Disclosure of Invention
The invention aims to provide a collagen-based artificial cornea with a double-layer structure and capable of being rapidly epithelialized and a preparation method thereof, so as to overcome the defects in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
The application discloses a preparation method of a collagen-based artificial cornea with a double-layer structure and capable of being rapidly epithelialized, which comprises the following steps:
S1, preparing a collagen solution, namely adding collagen into an acetic acid solution, stirring and dissolving, adding a plasticizer after the collagen is completely dissolved, stirring uniformly, and centrifugally defoaming to obtain a high-concentration collagen solution and a low-concentration collagen solution, wherein the concentration of the high-concentration collagen solution is 0.3% -0.5% w/v, and the concentration of the low-concentration collagen solution is 0.1% -0.25% w/v;
s2, performing primary electrochemical deposition, namely performing electrochemical deposition on a high-concentration collagen solution, wherein a platinum mesh is connected with an anode, a titanium sheet is connected with a cathode, and an electrode plate is connected with a direct current power supply to obtain a collagen film I;
S3, performing primary ultraviolet crosslinking, namely performing dehydration treatment on the collagen film I by using absolute ethyl alcohol, and performing photocrosslinking by using strong ultraviolet irradiation;
s4, chemical crosslinking, namely soaking the collagen film I subjected to primary ultraviolet crosslinking treatment in the step 3 in a 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride solution, and performing chemical crosslinking to obtain a compact layer;
S5, secondary electrochemical deposition, namely connecting a titanium sheet deposited with a compact layer on a negative electrode, connecting a platinum net on a positive electrode, pouring a low-concentration collagen solution into a deposition tank, immersing the compact layer, and performing secondary electrochemical deposition, wherein a collagen film II is obtained on the compact layer;
S6, secondary ultraviolet crosslinking, namely, carrying out dehydrated treatment on the collagen film II by absolute ethyl alcohol, and carrying out secondary photocrosslinking by weak ultraviolet irradiation to obtain a loose layer, wherein the loose layer and a compact layer are combined to form the collagen-based artificial cornea with the double-layer structure and capable of being rapidly epithelialized.
Preferably, the collagen is type I collagen.
Preferably, the plasticizer is selected from any one or a combination of a plurality of glycerol, polyethylene glycol, polypropylene glycol and citrate plasticizers, and the concentration of the plasticizer is 10% -20% v/v.
Preferably, the concentration of the acetic acid solution is 1% -3% v/v.
Preferably, the direct current power supply in the step S2 selects a constant current mode 1-4A, the deposition time is 30-60 min, and the direct current power supply in the step S4 selects the constant current mode 1-4A, the deposition time is 10-30 min.
Preferably, the intensity of the strong ultraviolet light in the step S3 is 600-1000 mu w/cm2 for 30-60 min, and the intensity of the weak ultraviolet light in the step S6 is 10-200 mu w/cm2 for 1-10 min.
Preferably, in the step S4, the concentration of the 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride solution is 0.01% -0.04% w/w, and the soaking time is 1-5 hours.
Preferably, the method further comprises a step S7 of washing the collagen-based artificial cornea which has the double-layer structure and can be rapidly epithelialized and is prepared in the step S6 through PBS and storing the collagen-based artificial cornea in PBS buffer solution containing recombinant human epidermal growth factor, wherein the concentration of the recombinant human epidermal growth factor is 0.01% -0.03% w/v.
The invention also discloses a collagen-based artificial cornea with a double-layer structure and capable of being rapidly epithelialized, which is prepared by adopting the preparation method of the collagen-based artificial cornea with the double-layer structure and capable of being rapidly epithelialized, and is applied to keratopathy caused by cornea injury, cornea infection and the like, and the artificial cornea with the double-layer structure is transplanted for a keratopathy patient through lamellar transplantation operation. The upper layer of the double-layer structure is a loose layer which is easy to degrade, is favorable for epithelial cell migration, accelerates the lamellar transplanting operation to finish autologous cornea epithelialization, and simultaneously has slow degradation rate of a lower layer compact layer and provides a certain thickness support and light penetration performance.
The invention has the beneficial effects that:
1. The collagen-based artificial cornea with the double-layer structure and rapid epithelialization has high light transmittance, and meets the clinical mechanical properties that the light transmittance at 600nm is higher than 90%, the tensile strength is higher than 1Mpa, and the elastic modulus is higher than 10Mpa. The swelling rate is low, the tissue compression of the product to the autologous cornea can be avoided, the mechanical stimulation to eye tissues is reduced, and the comfort after implantation is improved.
2. Excellent biocompatibility, the composition raw materials are I-type collagen and a small amount of medicine auxiliary plasticizer, and the composition has small stimulation to tissue cells and does not generate inflammatory reaction. The artificial cornea is of a double-layer structure, the upper layer is a loose layer and is easy to degrade, a movable and proliferation area is provided for epithelial cells, meanwhile, the artificial cornea is decomposed into micromolecular amino acids after degradation, nutritional ingredients are provided for tissue cells, the artificial cornea is also beneficial to rapidly completing epithelialization in lamellar transplantation operation, and the occurrence of infection caused by contact between a focus area and the outside is avoided.
3. The long-term stability and synergistic epithelialization promotion, wherein the lower layer is a compact layer, the degradation rate is low, and the cornea can be supported by a certain thickness and matched with other structures of the cornea to realize the refraction function. Meanwhile, the crosslinking strength is increased, so that the modified polyurethane can exist stably for a long time. The epidermal growth factor carried on the surface of the artificial cornea can cooperatively promote the rapid growth of corneal epithelial cells to finish epithelialization, ensure the isolation of the artificial cornea material main body from the external environment and avoid the adverse conditions of degradation, infection, inflammation and the like caused by the external environment to the material and surrounding tissues. Simple operation steps and convenient mass production.
The features and advantages of the present invention will be described in detail by way of example with reference to the accompanying drawings.
Detailed Description
The present invention will be further described in detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.
The invention relates to a preparation method of a collagen-based artificial cornea with a double-layer structure and capable of being rapidly epithelialized, which comprises the following steps:
S1, preparing a collagen solution, namely adding collagen into an acetic acid solution, stirring and dissolving, adding a plasticizer after the collagen is completely dissolved, stirring uniformly, and centrifugally defoaming to obtain a high-concentration collagen solution and a low-concentration collagen solution, wherein the concentration of the high-concentration collagen solution is 0.3% -0.5% w/v, and the concentration of the low-concentration collagen solution is 0.1% -0.25% w/v;
specifically, the collagen adopts type I collagen, the plasticizer is selected from any one or a combination of more of glycerol, polyethylene glycol, polypropylene glycol and citrate plasticizers, the concentration of the plasticizer is 10% -20% v/v, and the concentration of the acetic acid solution is 1% -3% v/v
S2, performing primary electrochemical deposition, namely performing electrochemical deposition on a high-concentration collagen solution, wherein a platinum mesh is connected with an anode, a titanium sheet is connected with a cathode, and an electrode plate is connected with a direct current power supply to obtain a collagen film I;
specifically, a direct current power supply selects a constant current mode 1-4A, and the deposition time is 30-60 min;
S3, performing primary ultraviolet crosslinking, namely performing dehydration treatment on the collagen film I by using absolute ethyl alcohol, and performing photocrosslinking by using strong ultraviolet irradiation;
specifically, the intensity of the strong ultraviolet light is 600-1000 mu w/cm2, and the time is 30-60 min;
s4, chemical crosslinking, namely soaking the collagen film I subjected to primary ultraviolet crosslinking treatment in the step 3 in a 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride solution, and performing chemical crosslinking to obtain a compact layer;
specifically, the concentration of the 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride solution is 0.01% -0.04% w/w, and the soaking time is 1-5 hours;
S5, secondary electrochemical deposition, namely connecting a titanium sheet deposited with a compact layer on a negative electrode, connecting a platinum net on a positive electrode, pouring a low-concentration collagen solution into a deposition tank, immersing the compact layer, and performing secondary electrochemical deposition, wherein a collagen film II is obtained on the compact layer;
specifically, the direct current power supply selects a constant current mode 1-4A, and the deposition time is 10-30 min.
S6, secondary ultraviolet crosslinking, namely, after the collagen film II is dehydrated by absolute ethyl alcohol, weak ultraviolet irradiation is carried out for secondary photocrosslinking to obtain a loose layer, and the loose layer and a compact layer are combined to form the collagen-based artificial cornea which has the double-layer structure and can be rapidly epithelialized, as shown in figure 5.
Wherein the intensity of the weak ultraviolet light is 10-200 mu w/cm2, and the time is 1-10 min.
The method also comprises a step S7 of cleaning the collagen-based artificial cornea which has the double-layer structure and can be rapidly epithelialized and is prepared in the step S6 through PBS and storing the collagen-based artificial cornea into PBS buffer solution containing recombinant human epidermal growth factor, wherein the concentration of the recombinant human epidermal growth factor is 0.01% -0.03% w/v.
Example 1
In this embodiment, a preparation method of an artificial cornea with a collagen-based bilayer structure is provided, which specifically includes the following steps:
(1) The preparation of the collagen solution comprises the steps of taking 1% v/v acetic acid solution as a solvent, preparing 0.3% w/v high-concentration collagen solution and 0.1% w/v low-concentration collagen solution, stirring and dissolving until no obvious massive substances exist in the solution, and adding 10% v/v plasticizer after the dissolution is completed and stirring uniformly.
(2) And (3) bubble removal, namely, carrying out centrifugal defoaming treatment on the collagen solution, wherein the centrifugal program is that the rotating speed is 8000rpm, and the time is 15min.
(3) And performing primary electrochemical deposition, namely performing electrochemical deposition on a 0.3% w/v high-concentration collagen solution at a low temperature of 4 ℃, connecting a platinum net with an anode, connecting a titanium sheet with a cathode, connecting an electrode plate with a direct current source, and performing electrochemical deposition to obtain a collagen film material on the titanium sheet, wherein the direct current source selects a constant current mode 1A, and the deposition time is 30min.
(4) And (3) performing primary ultraviolet crosslinking, namely soaking the collagen film on the titanium sheet in absolute ethyl alcohol for dehydration treatment, and performing ultraviolet crosslinking by irradiating 600 mu w/cm <2 > of ultraviolet light for 30 min.
(5) Chemical crosslinking, namely soaking the collagen membrane for 1h by using 0.01% w/w DMTMM solution, and performing chemical crosslinking to obtain a compact layer.
(6) And (3) secondary electrochemical deposition, namely connecting a titanium sheet deposited with a compact layer on a negative electrode in an 8 ℃ low-temperature environment, connecting a platinum net on a positive electrode, pouring a 0.1% w/v low-concentration collagen solution into a deposition tank, immersing the compact layer, performing secondary electrochemical deposition, and selecting a constant-current mode 1A by a direct-current power supply for 10min.
(7) And (3) secondary ultraviolet crosslinking, namely covering a new layer of collagen film on the compact layer, carrying out ethanol dehydration treatment, and irradiating 10min under 10 mu w/cm < 2> of ultraviolet light to carry out ultraviolet crosslinking.
(8) And (3) removing the film, namely soaking the titanium sheet and the collagen film in pure water together, and slowly stripping the collagen film from the titanium sheet after the collagen film is fully wetted to obtain the artificial cornea with a double-layer structure.
(9) PBS cleaning, namely immersing the artificial cornea in PBS buffer solution for vibration cleaning, and 1L PBS solution for vibration cleaning for 10min for 5 times.
(10) Preservation mode the prepared artificial cornea was preserved in PBS buffer containing 0.01% w/v rhEGF.
Example 2
In this embodiment, a preparation method of an artificial cornea with a collagen-based bilayer structure is provided, which specifically includes the following steps:
(1) The preparation of the collagen solution, namely, preparing a high-concentration collagen solution with the concentration of 0.38% w/v and a low-concentration collagen solution with the concentration of 0.12% w/v by taking a 2% v/v acetic acid solution as a solvent, stirring and dissolving until no obvious massive substances exist in the solution, and adding a 15% v/v plasticizer after the dissolution is completed and stirring uniformly.
(2) And (3) bubble removal, namely, carrying out centrifugal defoaming treatment on the collagen solution, wherein the centrifugal program is that the rotating speed is 8000rpm, and the time is 15min.
(3) And performing primary electrochemical deposition, namely performing electrochemical deposition on a 0.38% w/v high-concentration collagen solution at a low temperature of 5 ℃, connecting a platinum net with an anode, connecting a titanium sheet with a cathode, connecting an electrode plate with a direct current source, and performing electrochemical deposition to obtain a collagen film material on the titanium sheet, wherein the direct current source selects a constant current mode 2A, and the deposition time is 40min.
(4) And one-time ultraviolet crosslinking, namely immersing the collagen film on the titanium sheet in absolute ethyl alcohol for dehydration treatment, and irradiating 800 mu w/cm < 2 > of ultraviolet light for 40min.
(5) DMTMM crosslinking A0.02% w/w DMTMM solution soaked collagen membrane for 4h and chemically crosslinked to give a compact layer.
(6) And (3) secondary electrochemical deposition, namely connecting a titanium sheet deposited with a compact layer on a negative electrode in a low-temperature environment at 4 ℃, connecting a platinum net with a positive electrode, pouring 0.12% w/v low-concentration collagen solution into a deposition tank, immersing the compact layer, performing secondary electrochemical deposition, and selecting a constant-current mode 2A by a direct-current power supply for 15min.
(7) And (3) secondary ultraviolet crosslinking, namely covering a new layer of collagen film on the compact layer, carrying out ethanol dehydration treatment, and irradiating 2.5 min under 100 mu w/cm < 2 > of ultraviolet light to carry out ultraviolet crosslinking.
(8) And (3) removing the film, namely soaking the titanium sheet and the collagen film in pure water together, and slowly stripping the collagen film from the titanium sheet after the collagen film is fully wetted to obtain the artificial cornea with a double-layer structure.
(9) PBS cleaning, namely immersing the artificial cornea in PBS buffer solution for vibration cleaning, and performing vibration cleaning for 30min by using 3L PBS solution for 8 times.
(10) Preservation mode the prepared artificial cornea was preserved in PBS buffer containing 0.018% w/v rhEGF.
Example 3
In this embodiment, a preparation method of an artificial cornea with a collagen-based bilayer structure is provided, which specifically includes the following steps:
(1) The preparation of the collagen solution comprises the steps of taking 3% v/v acetic acid solution as a solvent, preparing 0.5% w/v high-concentration collagen solution and 0.25% w/v low-concentration collagen solution, stirring and dissolving until no obvious massive substances exist in the solution, and adding 20% v/v plasticizer after the dissolution is completed and stirring uniformly.
(2) And (3) bubble removal, namely, carrying out centrifugal defoaming treatment on the collagen solution, wherein the centrifugal program is that the rotating speed is 8000rpm, and the time is 15min.
(3) And performing primary electrochemical deposition, namely performing electrochemical deposition on a 0.5% w/v high-concentration collagen solution at a low temperature of 8 ℃, connecting a platinum net with an anode, connecting a titanium sheet with a cathode, connecting an electrode plate with a direct current source, and performing electrochemical deposition to obtain a collagen film material on the titanium sheet, wherein the direct current source selects a constant current mode 4A, and the deposition time is 60min.
(4) And one-time ultraviolet crosslinking, namely immersing the collagen film on the titanium sheet in absolute ethyl alcohol for dehydration treatment, and irradiating 1000 mu w/cm < 2 > of ultraviolet light for 60min.
(5) DMTMM crosslinking A0.04% w/w DMTMM solution soaked collagen membrane for 5h and chemically crosslinked to give a compact layer.
(6) And (3) secondary electrochemical deposition, namely connecting a titanium sheet deposited with a compact layer on a negative electrode in a low-temperature environment of 5 ℃, connecting a platinum net with a positive electrode, pouring 0.25% w/v low-concentration collagen solution into a deposition tank, immersing the compact layer, performing secondary electrochemical deposition, and selecting a constant-current mode 4A by a direct-current power supply for 30min.
(7) And (3) secondary ultraviolet crosslinking, namely covering a new layer of collagen film on the compact layer, carrying out ethanol dehydration treatment, and carrying out ultraviolet crosslinking by irradiating for 1min under 200 mu w/cm <2 > of ultraviolet light.
(8) And (3) removing the film, namely soaking the titanium sheet and the collagen film in pure water together, and slowly stripping the collagen film from the titanium sheet after the collagen film is fully wetted to obtain the artificial cornea with a double-layer structure.
(9) PBS cleaning, namely immersing the artificial cornea in PBS buffer solution for vibration cleaning, and 4L PBS solution for vibration cleaning for 60min for 10 times.
(10) Preservation mode the prepared artificial cornea was preserved in PBS buffer containing 0.03% w/v rhEGF.
Comparative example 1
This comparative example differs from example 2 only in that no plasticizer was added to the collagen solution.
Comparative example 2
This comparative example differs from example 2 only in that the artificial cornea was not photocrosslinked using ultraviolet light.
Comparative example 3
This comparative example differs from example 2 only in that the artificial cornea was not chemically crosslinked using DMTMM.
Comparative example 4
This comparative example differs from example 2 only in that no secondary electrochemical deposition was performed.
Experiment 1
The artificial cornea obtained in the above examples and comparative examples was subjected to light transmittance, moisture content, mechanical properties, swelling ratio and ion transmittance tests. Each group was tested in 5 replicates and averaged as follows:
1. Light transmittance is measured by taking a sample, wiping surface moisture by using absorbent paper, placing the sample at the center of a light transmittance meter, and selecting a 600nm wave band for measurement.
2. The water content is measured by taking a sample and adopting a second method drying method in four 0832 water measuring methods in pharmacopoeia (2020 edition).
3. Mechanical properties the width and thickness of the samples after special sample preparation are measured, and the samples are tested at room temperature and a stretching rate of 50 mm/min.
4. The swelling ratio is that the sample is weighed, the mass is recorded, then the sample is placed in a swelling tube, 20mL of solvent is added into the tube, the tube plug is covered tightly, the tube plug is placed in a constant temperature tank with the temperature of 25 ℃ plus or minus 0.1 ℃, the mass of the swelling body is weighed once every 3 hours by the same method, and the swelling body is considered to reach swelling balance when the difference between the two weighing results of the swelling body is not more than 0.019.
5. Osmotic Property the sample was held between an osmotic chamber (containing nutrient solution) and a receptor chamber (containing deionized water) and the solutions in the two chambers were then stirred evenly with an electromagnetic stirrer. The concentration of the nutrient solution (tryptophan solution) in the receptor chamber is then measured byThe osmotic coefficient is calculated, and the principle is shown in the figure 2:
Where P is the permeability coefficient, V and S represent the volume of solution in the chamber and the membrane area between the chambers, respectively, d represents the thickness of the wet sample, t is the diffusion time, C0 is the initial ion concentration of the permeation chamber, and C is the ion concentration of the acceptor chamber at the target times (1, 6, 12, 18, and 24 hours, respectively).
6. In-vitro enzymolysis performance under an ultra-clean workbench, 5mL of Tris-HCl solution is measured and filled into a 15mL centrifuge tube. The artificial cornea was grasped with forceps, placed in a sterile centrifuge tube containing 5mL Tris-HCl solution, sealed, and placed in a 37℃water bath for 1 hour. After the artificial cornea is finished, the artificial cornea is taken out again, the surface moisture is gently wiped off, and the artificial cornea is weighed as the original weight. And then placing mother liquor activated for 1 hour in a 37 ℃ water bath kettle into the centrifuge tube, placing 88.3 mu L of mother liquor in each tube, shaking, sealing, standing at 37 ℃, performing in-vitro enzymolysis experiment observation, changing enzyme liquor every 8 hours (1 hour before use) to keep enzyme activity, observing and recording the state and the residual weight of a sample until the enzymolysis of the sample is complete, and the degradation trend is shown in figure 3.
7. Artificial tear simulates in vitro degradation by weighing the artificial cornea as the original weight. Under an ultra-clean bench, 5mL of artificial tear was measured and loaded into a 15mL centrifuge tube. The artificial cornea is clamped by forceps, placed into the sterile centrifuge tube, sealed, placed into a shaking table with constant temperature of 37 ℃ for observation, and the state and the residual weight of the sample are recorded until the sample is completely degraded, and the degradation trend is shown in figure 4.
The results of the above test are shown in table 1 below.
TABLE 1
The result shows that the artificial cornea prepared by the method has the light transmittance of more than 90 percent at 600nm, the optical performance is excellent, the light blocking condition caused by a sample per se is avoided, the water content is more than 85 percent, the light transmittance is similar to that of a healthy human cornea, the tensile strength is more than 1.5MPa, the elastic modulus is higher than 10MPa, the mechanical performance is excellent, the swelling rate of the artificial cornea in examples 1-3 is lower than 15 percent, the nerve compression of the product on the autologous cornea can be avoided, the mechanical stimulation on eye tissues is reduced, the comfort after implantation is improved, meanwhile, the tryptophan permeability coefficient is similar to that of the healthy human cornea, the product can support the transmission of small molecular nutrients, the absorption of nutrients of surrounding tissues after operation is ensured, the wound repair is promoted, the period is about 80-100 days on the simulated degradation time of the artificial tears in vitro, the upper layer of the artificial cornea begins to degrade after one week, and the lower layer keeps the complete form unchanged. In-vitro enzymolysis time is more than 90 hours in the examples, and enzymolysis resistance of the comparative examples is obviously weakened, which proves that the crosslinking mode of the product has obvious effect of improving enzymolysis resistance. The upper layer of the double-layer structure is a loose layer under the simulated degradation of two conditions, the degradation is faster than that of a compact layer, the migration and proliferation of epithelial cells are facilitated, collagen of the loose layer is degraded into micromolecular amino acids, nutrition required by the cells is given, the epithelialization can be promoted, the rapid sealing and closing of cornea tissues are ensured, the subsequent infection caused by external bacteria entering the cornea tissues is avoided, the lower layer is the compact layer, the degradation rate is slower, and the collagen is used as tissue regeneration guide, so that a certain thickness support and light conduction performance can be provided for the cornea.
Experiment 2
Cytotoxicity test under aseptic conditions, the cornea of example 2 was taken, 15.00mL of a leaching medium (MEM (1 xMEM) of 10% fetal bovine serum) was added in a proportion of 6cm2/mL, and after 24 hours leaching at 37℃a stock solution was obtained, and before use, a cell suspension having a density of 1X 104 cells/mL was inoculated into each group of parallel wells by filtration through a 0.22 μm sterile filter, 100mL of the cell suspension was inoculated into each well, and cultured in an incubator containing 5% CO2 at 37℃for 24 hours, the stock solution was discarded after the completion of the culture, and the sample leaching solution was added and 100mL of each well was inoculated. Then, the culture was continued for 72 hours, and after the completion of the culture, the morphology of the cultured cells was observed under a microscope, and the result was shown in FIG. 1.
The result shows that the artificial cornea prepared by the method has good cell affinity and biocompatibility, small stimulation to cells and can ensure the normal growth of the cells.
Experiment 3
And (3) carrying out epithelialization observation on the rabbit cornea lamellar transplanting operation, namely selecting New Zealand rabbits with the weight of 2.5-3 kg for carrying out in-vivo animal experiments, carrying out local anesthesia by using obukaine hydrochloride eye drops after general anesthesia, and carrying out operation in a sterile environment. The beds having a depth of 150 to 200 μm were trephine-drilled at the center of rabbit cornea with 4.5 mm, and the cornea of example 2 and the cornea of comparative example 4 were taken out of the preservation solution at the time of surgical use and sutured with 10-0 surgical sutures, respectively. On the following day and week after surgery, the defect area was stained with fluorescein sodium ophthalmic paper wetted at the implantation site and examined for corneal epithelialization using cobalt blue slit lamp fluorescence staining photography, see fig. 6.
The experimental result shows that the artificial cornea with the double-layer structure prepared by the method has higher epithelial rate, on one hand, the release of rhEGF has very remarkable promotion effect on the healing of the corneal epithelial injury in clinical experiments, and on the other hand, the upper layer of the implant is thinner and loose, so that the material is more beneficial to cell adhesion, can promote the migration of epithelial cells, and further promote the repair process of epithelialization. Other biosynthesis artificial cornea has only one layer of compact surface structure on structural design, the smooth surface is unfavorable for cell climbing, the epithelial rate is slower, and finally the clinical effect is not satisfied.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.