CN114796604B - 3D printing ink for cornea regeneration and preparation method and application thereof - Google Patents

Abstract

The invention provides a 3D printing ink for corneal regeneration and its preparation method and application. A phosphate buffer solution is used to prepare a polyethylene glycol diacrylate solution and a methacrylic gelatin solution, and the polyethylene glycol diacrylate Ester solution, methacrylated gelatin solution, photoinitiator phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and light blocking agent tartrazine were filtered through a 0.22 μm filter membrane at 37 Under the environment of ℃, polyethylene glycol diacrylate solution, methacrylated gelatin solution, photoinitiator phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and light blocking agent lemon yellow Mix to get 3D printing ink. The preparation method of the invention is simple, the source of materials is wide, and the practicability is strong.

Description

A 3D printing ink for corneal regeneration and its preparation method and application

Technical Field

The present invention relates to the technical field of biomedical materials, and more specifically to a 3D printing ink for corneal regeneration, and a preparation method and application thereof.

Background Art

The cornea is a transparent, multi-layered structure on the surface of the eyeball. Its main function is to focus light onto the lens and then direct it to the retina. It is composed of the corneal epithelium, corneal stroma, and corneal endothelium. The epithelium is composed of stratified epithelial cells, which are differentiated from limbal stem cells. It allows oxygen and essential nutrients to penetrate and prevents pathogens and dust from entering the eye. The stroma layer constitutes 90% of the thickness of the cornea and is composed of highly orthogonally arranged collagen fibers, which contain corneal stromal cells. The innermost layer is the corneal endothelium, which is composed of a layer of endothelial cells and has poor regeneration ability.

When the cornea is damaged, corneal diseases may occur. Nowadays, corneal diseases have become one of the main causes of blindness. According to statistics, there are nearly 10 million patients with corneal diseases every year, but due to the lack of donors, only less than 200,000 patients can undergo corneal transplantation. Therefore, research on artificial corneal substitutes is imminent.

Currently, the artificial corneas that have been used in corneal transplant surgeries include the Boston artificial cornea and the AlphaCor artificial cornea. Although they have successfully improved the vision of many patients, the complications that follow, such as the increased risk of glaucoma and endophthalmitis, make it difficult to meet the requirements of an ideal corneal substitute.

Tissue engineering technology has brought good news to corneal patients, and as a key part of the three elements of tissue engineering, the selection of scaffold materials is particularly critical. Hydrogel is a swollen body of a water-rich cross-linked polymer network. Due to its good biocompatibility, high porosity, high water content and suitable viscoelasticity, it is expected to be used as a scaffold material in corneal regeneration. Nowadays, a variety of polymer materials and their composites have been used to prepare hydrogels, such as collagen, gelatin, chitosan, silk fibroin, etc. in natural polymers, and polyethylene glycol (PEG), polycaprolactone (PCL), polyhydroxyethyl methacrylate (PHEMA), etc. in synthetic polymers. Natural polymers usually have excellent biocompatibility, but their mechanical properties are difficult to meet the requirements. Synthetic polymers can achieve ideal properties by improving the synthesis process, but their biological activity is low, and it is difficult to support cell adhesion and proliferation, so it is difficult to fuse with their own tissues to achieve the purpose of regeneration. Therefore, it is very important to develop a biological scaffold material that has both sufficient biocompatibility and sufficient mechanical properties.

3D bioprinting technology is one of the effective ways to create scaffold materials with a suitable cell growth microenvironment for tissue engineering and regenerative medicine. In the past few years, digital light processing (DLP) technology has been favored by more and more researchers as a light-assisted 3D printing technology. It can solve some shortcomings in inkjet printing and extrusion printing, such as the cells are easily damaged by shear force during the extrusion process, the high requirements for ink viscosity, and the decrease in cell viability caused by long printing time. DLP can prepare products of various shapes by layered printing and layer-by-layer photocuring, and its ink source is wide. Compared with traditional ultraviolet light initiation, DLP's light source uses a 405nm wavelength, which belongs to the visible light band, so it is less damaging to cells.

Summary of the invention

The present invention overcomes the deficiencies in the prior art. The existing corneal regeneration scaffold materials have poor mechanical properties and low biological activity, and are difficult to support cell adhesion and proliferation, and are further difficult to fuse with self-tissue to achieve the purpose of regeneration. A 3D printing ink for corneal regeneration and a preparation method and application thereof are provided. The 3D printing ink is mainly composed of polyethylene glycol diacrylate (PEGDA) and methacryloyl gelatin (GelMA). Methacryloyl gelatin (GelMA) can enhance the biocompatibility of the gel, and polyethylene glycol diacrylate (PEGDA) can enhance the mechanical properties of the gel. A hydrogel with a corneal shape is prepared by using digital light processing (DLP) 3D printing technology, and its optical properties, rheological properties, mechanical properties, degradation and swelling properties and cell compatibility are tested, preliminarily proving its application potential in corneal regeneration.

The purpose of the present invention is achieved through the following technical solutions.

A 3D printing ink for corneal regeneration and a preparation method thereof, wherein a polyethylene glycol diacrylate (PEGDA) solution and a methacrylated gelatin (GelMA) solution are prepared using a phosphate buffer solution (PBS), and the polyethylene glycol diacrylate (PEGDA) solution, the methacrylated 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 μm filter membrane, and the polyethylene glycol diacrylate (PEGDA) solution, the methacrylated gelatin (GelMA) solution, the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and the light-blocking agent lemon yellow (UV absorber) are filtered under a 37°C environment. absorber) to obtain a 3D printing ink, wherein the concentration of polyethylene glycol diacrylate (PEGDA) solution is 10-20wt%, the concentration of methacryloyl gelatin (GelMA) solution is 5-10wt%, the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) is 0.25-0.5wt%, the concentration of lemon yellow (UV absorber) is 0.05-0.15wt%, and the mass ratio of polyethylene glycol diacrylate (PEGDA), methacryloyl gelatin (GelMA), phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and lemon yellow (UV absorber) is (1-5): (1-2): 0.05: 0.01, and the DLP The printing parameters were set in the 3D printer: exposure time 20-80s, printing layer height 20-60μm, printing temperature 37°C. After printing was completed, the printed product (PEGDA-GelMA) was obtained by peeling it off from the base.

The concentrations of polyethylene glycol diacrylate (PEGDA) solution were 10, 15, and 20 wt %, the concentration of methacrylated gelatin (GelMA) solution was 5 wt %, the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) was 0.25 wt %, and the concentration of tartrazine (UV absorber) was 0.05 wt %.

The mass ratio of polyethylene glycol diacrylate (PEGDA), methacryloyl gelatin (GelMA), phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and tartrazine (UV absorber) is (2-4): 1: 0.05: 0.01.

Printing parameters: exposure time is 20s, printing layer height is 50μm, and printing temperature is 37℃.

Preparation method of polyethylene glycol diacrylate (PEGDA): polyethylene glycol (PEG) (molecular weight of 8000) is dissolved in dichloromethane to obtain a polyethylene glycol (PEG) solution, and then triethylamine is added to the polyethylene glycol (PEG) solution, and an ice bath is placed. After cooling, a mixed solution is obtained, and dichloromethane and acryloyl chloride are added to a constant pressure dropping funnel. The mixed solution of dichloromethane and acryloyl chloride is slowly dripped into the mixed solution at a rate of 5s/drop. After reacting at room temperature of 20-25°C for 24h, the solution is dripped dropwise into ice ether, and after sedimentation, filtration and drying, polyethylene glycol diacrylate (PEGDA) is obtained, wherein the mass ratio of polyethylene glycol (PEG), triethylamine, dichloromethane and acryloyl chloride is 10:1:70:0.6.

The preparation method of methacryloyl gelatin (GelMA) is as follows: gelatin is dissolved in water at 40°C to obtain a gelatin aqueous solution, sodium hydroxide (NaOH) is added to the gelatin aqueous solution, N,N'-dimethylformamide (DMF) is added after stirring to dissolve, methacrylic anhydride is added after stirring to clarify, and the solution is reacted at 40°C for 2 hours, and then the solution is quickly poured into anhydrous ethanol to obtain a precipitate by sedimentation. The precipitate is cut into pieces and then added to anhydrous ethanol for washing. Finally, the precipitate is dissolved in ultrapure water in an oven at 37°C, dialyzed for three days, and freeze-dried to obtain methacryloyl gelatin (GelMA), wherein the mass ratio of gelatin, sodium hydroxide (NaOH), N,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) obtained by printing using a 3D printing ink for corneal regeneration are 82-83 kPa, 77-78 kPa and 100-104%, respectively.

At a wavelength of 550nm, the transmittance of the printed product (PEGDA-GelMA) obtained using a 3D printing ink for corneal regeneration is 82-91%, which is greater than the transmittance of the natural cornea of 71.1%.

In the in vitro cytotoxicity test of the hydrogel, the cell survival rate of the printed product (PEGDA-GelMA) printed using a 3D printing ink for corneal regeneration was above 90% compared with the control group, indicating that the printed product (PEGDA-GelMA) had better cell compatibility. After the cells were co-cultured with the printed product (PEGDA-GelMA) for 1-3 days, the cells were able to maintain a normal morphology and had a high survival rate.

Without cells, the conditions for printing the cornea were: exposure time of 40 s, lemon yellow concentration of 0.15 wt%, while with cells, the conditions for printing the cornea were: exposure time of 80 s, lemon yellow concentration of 0.1 wt%.

The beneficial effects of the present invention are as follows: 3D printing ink is mainly composed of polyethylene glycol diacrylate (PEGDA) and methacryloyl gelatin (GelMA), methacryloyl gelatin (GelMA) can enhance the biocompatibility of the gel, polyethylene glycol diacrylate (PEGDA) can enhance the mechanical properties of the gel, digital light processing technology (DLP) 3D printing technology is used to prepare a hydrogel in the shape of a cornea, and its optical properties, rheological properties, mechanical properties, degradation and swelling properties and cell compatibility are tested, preliminarily proving its application potential in corneal regeneration, the preparation method of the present invention is simple, the material source is wide, and the practicability is strong.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a NMR image of polyethylene glycol (PEG) and polyethylene glycol diacrylate (PEGDA);

FIG2 is a NMR image of gelatin and methacryloyl gelatin (GelMA);

FIG3A is an XRD pattern of polyethylene glycol diacrylate (PEGDA), methacryloyl gelatin (GelMA), and a printed product (PEGDA-GelMA-20-5), and FIG3B is an infrared spectrum of polyethylene glycol diacrylate (PEGDA) and methacryloyl gelatin (GelMA);

FIG4 is a tensile diagram of a methacrylated gelatin (GelMA) hydrogel, a printed product (PEGDA-GelMA);

Figure 5 is a compression diagram of methacrylated gelatin (GelMA) hydrogel, printed product (PEGDA-GelMA)

Fig. 6A is the swelling degree of the printed product (PEGDA-GelMA) in PBS, Fig. 6B is the degradation diagram of the printed product (PEGDA-GelMA) in type I collagenase, Fig. 6C is the transmittance of the printed product (PEGDA-GelMA), Fig. 6D is a transparency display diagram of the natural cornea, the printed cornea and the printed sheet, Fig. 6E is a frequency scan diagram of the printed product (PEGDA-GelMA), and Fig. 6F is a modulus statistical diagram of the printed product (PEGDA-GelMA);

Figure 7 shows the adhesion and survival rate of L929 cells on the printed product (PEGDA-GelMA), wherein (A) is a bright field photo of cells on the surface of the hydrogel, (B) is a fluorescent photo of live and dead cells on the surface of the hydrogel, (C) is an in vitro cytotoxicity test, (D, E) are live and dead staining images of cells and hydrogels after co-culture for 1 and 3 days, and the control group is a blank control without gel;

FIG8A is a live-dead staining image of rabbit corneal epithelial cells (SIRC) co-cultured with printed products (PEGDA-GelMA) for 1, 3, and 5 days, FIG8B is MTT test data of SIRC cells co-cultured with polyethylene glycol diacrylate (PEGDA), methacryloyl gelatin (GelMA) solution, and printed products (PEGDA-GelMA) for 1, 3, and 5 days, and the control group is a blank control without cells, and FIG8C is an in vitro cytotoxicity test of a photoinitiator (LAP) and tartrazine (UV absorber);

Figures 9A and 9B are live-dead staining images of SIRC cells migrating on the surface of printed products (PEGDA-GelMA) and the corresponding migration rates, and Figures 9C and 9D are live-dead staining images of SIRC cells adhering to the surface of printed products (PEGDA-GelMA) and proliferation data;

FIG. 10 is a live-death staining image of SIRC cells cultured in the printed product (PEGDA-GelMA-20-5) for 3 days.

DETAILED DESCRIPTION

The technical solution of the present invention is further described below through specific embodiments.

Preparation of polyethylene glycol diacrylate (PEGDA): First, weigh 10g PEG (Mw = 8k) in a 250mL round-bottom flask, stir and dissolve it using 50mL dichloromethane as a solvent, then add 1mL triethylamine to the round-bottom flask, ice bath for 30min, add 20mL dichloromethane and 0.6mL acryloyl chloride to a constant pressure dropping funnel, slowly drip the above solution at a rate of one drop per 5s, and react at room temperature for 24h. The solution after the reaction is added dropwise to a large amount of ice ether, precipitated, filtered, and dried to obtain the product PEGDA.

Preparation of methacryloyl gelatin (GelMA): First, add 4g gelatin to 200mL water, stir and dissolve at 40℃, add a few grains of sodium hydroxide (NaOH) after dissolving, stir and dissolve, add 132mL N,N'-dimethylformamide (DMF), add 290μL methacrylic anhydride after stirring and clarifying, wait for 10min, add 292μL methacrylic anhydride again, and react at 40℃ for 2h. The solution after the reaction is quickly poured into a large amount of anhydrous ethanol for sedimentation, cut the precipitate into pieces and continue to add anhydrous ethanol for washing, and after 10min, use 180mL ultrapure water to dissolve the precipitate in a 37℃ oven, dialyze for three days, and freeze-dry to obtain the product GelMA.

Example 1

Preparation of 3D printing ink and printed products (PEGDA-GelMA): Use PBS to prepare a certain concentration of PEGDA solution (10wt%) and GelMA solution (5wt%) and filter the bacteria through a 0.22μm filter membrane. Add a certain amount of sterile photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP, 0.25wt%) and light-blocking agent lemon yellow (UV absorber, 0.05wt%) at 37°C. Set the printing parameters in the DLP 3D printer, exposure time 20s, layer height 50μm, printing temperature 37°C.

Example 2

Preparation of 3D printing ink and printed products (PEGDA-GelMA): Use PBS to prepare a certain concentration of PEGDA solution (15wt%) and GelMA solution (5wt%) and filter the bacteria through a 0.22μm filter membrane. Add a certain amount of sterile photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP, 0.5wt%) and light-blocking agent lemon yellow (UV absorber, 0.1wt%) at 37°C. Set the printing parameters in the DLP 3D printer, exposure time 10s, layer height 20μm, printing temperature 37°C.

Example 3

Preparation of 3D printing ink and printed products (PEGDA-GelMA): Use PBS to prepare a certain concentration of PEGDA solution (20wt%) and GelMA solution (5wt%) and filter the bacteria through a 0.22μm filter membrane. Add a certain amount of sterile photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP, 0.25wt%) and light-blocking agent lemon yellow (UV absorber, 0.05wt%) at 37°C. Set the printing parameters in the DLP 3D printer, exposure time 30s, layer height 60μm, printing temperature 37°C.

Characterization of printed products (PEGDA-GelMA):

(1) The synthesized PEGDA and GelMA were characterized by NMR and IR. As shown in FIG1 , an obvious double bond peak appeared at 5.5-6.5 ppm, indicating that acryloyl chloride has successfully reacted with the hydroxyl groups at both ends of PEG to form double bonds, proving the successful synthesis of PEGDA. As shown in FIG2 , an obvious double bond peak appeared at 5.0-6.0 ppm, indicating that the double bond has been successfully grafted onto the gelatin side chain. The peak at 3.0 ppm is on the methylene group of gelatin lysine. By integration, it can be calculated that the double bond substitution degree of GelMA is 75%. The crystal structure of PEGDA, GelMA and the printed product (PEGDA-GelMA) was characterized by X-ray diffractometer. As shown in FIG3 , GelMA has no obvious diffraction peak, but PEGDA and the hydrogel both have diffraction peaks at 2θ of 19 and 23°, proving that the hydrogel contains a crystalline structure caused by PEGDA. The hydroxyl peak that should have existed in PEGDA disappears, and an ester bond absorption peak appears at 1726 cm ^-1. The absorption peak of GelMA is at 3300 cm ^-1 showed a peak due to hydrogen bonding, which may be due to the presence of hydroxyl and amide bonds. The peak of amide I band appeared at 1643 cm ^-1 . The above results once again demonstrated the successful synthesis of PEGDA and GelMA.

(2) Mechanical tests were performed on the printed products (PEGDA-GelMA) with different proportions: first, a 1 mm thick sheet of hydrogel was printed and pressed into a dumbbell shape using a tablet press, or a 5*5*5 mm cylindrical hydrogel was printed and subjected to tensile and compression tests using a universal tensile testing machine, as shown in Figure 4. The results show that the introduction of PEGDA can improve the tensile strength and tensile modulus of GelMA, and the elongation at break is also improved to a certain extent. This may be because the long chain of PEGDA gives the hydrogel a certain toughness, and its crystalline structure can also effectively improve the strength of the hydrogel. The tensile strength, tensile modulus and elongation at break of PEGDA-GelMA-20-5 hydrogel can reach 82.2 kPa, 77.2 kPa and 101%, respectively. As shown in Figure 5, the introduction of PEGDA slightly improves the compression modulus of GelMA, but improves the compression strength and toughness. In the compression display diagram, the GelMA gel is broken when it is lightly pressed by hand, but the PEGDA-GelMA-20-5 hydrogel can quickly recover its shape without breaking.

(3) Swelling and degradation tests were performed on the printed products (PEGDA-GelMA) with different ratios: First, a 5*5*5 mm cylindrical hydrogel was printed and the mass was weighed. The hydrogels with different ratios were immersed in PBS and the mass was weighed again at 1, 2, 3, 5, 7, 9, 11, and 14 days. The swelling degree was calculated using the increased mass ratio. The printed columnar hydrogel was dried and weighed, or placed in collagenase, taken out and dried and weighed after 7, 14, and 28 hours, and the remaining mass ratio was calculated. As shown in Figure 6A-D, the printed product (PEGDA-GelMA) can reach swelling equilibrium within 1 day, and the higher the PEGDA content, the lower the swelling degree. Within one month, the mass of the three ratios of gels decreased to varying degrees, indicating that this material can be degraded under the action of enzymes. Among them, PEGDA-GelMA-20-5 hydrogel finally had 41% of the mass remaining. Due to its low strength, PEGDA-GelMA-10-5 hydrogel gradually softened during the degradation process, so only the mass at 7 days was measured.

(4) Transmittance and rheological tests were performed on the printed products (PEGDA-GelMA) with different ratios. First, a 1 mm thick sheet of hydrogel was printed, cut into a suitable shape and placed in a cuvette. PBS was used as a blank control to test the transmittance at 400-800 nm. A disc with a diameter of 25 mm was cut and placed on the stage of the rheometer for a frequency sweep test with a sweep range of 0.1-10 Hz, a strain of 1%, and a temperature of 37°C. As shown in Figures 6E-F, at a wavelength of 550 nm, the transmittances of PEGDA-GelMA-10, 15, and 20-5 hydrogels can reach 90.7%, 86.3%, and 82.5%, respectively, which are all greater than the transmittance of the natural cornea (71.1%). With the increase of the PEGDA content, the storage modulus and loss modulus of the hydrogel both increase to a certain extent, among which the storage modulus of the PEGDA-GelMA-20-5 hydrogel is close to that of the natural cornea (~4 kPa).

(5) The printed product (PEGDA-GelMA) was subjected to in vitro cell adhesion and toxicity tests of mouse fibroblasts (L929): For the adhesion test, a 10 mm diameter, 1 mm thick disc hydrogel was first printed and placed in a 24-well plate, and L929 cells were inoculated at a concentration of 5*10 ⁴ cells/well. After 48 hours of culture, photos were taken using a microscope. In order to observe the cell morphology more clearly, the cells were stained with a live cell/dead cell stain (Calcein-AM/PI), and finally, photos were taken using an inverted fluorescence microscope. As shown in Figure 7A-B, due to the cell adhesion sites in GelMA, L929 cells can adhere to the surface of this PEGDA-GelMA hydrogel and maintain normal cell morphology and activity; For the cytotoxicity test, several 5 mm diameter, 1 mm thick disc hydrogels were first printed, and L929 cells were inoculated at a concentration of 2*10 ⁴ cells/well were inoculated into a 96-well plate, cultured for 24 hours, the above hydrogel was added to each well, co-cultured for 1 day, fresh culture medium was added after 3 days, and cultured for 24 hours, then 20 μL 5 mg/mL 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) solution and 180 μL fresh culture medium were added to each well, cultured for 4 hours, N,N'-dimethyl sulfoxide (DMSO) was added, and the absorbance at 570 nm was measured. Cells without hydrogel were used as the control group, and the cell survival rate was calculated by the ratio of absorbance. While using MTT to quantitatively determine the cell survival rate, the cells were stained using the above live-dead staining method, and photographed using an inverted fluorescence microscope. As shown in Figure 7C-D, the cell survival rate was above 90% compared with the control group, indicating that the cell compatibility of this hydrogel was good. After the cells were co-cultured with the hydrogel for 1 and 3 days, the cells could maintain normal morphology and had a high survival rate.

(6) The cytocompatibility of the printed product (PEGDA-GelMA) was evaluated using rabbit corneal epithelial cells (SIRC). Using the above-mentioned cytotoxicity test method, different concentrations of PEGDA solution (10, 15, 20wt%), GelMA solution (5wt%), and different proportions of the printed product (PEGDA-GelMA) were co-cultured with SIRC cells for 1 day, 3 days, and 5 days, and then MTT and live-dead staining tests were performed. At the same time, in order to evaluate the cytotoxicity of the photoinitiator LAP (0.25wt%) and the light-blocking agent lemon yellow (0.05wt%), they were co-cultured with SIRC cells for 1 day, 2 days, and 3 days, and then the cell survival rate was tested using the MTT method. The migration ability of cells on the gel surface was tested. The cells were inoculated in a 24-well plate at a concentration of 5*10 ⁴ cells/well. When the cell healing rate reached about 80%, a 200μL pipette tip was used to scratch the middle of each well, and then a layer of hydrogel was attached thereon. The cells were cultured for 1 day, and live-dead staining was performed after 3 days. Photos were taken and the cell migration rate was calculated. The adhesion and proliferation ability of cells on the gel surface were tested. The printed gel discs were placed in a 24-well plate and SIRC cells were inoculated at a concentration of 5*10 ⁴ cells/well. After culturing for 2, 4, and 6 days, live and dead staining was performed and photographs were taken. The proliferation of cells on the gel surface was tested using a cell proliferation kit (CCK-8). As shown in Figure 8, after co-culturing SIRC cells with PEGDA-GelMA hydrogel for 1, 3, and 5 days, compared with the control group, SIRC cells can maintain normal cell proliferation ability, morphology and metabolic activity, and in the in vitro cytotoxicity test of photoinitiator (LAP) and lemon yellow (UV absorber), the cell survival rate was above 80%, with no obvious cytotoxicity. As shown in Figure 9, SIRC cells can migrate on the hydrogel surface, and SIRC cells can adhere to and proliferate on the hydrogel, which once again proves that this hydrogel is expected to be used in the field of tissue engineering regeneration.

(7) The printed product (PEGDA-GelMA) encapsulated cell-printed cornea was investigated. The concentrations of PEGDA and GelMA were fixed at 20.5wt%, LAP at 0.25wt%, and the printing layer height was 50μm. The exposure time and the concentration of lemon yellow were changed for printing. First, the cornea was modeled, converted into stl format and input into the printer. By adjusting the exposure time and the concentration of lemon yellow, corneas with different morphologies were printed. The thickness and strength of the cornea were evaluated and the optimal printing parameters were selected. The specific data are shown in Tables 1 and 2:

Table 1 Results of printing cornea without cells

Table 2 Results of printing cornea with corneal epithelial cells

It can be seen from Tables 1 and 2 that, without cells, using an exposure time of 40 seconds and a lemon yellow concentration of 0.15wt% can produce a better-shaped cornea, while with cells, using an exposure time of 80 seconds and a lemon yellow concentration of 0.1wt% can produce a better-shaped cornea.

The printed cornea was placed in a special culture medium for 3 days, subjected to live-dead staining, and photographed using an inverted fluorescence microscope. As shown in FIG10 , the cells also had a high survival rate inside the hydrogel.

The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive labor falls within the protection scope of the present invention.

Claims (6)

1. A 3D printing ink for corneal regeneration, characterized in that: a polyethylene glycol diacrylate (PEGDA) solution and a methacrylated gelatin (GelMA) solution are prepared using a phosphate buffer solution (PBS), and the polyethylene glycol diacrylate (PEGDA) solution, the methacrylated gelatin (GelMA) solution, a photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and a light-blocking agent lemon yellow (UVabsorber) are filtered through a 0.22 μm filter membrane, and at 37°C, the polyethylene glycol diacrylate (PEGDA) solution, the methacrylated gelatin (GelMA) solution, the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and The light-blocking agent tartrazine (UVabsorber) is mixed to obtain a 3D printing ink, wherein the concentration of polyethylene glycol diacrylate (PEGDA) solution is 10, 15, and 20wt%, the concentration of methacryloyl gelatin (GelMA) solution is 5wt%, the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) is 0.25wt%, the concentration of tartrazine (UVabsorber) is 0.05wt%, and the mass ratio of polyethylene glycol diacrylate (PEGDA), methacryloyl gelatin (GelMA), phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and tartrazine (UVabsorber) is (2-4):1:0.05:0.01. 2. A method for preparing a 3D printing ink for corneal regeneration, characterized in that: a phosphate buffer solution (PBS) is used to prepare a polyethylene glycol diacrylate (PEGDA) solution and a methacrylic gelatin (GelMA) solution, and the polyethylene glycol diacrylate (PEGDA) solution, the methacrylic gelatin (GelMA) solution, a photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and a light-blocking agent lemon yellow (UVabsorber) are filtered through a 0.22 μm filter membrane, and the polyethylene glycol diacrylate (PEGDA) solution, the methacrylic gelatin (GelMA) solution, the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and the light-blocking agent lemon yellow (UVabsorber) are filtered under a 37°C environment. ) and a light-blocking agent, tartrazine (UVabsorber), are mixed to obtain a 3D printing ink, wherein the concentration of the polyethylene glycol diacrylate (PEGDA) solution is 10, 15, and 20 wt %, the concentration of the methacryloyl gelatin (GelMA) solution is 5 wt %, the concentration of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) is 0.25 wt %, the concentration of tartrazine (UVabsorber) is 0.05 wt %, and the mass ratio of polyethylene glycol diacrylate (PEGDA), methacryloyl gelatin (GelMA), phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and tartrazine (UVabsorber) is (2-4):1:0.05:0.01. 3. The use of a printed product (PEGDA-GelMA) obtained by printing with a 3D printing ink for corneal regeneration according to claim 1 on a corneal regeneration scaffold material, characterized in that: the 3D printing ink is placed in a DLP3D printer, and the printing parameters are set, the printing parameters are: exposure time is 20-80s, printing layer height is 20-60μm, printing temperature is 37°C, and after printing is completed, the printed product (PEGDA-GelMA) is peeled off from the base to obtain. 4. The use according to claim 3 is characterized in that: the tensile strength, tensile modulus and elongation at break of the printed product (PEGDA-GelMA) are 82-83 kPa, 77-78 kPa and 100-104% respectively; at a wavelength of 550 nm, the transmittance of the printed product (PEGDA-GelMA) printed using a 3D printing ink for corneal regeneration is 82-91%, which is greater than the transmittance of 71.1% of the natural cornea. 5. The use according to claim 3, characterized in that: in the case of no cells, the conditions for printing the cornea are: exposure time is 40s, and the concentration of lemon yellow is 0.15wt%. 6. The use according to claim 3, characterized in that: in the case of cells, the conditions for printing the cornea are: exposure time is 80s, and the concentration of lemon yellow is 0.1wt%.

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