CN114796604B - 3D printing ink for cornea regeneration and preparation method and application thereof - Google Patents
3D printing ink for cornea regeneration and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y80/00—Products made by additive manufacturing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P10/00—Technologies related to metal processing
- Y02P10/25—Process efficiency
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Abstract
The invention provides 3D printing ink for cornea regeneration and a preparation method and application thereof.A phosphoric acid buffer solution is used for preparing a polyethylene glycol diacrylate solution and a methacrylic acylated gelatin solution, the polyethylene glycol diacrylate solution, the methacrylic acylated gelatin solution, a photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate and a light-resistant agent lemon yellow are filtered through a 0.22 mu m filter membrane, and the polyethylene glycol diacrylate solution, the methacrylic acylated gelatin solution, the photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate and the light-resistant agent lemon yellow are mixed at 37 ℃ to obtain the 3D printing ink. The preparation method is simple, the material source is wide, and the practicability is high.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to 3D printing ink for cornea regeneration and a preparation method and application thereof.
Background
The cornea is a transparent, multilayered structure on the surface of the eye, whose primary function is to concentrate light onto the lens and then direct it toward the retina. It consists of corneal epithelium, corneal stroma, and corneal endothelial layer, which consists of stratified epithelial cells differentiated from limbal stem cells that allow oxygen and essential nutrients to penetrate and block pathogens and dust from entering the eye. The stromal layer constitutes 90% of the corneal thickness and is composed of highly orthogonally arranged collagen fibers with corneal stromal cells therebetween. The innermost layer is the corneal endothelial layer, which is composed of a layer of endothelial cells and has poor regeneration ability.
Corneal disease may develop when the cornea is damaged, and nowadays, corneal disease becomes one of the main causes of blindness. Statistically, there are nearly ten million patients with corneal disease each year, but due to the lack of donors, only less than 20 ten thousand patients can undergo corneal transplantation surgery. Therefore, research on artificial cornea substitutes has been in demand.
Including boston's keratoprosthesis, alphaCor keratoprosthesis, etc., which have been used in corneal transplantation operations to date, although they have succeeded in improving the vision of many patients, the accompanying complications, such as glaucoma, increased risk of endophthalmitis, and difficulty in achieving the desired corneal substitute.
The tissue engineering technology brings good news to corneal patients, and as a key part of three elements of tissue engineering, the selection of a scaffold material is particularly critical. The hydrogel is a swelling body of a cross-linked polymer network rich in water, and is expected to be applied to a scaffold material in corneal regeneration due to good biocompatibility, high porosity, high water content and proper viscoelasticity. At present, various polymer materials and composites thereof are used for preparing hydrogel, such as collagen, gelatin, chitosan, fibroin and the like in natural polymers, and polyethylene glycol (PEG), polycaprolactone (PCL), polyhydroxyethylmethacrylate (PHEMA) and the like in synthetic polymers, the natural polymers generally have excellent biocompatibility, but the mechanical properties of the natural polymers are difficult to meet the requirements, and the synthetic polymers can achieve ideal properties by improving a synthesis process, but have low bioactivity and are difficult to support the adhesion and proliferation of cells, so that the synthetic polymers are difficult to fuse with self tissues to achieve the regeneration purpose. Therefore, it is very important to develop a bioscaffold material having both sufficient biocompatibility and sufficient mechanical properties.
3D bioprinting technology is one of the effective ways to create scaffold materials with appropriate cell growth microenvironments for tissue engineering and regenerative medicine. In the last few years, digital Light Processing (DLP) has been favored as a light-assisted 3D printing technique, which can solve some disadvantages in inkjet printing and extrusion printing methods, such as the damage of cells due to shearing force during extrusion, high requirement on ink viscosity, and cell viability decrease due to long printing time. DLP can be prepared into products in various shapes by utilizing a layered printing and layer-by-layer photocuring method, and the ink source of the DLP is wide, compared with the traditional ultraviolet light initiation, the DLP light source uses 405nm wavelength and belongs to a visible light wave band, so that the DLP ink has small damage to cells.
Disclosure of Invention
The invention overcomes the defects in the prior art, the existing cornea regeneration scaffold material has poor mechanical property and low bioactivity, is difficult to support the adhesion and proliferation of cells and further difficult to fuse with self tissues to achieve the regeneration purpose, and provides 3D printing ink for cornea regeneration and a preparation method and application thereof.
The purpose of the invention is realized by the following technical scheme.
A3D printing ink for cornea regeneration and a preparation method thereof are disclosed, wherein a phosphoric acid buffer solution (PBS) is used for preparing a polyethylene glycol diacrylate (PEGDA) solution and a methacrylic acylated gelatin (GelMA) solution, the polyethylene glycol diacrylate (PEGDA) solution, the methacrylic acylated gelatin (GelMA) solution, a photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP) and a light-blocking agent lemon yellow (UV absorber) are filtered through a 0.22 mu m filter membrane, the polyethylene glycol diacrylate (PEGDA) solution, the methacrylic acylated gelatin (GelMA) solution, the photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP) and the light-blocking agent lemon yellow (UV absorber) are mixed under the environment of 37 ℃, obtaining the 3D printing ink, wherein the concentration of the polyethylene glycol diacrylate (PEGDA) solution is 10-20wt%, the concentration of the methacrylated gelatin (GelMA) solution is 5-10wt%, the concentration of the phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate salt (LAP) is 0.25-0.5wt%, the concentration of the lemon yellow (UV abs) is 0.05-0.15wt%, and the mass ratio of the polyethylene glycol diacrylate (PEGDA), the methacrylated gelatin (GelMA), the phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate salt (LAP) and the lemon yellow (UV abs) is (1-5): (1-2): 0.05:0.01, set up the printing parameter in DLP3D printer, the printing parameter: the exposure time is 20-80s, the printing layer height is 20-60 μm, the printing temperature is 37 ℃, and the printing product (PEGDA-GelMA) is obtained by peeling from the base station after printing.
The concentration of polyethylene glycol diacrylate (PEGDA) solution was 10, 15, 20wt%, the concentration of methacrylated gelatin (GelMA) solution was 5wt%, the concentration of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate salt (LAP) was 0.25wt%, and the concentration of lemon yellow (UV absorber) was 0.05wt%.
The mass ratio of polyethylene glycol diacrylate (PEGDA), methacrylated gelatin (GelMA), phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP) and lemon yellow (UV absorber) is (2-4): 1:0.05:0.01.
printing parameters: the exposure time was 20s, the print layer height was 50 μm, and the print temperature was 37 ℃.
The preparation method of the polyethylene glycol diacrylate (PEGDA) comprises the following steps: dissolving polyethylene glycol (PEG) (molecular weight of 8000) in dichloromethane to obtain polyethylene glycol (PEG) solution, adding triethylamine into the polyethylene glycol (PEG) solution, carrying out ice bath, cooling to obtain mixed solution, adding dichloromethane and acryloyl chloride into a constant-pressure dropping funnel, slowly dropping the mixed solution of dichloromethane and acryloyl chloride into the mixed solution at a speed of 5 s/drop, reacting at room temperature of 20-25 ℃ for 24 hours, dropwise dropping the solution into glacial ethyl ether, and settling, filtering and drying to obtain polyethylene glycol diacrylate (PEGDA), wherein the mass ratio of polyethylene glycol (PEG), triethylamine, dichloromethane and acryloyl chloride is 10:1:70:0.6.
preparation of methacrylated gelatin (GelMA): dissolving gelatin in water at 40 ℃ to obtain a gelatin aqueous solution, adding sodium hydroxide (NaOH) into the gelatin aqueous solution, stirring for dissolving, then adding N, N '-Dimethylformamide (DMF), stirring for clarifying, then adding methacrylic anhydride, reacting at 40 ℃ for 2 hours, then quickly pouring the solution into absolute ethyl alcohol, settling to obtain a precipitate, shearing the precipitate, then continuously adding the precipitate into absolute ethyl alcohol for washing, finally dissolving the precipitate in an oven at 37 ℃, dialyzing for three days, and freeze-drying by using ultrapure water to obtain the methacrylated gelatin (GelMA), wherein the mass ratio of gelatin, sodium hydroxide (NaOH), N' -Dimethylformamide (DMF) and methacrylic anhydride is 4:0.1:132:0.6.
the tensile strength, tensile modulus and elongation at break of the printed product (PEGDA-GelMA) printed with a 3D printing ink for corneal regeneration were 82-83kPa,77-78kPa and 100-104%, respectively.
The printed product (PEGDA-GelMA) printed with a 3D printing ink for corneal regeneration has a light transmittance of 82-91% at a wavelength of 550nm, which is greater than 71.1% of the light transmittance of a natural cornea.
In an in vitro cytotoxicity test of hydrogel, the cell survival rate of a printed product (PEGDA-GelMA) printed by using a 3D printing ink for corneal regeneration is more than 90% compared with that of a control group, which shows that the printed product (PEGDA-GelMA) has better cell compatibility, and the cells can keep normal shape and have high survival rate after being cultured with the printed product (PEGDA-GelMA) for 1-3 days.
In the case of no cells, the conditions for printing the cornea were: the exposure time was 40s and the lemon yellow concentration was 0.15wt%, whereas in the case of the cells, the conditions for printing the cornea were: the exposure time was 80s and the lemon yellow concentration was 0.1wt%.
The invention has the beneficial effects that: the 3D printing ink mainly comprises polyethylene glycol diacrylate (PEGDA) and methacrylated gelatin (GelMA), the methacrylated gelatin (GelMA) can enhance the biocompatibility of gel, the polyethylene glycol diacrylate (PEGDA) can enhance the mechanical property of the gel, the hydrogel with the shape of the cornea is prepared by using a Digital Light Processing (DLP) 3D printing technology, and the optical property, rheological property, mechanical property, degradation swelling property and cell compatibility of the hydrogel are tested, so that the application potential of the hydrogel in cornea regeneration is preliminarily proved.
Drawings
FIG. 1 is a nuclear magnetic diagram of polyethylene glycol (PEG) and polyethylene glycol diacrylate (PEGDA);
FIG. 2 is a nuclear magnetic map of gelatin with methacrylated gelatin (GelMA);
FIG. 3A is an XRD pattern of polyethylene glycol diacrylate (PEGDA), methacrylated gelatin (GelMA), and printed product (PEGDA-GelMA-20-5), and FIG. 3B is an infrared spectrum of polyethylene glycol diacrylate (PEGDA) and methacrylated gelatin (GelMA);
FIG. 4 is a drawing of a methacrylated gelatin (GelMA) hydrogel, printed product (PEGDA-GelMA);
FIG. 5 is a compression diagram of a methacrylated gelatin (GelMA) hydrogel, printed product (PEGDA-GelMA)
Fig. 6A is a swelling degree of a printed product (PEGDA-GelMA) in PBS, fig. 6B is a degradation graph of the printed product (PEGDA-GelMA) in collagenase type I, fig. 6C is a transmittance of the printed product (PEGDA-GelMA), fig. 6D is a transparency illustration of a natural cornea, a printed cornea, and a printed sheet, fig. 6E is a frequency scan graph of the printed product (PEGDA-GelMA), and fig. 6F is a modulus statistic graph of the printed product (PEGDA-GelMA);
FIG. 7 is a photograph showing the adhesion and survival rate of L929 cells on the printed product (PEGDA-GelMA), wherein (A) is a photograph of a bright field of cells on the surface of hydrogel, (B) is a photograph of live-dead staining fluorescence of cells on the surface of hydrogel, (C) is a test for in vitro cytotoxicity, (D, E) is a photograph of live-dead staining after 1,3 days of co-culture of cells and hydrogel, and a control group is a blank control without gel;
FIG. 8A is a graph of live-dead staining of rabbit corneal epithelial cells (SIRC) co-cultured with printed product (PEGDA-GelMA) for 1,3,5 days, FIG. 8B is MTT test data of SIRC cells co-cultured with polyethylene glycol diacrylate (PEGDA), methacrylated gelatin (GelMA) solution, printed product (PEGDA-GelMA) for 1,3,5 days, control group is blank control without cells, and FIG. 8C is an in vitro cytotoxicity test of photoinitiator (LAP) with lemon yellow (UV absorber);
FIGS. 9A and 9B are the live-dead staining pattern and corresponding mobility of SIRC cells on the surface of the printed product (PEGDA-GelMA), and FIGS. 9C and 9D are the live-dead staining pattern and proliferation data of SIRC cells on the surface of the printed product (PEGDA-GelMA);
FIG. 10 is a graph of the live and dead staining of SIRC cells after 3 days of culture in printed product (PEGDA-GelMA-20-5).
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
Preparation of polyethylene glycol diacrylate (PEGDA): first, 10g of PEG (Mw =8 k) was weighed in a 250mL round-bottom flask, and stirred and dissolved using 50mL of dichloromethane as a solvent, then 1mL of triethylamine was added to the round-bottom flask, ice-cooled for 30min, 20mL of dichloromethane and 0.6mL of acryloyl chloride were added to a constant pressure dropping funnel, and the above solution was slowly dropped at a rate of 5s one drop, and reacted at room temperature for 24 hours. And (3) dropwise adding the solution after the reaction into a large amount of ethyl glacial ether, settling, filtering and drying to obtain the product PEGDA.
Preparation of methacrylated gelatin (GelMA): firstly, 4g of gelatin is added into 200mL of water, stirred and dissolved at 40 ℃, after the gelatin is dissolved, a plurality of sodium hydroxide (NaOH) particles are added, stirred and dissolved, 132mL of N, N' -Dimethylformamide (DMF) is added, after stirring and clarification, 290 mu L of methacrylic anhydride is added, waiting for 10min, 292 mu L of methacrylic anhydride is added again, and reaction is carried out for 2h at 40 ℃. And (3) quickly pouring the solution after the reaction is finished into a large amount of absolute ethyl alcohol for sedimentation, shearing the precipitate, continuously adding the crushed precipitate into the absolute ethyl alcohol for washing, dissolving the precipitate in a 37 ℃ oven by using 180mL of ultrapure water after 10min, dialyzing for three days, and freeze-drying to obtain the product GelMA.
Example 1
Preparation of 3D printing inks and printed products (PEGDA-GelMA): a PEGDA solution (10 wt%) and a GelMA solution (5 wt%) were prepared at a given concentration using PBS and filtered through a 0.22 μm filter, to which were added a given amount of a sterile photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate salt (LAP, 0.25 wt%) and a light-blocking agent lemon yellow (UV absorber,0.05 wt%) at 37 ℃, printing parameters were set in a DLP3D printer for an exposure time of 20s, a layer height of 50 μm, and a printing temperature of 37 ℃.
Example 2
Preparation of 3D printing inks and printed products (PEGDA-GelMA): a PEGDA solution (15 wt%) and a GelMA solution (5 wt%) were prepared at a given concentration using PBS and filtered through a 0.22 μm filter, to which were added a given amount of a sterile photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate salt (LAP, 0.5 wt%) and a light-blocking agent lemon yellow (UV absorber,0.1 wt%) at 37 ℃, printing parameters were set in a DLP3D printer, an exposure time of 10s, a layer height of 20 μm, and a printing temperature of 37 ℃.
Example 3
Preparation of 3D printing inks and printed products (PEGDA-GelMA): a PEGDA solution (20 wt%) and a GelMA solution (5 wt%) were prepared at a given concentration using PBS and filtered through a 0.22 μm filter, to which were added a given amount of a sterile photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate salt (LAP, 0.25 wt%) and a light-blocking agent lemon yellow (UV absorber,0.05 wt%) at 37 ℃, printing parameters were set in a DLP3D printer for an exposure time of 30s, a layer height of 60 μm, and a printing temperature of 37 ℃.
Characterization of the printed product (PEGDA-GelMA):
(1) Performing nuclear magnetic and infrared structural characterization on the synthesized PEGDA and GelMA, as shown in figure 1, showing that an obvious double bond peak appears at 5.5-6.5ppm in the figure, which indicates that acryloyl chloride has successfully reacted with hydroxyl groups at two ends of PEG to generate double bonds, and proving that the synthesis of PEGDA is successful, as shown in figure 2, showing that an obvious double bond peak appears at 5.0-6.0ppm in the figure, indicating that the double bonds have successfully grafted to gelatin side chains, and a peak on lysine methylene is at 3.0ppm, and calculating by integration to obtain the double bond substitution degree of GelMA to be 75%, and using an X-ray diffractometer to perform the characterization of the crystal structures of PEGDA, gelMA and a printed product (PEGDA-GelMA), as shown in figure 3, gelMA has no obvious diffraction peak, but PEGDA and hydrogel both have diffraction peaks at 2 theta of 19 and 23 degrees, and proving that the inside of the hydrogel contains the crystal structure caused by PEGDA, and the hydroxyl group peak in PEA should disappear and 1726cm should disappear -1 An absorption peak of an ester bond appears, and GelMA is 3300cm -1 An envelope due to hydrogen bonding was observed, probably due to the presence of hydroxyl and amide bonds, at 1643cm -1 The peak of the amide I band appears there, and the above results again illustrate the successful synthesis of PEGDA and GelMA.
(2) Mechanical testing was performed on different proportions of the printed product (PEGDA-GelMA): firstly, sheet-shaped hydrogel with the thickness of 1mm is printed, a dumbbell shape is pressed by a tablet press, or cylindrical hydrogel with the thickness of 5 to 5mm is printed, and a universal tensile machine is used for respectively carrying out tensile and compression tests on the hydrogel, as shown in figure 4, the result shows that the tensile strength and the tensile modulus of GelMA can be improved by introducing PEGDA, the elongation at break is also improved to a certain extent, probably because the long chain of the PEGDA endows the hydrogel with certain toughness, and the strength of the hydrogel can also be effectively improved by the crystal structure of the PEGDA. The tensile strength, tensile modulus and elongation at break of the PEGDA-GelMA-20-5 hydrogel can reach 82.2kPa,77.2kPa and 101 percent respectively, as shown in figure 5, the compressive modulus of GelMA is slightly improved by introducing PEGDA, but the compressive strength and toughness are improved, in a compression display diagram, the GelMA gel is cracked when being lightly pressed by hands, but the PEGDA-GelMA-20-5 hydrogel can be quickly restored to the shape without being cracked.
(3) Swelling and degradation tests were carried out on different proportions of the printed product (PEGDA-GelMA): first, 5 × 5mm cylindrical hydrogel was printed out, the mass was weighed, hydrogels of different proportions were soaked in PBS, the mass was weighed again at 1,2,3,5,7,9, 11, 14 days, and the swelling degree was calculated using the increased mass ratio. Drying and weighing the printed columnar hydrogel, or placing the printed columnar hydrogel in collagenase, taking out and drying and weighing the hydrogel after 7, 14 and 28, and calculating the residual mass ratio, as shown in fig. 6A-D, the printed product (PEGDA-GelMA) can reach swelling equilibrium within 1 day, and the more the PEGDA content is, the smaller the swelling degree is, and the mass of the gel in three proportions is reduced in different degrees within one month, which shows that the material can be degraded under the action of enzyme, wherein 41% of the mass of the PEGDA-GelMA-20-5 hydrogel can be remained finally, and the PEGDA-GelMA-10-5 hydrogel is gradually softened in the degradation process due to lower strength, so that the mass of 7 days is only measured.
(4) The transmittance and rheology tests were carried out on printed products (PEGDA-GelMA) in different proportions. First, a sheet-like hydrogel having a thickness of 1mm was printed, cut into an appropriate shape, placed in a cuvette, and tested for light transmittance at 400 to 800nm using PBS as a blank control. A25 mm diameter wafer is cut and placed on a stage of a rheometer to carry out frequency scanning test, the scanning range is 0.1-10Hz, the strain is 1%, the temperature is 37 ℃, as shown in FIGS. 6E-F, at the wavelength of 550nm, PEGDA-GelMA-10, 15, 20-5 hydrogel can respectively reach the light transmittance of 90.7, 86.3 and 82.5 percent, which are all larger than the light transmittance of a natural cornea (71.1%), the storage modulus and the loss modulus of the hydrogel are increased to a certain degree along with the increase of the content of PEGDA, wherein the storage modulus of the PEGDA-GelMA-20-5 hydrogel is close to the storage modulus (4 kPa) of the natural cornea.
(5) In vitro cell adhesion and toxicity testing of mouse fibroblasts (L929) was performed on the printed product (PEGDA-GelMA): for the adhesion test, a 10mm diameter, 1mm thick disc of hydrogel was first printed and placed in a 24-well plate at 5 x 10 4 Cell/well concentrations were seeded with L929 cells. After 48h incubation, pictures were taken using a microscope. For more clear observation of cell morphology, staining the cells with live/dead cell stain (Calcein-AM/PI), and finally taking pictures with inverted fluorescence microscope, as shown in fig. 7A-B, L929 cells can adhere to the PEGDA-GelMA hydrogel surface due to the cell adhesion sites in GelMA, and maintain normal cell morphology and activity; for cytotoxicity testing, first several 5mm diameter, 1mm thick discs of hydrogel were printed, and L929 cells were treated at 2 x 10 4 Inoculating the cells/well into a 96-well plate, culturing for 24h, adding the hydrogel into each well, culturing for 1 day, adding a fresh culture medium after 3 days, continuing culturing for 24h, adding 20 mu L of 5mg/mL 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide (MTT) solution and 180 mu L of the fresh culture medium into each well, culturing for 4h, adding N, N' -dimethyl sulfoxide (DMSO), shaking in a microplate reader for 3min, measuring the absorbance at 570nm, using the cells without the hydrogel as a control group, and calculating the cell survival rate according to the ratio of the absorbance. When the cell survival rate is quantitatively determined by using MTT, the cells are stained by using the live-dead staining method, and photographed by using an inverted fluorescence microscope, as shown in FIGS. 7C-D, the cell survival rate is over 90% compared with that of a control group, which shows that the hydrogel has good cell compatibility, and the cells can keep normal shape and have high survival rate after being cultured with the hydrogel for 1,3 days.
(6) The cellular compatibility of the printed product (PEGDA-GelMA) was evaluated using rabbit corneal epithelial cells (SIRC). Using the cytotoxicity test method described above, different concentrations of PEGDA solution (10, 15, 20 wt%), gelMA solution (5 wt%), different proportions of printed product (PEGDA-GelMA) were co-cultured with SIRC cells for 1 day, 3 days, 5 days before MTT and vital stain tests were performed. Meanwhile, in order to evaluate the cytotoxicity of the photoinitiator LAP (0.25 wt%) and the light-blocking agent lemon yellow (0.05 wt%), the photoinitiator LAP and the SIRC cells were co-cultured for 1 day, 2 days and 3 days, and then the cell survival rate was tested by using the MTT method. The migration ability of the cells on the gel surface was tested by dividing the cells by 5 x 10 4 The cell/well concentration was seeded in a 24-well plate, when the degree of cell union reached about 80%, the middle of each well was scratched using a 200 μ L pipette tip, a layer of hydrogel was then attached thereto, cultured for 1 day, stained for viability and death after 3 days, photographed and the cell mobility was calculated. The adhesion and proliferation capacity of cells on the gel surface was tested by placing the printed gel discs in 24-well plates at 5 x 10 4 The cell/hole concentration is inoculated with SIRC cells, after 2 days, 4 days and 6 days of culture, the cells are subjected to live-dead staining and photographed, and the proliferation of the cells on the gel surface is tested by using a cell proliferation kit (CCK-8), as shown in figure 8, after the SIRC cells are co-cultured with PEGDA-GelMA hydrogel for 1,3,5 days, compared with a control group, the SIRC cells can maintain normal cell proliferation capacity, morphology and metabolic activity, and the cell survival rate is over 80 percent in an in vitro cytotoxicity test of a photoinitiator (LAP) and lemon yellow (UV absorber), and no obvious cytotoxicity exists; as shown in figure 9, the SIRC cells can migrate on the surface of the hydrogel, and the SIRC cells can adhere to the hydrogel and proliferate, thereby proving that the hydrogel is expected to be applied to the field of tissue engineering regeneration.
(7) The printed product (PEGDA-GelMA) cell-packed cornea was explored. The concentrations of PEGDA and GelMA were fixed at 20,5wt%, respectively, the concentration of LAP was 0.25wt%, the height of the printed layer was 50 μm, and printing was performed with varying exposure time and the concentration of lemon yellow. The cornea was first modeled, converted to stl format and input into the printer. By adjusting the exposure time and the concentration of the lemon yellow, corneas with different forms are printed, the corneas are evaluated according to the thickness and the strength of the corneas, and the optimal printing parameters are screened, and the specific data are detailed in tables 1 and 2:
table 1 corneal results presentation without cell printing
Table 2 corneal results display by corneal epithelial cell printing
From tables 1 and 2, it can be seen that 0.15wt% concentration of lemon yellow can achieve better shaped corneas without cells using an exposure time of 40s, while 0.1wt% concentration of lemon yellow can achieve better shaped corneas with cells using an exposure time of 80 s.
The cornea obtained by printing was cultured in a special medium for 3 days, stained for alive and dead, and photographed by an inverted fluorescence microscope, as shown in fig. 10, and the cells also had a high survival rate inside the hydrogel.
The invention being thus described by way of example, it should be understood that any simple alterations, modifications or other equivalent alterations as would be within the skill of the art without the exercise of inventive faculty, are within the scope of the invention.
Claims (6)
1. A 3D printing ink for corneal regeneration, characterized by: preparing a polyethylene glycol diacrylate (PEGDA) solution and a methacrylated gelatin (GelMA) solution by using a Phosphate Buffer Solution (PBS), filtering the polyethylene glycol diacrylate (PEGDA) solution, the methacrylated gelatin (GelMA) solution, a photoinitiator lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate (LAP) and a light-blocking agent lemon yellow (UVabs) through a 0.22 mu m filter membrane, and mixing the polyethylene glycol diacrylate (PEGDA) solution, the methacrylated gelatin (GelMA) solution, the photoinitiator lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate (LAP) and the light-blocking agent lemon yellow (UVabs) at 37 ℃ to obtain a 3D printing ink, wherein the concentration of the polyethylene glycol diacrylate (PEGDA) solution is 10, 15, 20wt%, the concentration of the methacrylated gelatin (GelMA) solution is 5wt%, the concentration of the lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate (LAP) solution is 25wt%, the concentration of the photoinitiator lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate (LAP) solution is 0.05wt%, the concentration of the photoinitiator lithium phenyl (2, the light-4-trimethylbenzoyl) phosphate (UVabs) is 0.05wt%, and the mass ratio of the polyethylene glycol to the lithium citrate (UVabs) to the 3D printing ink is: 1:0.05:0.01.
2. a preparation method of 3D printing ink for cornea regeneration is characterized in that: preparing a polyethylene glycol diacrylate (PEGDA) solution and a methacrylated gelatin (GelMA) solution using a Phosphate Buffer Solution (PBS), and filtering the polyethylene glycol diacrylate (PEGDA) solution, the methacrylated gelatin (GelMA) solution, a photo-initiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP), and a light-blocking agent lemon yellow (UVabsorber) through a 0.22 μm filter, mixing the polyethylene glycol diacrylate (PEGDA) solution, the methacrylated gelatin (GelMA) solution, the photo-initiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP), and the light-blocking agent lemon yellow (UVabsorber) at 37 ℃ to obtain a 3D printing ink, wherein the concentration of the polyethylene glycol diacrylate (PEGDA) solution is 10, 15, 20wt%, the concentration of the methacrylated gelatin (GelMA) solution is 5wt%, the concentration of the phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP) solution is 0.25wt%, the concentration of the photo-benzyl yellow (UVabsorber) is 0.05wt%, the ratio of the phenyl (2, the 4-4 wt%, the weight of the 4 wt% of the 4-trimethylbenzoyl) lithium phosphate (UVabsorber) to the 4 wt%, the 4 wt% of the Phenyl (PEGDA) is greater than the 4 wt%, the 3D printing ink: 1:0.05:0.01.
3. use of a printed product (PEGDA-GelMA) printed with a 3D printing ink for corneal regeneration according to claim 1, in a corneal regeneration scaffold material, characterized in that: arrange 3D printing ink in DLP3D printer, set up printing parameter, print the parameter: exposing for 20-80s, printing at 37 deg.C with a printing layer height of 20-60 μm, and peeling off from the base to obtain printed product (PEGDA-GelMA).
4. Use according to claim 3, characterized in that: the tensile strength, tensile modulus and elongation at break of the printed product (PEGDA-GelMA) are 82-83kPa,77-78kPa and 100-104% respectively; the printed product (PEGDA-GelMA) printed with a 3D printing ink for corneal regeneration had a light transmittance of 82-91% at a wavelength of 550nm, which was greater than the light transmittance of 71.1% of the natural cornea.
5. Use according to claim 3, characterized in that: in the case of no cells, the conditions for printing the cornea were: the exposure time was 40s and the lemon yellow concentration was 0.15wt%.
6. Use according to claim 3, characterized in that: in the case of cells, the conditions for printing the cornea were: the exposure time was 80s and the lemon yellow concentration was 0.1wt%.
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