | 2018-04-27 | 2019-10-31 | Regents Of The University Of Michigan | Aligned hydrogel tubes for tissue repair and regeneration |
| EP3666294A1 (en) * | 2018-12-13 | 2020-06-17 | Valeopharm GmbH | Porous object based on biopolymers having a flexible pore size, its preparation and use thereof |
| CN111040199A (en) * | 2019-12-31 | 2020-04-21 | 华南理工大学 | Photo-crosslinking porous hydrogel based on two water-phase immiscible emulsions and preparation method and application thereof |
| CN112409553A (en) * | 2020-11-25 | 2021-02-26 | 杭州术道生物科技有限公司 | Method for preparing injectable porous hydrogel microspheres by micro-fluidic ice crystal method and application thereof |
| CN112813016A (en) * | 2021-01-20 | 2021-05-18 | 温州医科大学附属第一医院 | Porous hydrogel array for three-dimensional cell sphere generation and preparation method thereof |
| CN113041215A (en) * | 2021-03-17 | 2021-06-29 | 清华大学深圳国际研究生院 | Eye surface in-situ medicine and preparation method thereof |
| CN113274554A (en) * | 2021-05-14 | 2021-08-20 | 清华大学 | Gel microsphere-based 3D printing biological ink and application thereof |
| CN113398330A (en) * | 2021-05-17 | 2021-09-17 | 四川大学 | 3D printing biological ink capable of constructing multi-level bionic pore structure and preparation method and printing method thereof |
Non-Patent Citations (4)
| Title |
|---|
| GOU MALIN 等: "3D-printed nerve conduits with live platelets for effective perpheral nerve repair", ADVANCED FUNCTIONAL MATERIALS, 21 August 2020 (2020-08-21), pages 1 - 10 * |
| JIANG, Y等: "Encapsulating chromogenic reaction substrates with porous hydrogel scaffolds onto arrayed capillary tubes toward a visual and high-throughput colorimetric strategy for rapid occult blood tests", JOURNAL OF MATERIALS CHEMISTRY B, 31 December 2017 (2017-12-31), pages 1159 - 1165 * |
| 苟马玲 等: "一种聚己内酯 -聚乙二醇-聚己内酯磁 性共聚物微球的制备", 高分子材料科学与工程, vol. 23, no. 4, 31 July 2007 (2007-07-31), pages 235 - 239 * |
| 黄宇阳;白波;姚咏嫦;钱东阳;陈艺;张姝江;: "微孔水凝胶的构建及其原代猪软骨细胞培养的研究", 中华关节外科杂志(电子版), no. 05, 1 October 2017 (2017-10-01), pages 63 - 67 * |
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| CN115926198A (en) | 2023-04-07 |
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| CN115850730A (en) | 2023-03-28 |
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