阅读说明：本技术 一种人工角膜 (Artificial cornea ) 是由 陈百华 韩凯 彭立 马巍 彭晗晗 闫滨 于 2021-06-03 设计创作，主要内容包括：本发明提供了一种人工角膜,包括中央光学部位与裙边；所述中央光学部位包括相连接的圆柱体与伞状部位；所述伞状部位的直径大于圆柱体；所述裙边套于所述圆柱体外；所述裙边由复合材料制备得到；所述复合材料包括氧化石墨烯与碳化二铌。与现有技术相比,本发明以氧化石墨烯与碳化二铌复合材料制备裙边,其具有良好的亲水性、生物相容性、较好的孔隙率、易于修饰和功能化,并且也具有高强度的韧性与耐腐蚀性,使人工角膜与受体角膜组织具有良好的相容性,减少了人工角膜相关并发症的发生,提高了其应用安全性,降低了角膜疾病的致盲率。(The invention provides an artificial cornea, which comprises a central optical part and a skirt edge; the central optical part comprises a cylinder and an umbrella-shaped part which are connected; the diameter of the umbrella-shaped part is larger than that of the cylinder; the skirt edge is sleeved outside the cylinder; the skirt is prepared from a composite material; the composite material comprises graphene oxide and niobium carbide. Compared with the prior art, the skirt edge is prepared from the graphene oxide and niobium carbide composite material, has good hydrophilicity, biocompatibility and porosity, is easy to modify and functionalize, and also has high-strength toughness and corrosion resistance, so that the artificial cornea has good compatibility with receptor corneal tissues, the occurrence of complications related to the artificial cornea is reduced, the application safety of the artificial cornea is improved, and the blindness rate of corneal diseases is reduced.)

1. An artificial cornea comprising a central optical portion and a skirt; the central optical part comprises a cylinder and an umbrella-shaped part which are connected; the diameter of the umbrella-shaped part is larger than that of the cylinder; the skirt edge is sleeved outside the cylinder;

the skirt is prepared from a composite material; the composite material comprises graphene oxide and niobium carbide.

2. The keratoprosthesis of claim 1, wherein the niobium carbide is formed from niobium aluminum carbide by hydrofluoric acid etching to remove aluminum atoms.

3. The keratoprosthesis of claim 2, wherein the niobium carbide is prepared by the following method:

reacting niobium aluminum carbide with hydrofluoric acid, washing, centrifuging, and ultrasonically stripping to obtain niobium carbide.

4. The artificial cornea of claim 1, wherein the mass ratio of graphene oxide to niobium carbide is 1: (1.5-2.5).

5. The artificial cornea of claim 1, wherein the mass ratio of graphene oxide to niobium carbide is 1: 2.

6. the keratoprosthesis of claim 1, wherein a side of the skirt opposite the umbrella is coated with a nanocoating.

7. The artificial cornea of claim 6, wherein the thickness of the nanocoating is 100-300 nm.

8. The keratoprosthesis of claim 1, wherein a side of the skirt not opposite the umbrella is provided with a vascular endothelial growth factor coating.

9. The artificial cornea of claim 8, wherein said vascular endothelial growth factor coating has a thickness of 100-300 nm.

10. The keratoprosthesis of claim 1, wherein the cylinder is provided with a thread; the skirt is sleeved outside the cylinder body through threads.

Technical Field

The invention belongs to the technical field of medical instruments, and particularly relates to an artificial cornea.

Background

The cornea blindness is the second most serious eye disease next to cataract, about 400 ten thousand corneal blindness are currently seen in China, and 10 ten thousand cases are newly found each year, and about 5000 patients who obtain corneal transplantation operation each year are in shortage of cornea donors. In addition, a great number of patients with corneal blindness are accompanied by serious dry eye, serious burn, serious immune diseases, multiple times of corneal transplantation operation failure and other pathological changes, even the transplantation of a common fresh corneal donor from the thousands of bitter leaves and the like is not helpful for the patients, the transplantation of the artificial cornea is often failed due to serious rejection, and the artificial cornea transplantation is the only effective way for the patient to be remitted.

However, at present, there is no such product at home, and although there are some artificial cornea products at home and abroad, such as boston artificial cornea (also known as Dohlman-duane), bone-tooth artificial cornea, russian Moroz artificial cornea, etc., these products all need to use donor cornea as skirt, which is not practical for countries such as China where the cornea donor is seriously in short supply, and moreover, such cornea has a great deal of serious complications, such as endophthalmitis, wound leakage, glaucoma, retrobulbar cornea, etc., and thus its long-term effect is not ideal, and thus it is not widely popularized and applied.

The artificial cornea is composed of two parts, namely a central optical part and a skirt connected with receptor tissues, so that the application of the artificial cornea is limited and the complications are more caused mainly because the skirt material has insufficient biocompatibility with a central lens column and the receptor tissues, and the material has defects of hydrophilicity, porosity and the like.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide an artificial cornea with good biocompatibility.

The invention provides an artificial cornea which is characterized by comprising a central optical part and a skirt edge; the central optical part comprises a cylinder and an umbrella-shaped part which are connected; the diameter of the umbrella-shaped part is larger than that of the cylinder; the skirt edge is sleeved outside the cylinder;

the skirt is prepared from a composite material; the composite material comprises graphene oxide and niobium carbide.

Preferably, the niobium carbide is obtained by etching niobium aluminum carbide with hydrofluoric acid to remove aluminum atoms.

Preferably, the niobium carbide is prepared by the following method:

reacting niobium aluminum carbide with hydrofluoric acid, washing, centrifuging, and ultrasonically stripping to obtain niobium carbide.

Preferably, the mass ratio of the graphene oxide to the niobium carbide is 1: (1.5-2.5).

Preferably, the mass ratio of the graphene oxide to the niobium carbide is 1: 2.

preferably, the side of the skirt edge opposite to the umbrella-shaped part is coated with a nano coating.

Preferably, the thickness of the nano coating is 100-300 nm.

Preferably, the side of the skirt edge, which is not opposite to the umbrella-shaped part, is provided with a vascular endothelial growth factor coating.

Preferably, the thickness of the vascular endothelial growth factor coating is 100-300 nm.

Preferably, the cylinder is provided with a thread; the skirt is sleeved outside the cylinder body through threads.

The invention provides an artificial cornea, which comprises a central optical part and a skirt edge; the central optical part comprises a cylinder and an umbrella-shaped part which are connected; the diameter of the umbrella-shaped part is larger than that of the cylinder; the skirt edge is sleeved outside the cylinder; the skirt is prepared from a composite material; the composite material comprises graphene oxide and niobium carbide. Compared with the prior art, the skirt edge is prepared from the graphene oxide and niobium carbide composite material, has good hydrophilicity, biocompatibility and porosity, is easy to modify and functionalize, and also has high-strength toughness and corrosion resistance, so that the artificial cornea and receptor corneal tissue have good compatibility, the occurrence of complications related to the artificial cornea is reduced, the application safety of the artificial cornea is improved, and the blindness rate of corneal diseases is reduced.

Drawings

FIG. 1 is a schematic diagram of the central optical portion of a keratoprosthesis according to the present invention;

FIG. 2 is a schematic diagram of the structure of the skirt of the artificial cornea provided by the present invention;

FIG. 3 is a diagram showing the results of the detection of Calcein/PI cell activity and cytotoxicity in example 1 of the present invention;

FIG. 4 is a graph showing the results of apoptosis detection in example 1 of the present invention;

FIG. 5 is a graph showing the results of the proliferation assay of EdU-488 cells according to example 1 of the present invention;

FIG. 6 is a scanning electron microscope image of the disc, i.e., skirt of artificial cornea, obtained in example 1 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides an artificial cornea, which comprises a central optical part and a skirt edge; the central optical part comprises a cylinder and an umbrella-shaped part which are connected; the diameter of the umbrella-shaped part is larger than that of the cylinder; the skirt edge is sleeved outside the cylinder; the skirt is prepared from a composite material; the composite material comprises graphene oxide and niobium carbide.

Referring to fig. 1 and 2, fig. 1 is a schematic view of a central optical portion of an artificial cornea provided by the present invention, wherein 1 is an umbrella-shaped portion, 2 is a cylinder, and 3 is a thread structure; fig. 2 is a schematic structural diagram of a skirt of an artificial cornea provided by the present invention.

The artificial cornea provided by the invention comprises a central optical part; the central optical part comprises a cylinder and an umbrella-shaped part which are connected; the diameter of the cylinder is preferably 2-4 mm, more preferably 2.5-3.5 mm, and further preferably 3 mm; the diameter of the umbrella-shaped part is larger than that of the cylinder, and the diameter of the umbrella-shaped part is preferably 6-10 mm; the thickness of the center of the umbrella-shaped part is preferably 0.5 mm-1.5 mm; the thickness of the edge of the umbrella-shaped part is preferably 0.5 mm-1 mm; the material of the central optical part is preferably one or more of polyhydroxyethyl methacrylate, polyhydroxymethyl methacrylate, polyvinyl alcohol, silica gel polymer and polyethylene glycol diacrylate.

The cylinder is sleeved with a skirt edge; the cylinder and the surface of the skirt edge, which is in contact with the cylinder, are preferably provided with threads; the skirt edge is sleeved outside the cylinder body through threads; the thickness of the skirt is preferably 2-6 mm, more preferably 2.5-5.5 mm, and still more preferably 5 mm.

In the invention, the skirt is made of a composite material; the composite material comprises graphene oxide and niobium carbide.

The graphene oxide is preferably prepared by a modified Hummers method, and more preferably is prepared according to the following steps: pre-oxidizing graphite, deeply oxidizing, centrifugally washing, and ultrasonically stripping to obtain graphene oxide; the pre-oxidation adopts potassium persulfate and phosphorus pentoxide; the mass ratio of the graphite to the graphite is 2.5:2.5: 3.0; the pre-oxidation temperature is 35 ℃, and the duration is 4 hours; the deep oxidation oxidant is potassium persulfate and phosphorus pentoxide; the mass ratio of the addition amount to the graphite is 2.5:2.5: 3; the deep oxidation temperature is preferably 80 ℃, and the duration is 1 hour; the rotating speed of the centrifugation is 2500 r.min^-1For 30 minutes; the solvent adopted for washing is a 1:10 HCl solution; the power and time of ultrasonic stripping were 150W and 40 minutes, respectively.

The niobium carbide is preferably obtained by removing aluminum atoms from niobium aluminum carbide through hydrofluoric acid etching, and is more preferably prepared according to the following steps: reacting niobium aluminum carbide with hydrofluoric acid, washing, centrifuging, and ultrasonically stripping to obtain niobium carbide; the grain size of the niobium aluminum carbide is preferably 0.01-0.1 mm, more preferably 0.01-0.08 mm, and still more preferably 0.01-0.08 mm-0.06 mm, more preferably 0.02-0.05 mm, most preferably 0.02 mm; the concentration of the hydrofluoric acid is preferably 1-1.5 g/mL, more preferably 1-1.3 g/mL, still more preferably 1-1.2 g/mL, and most preferably 1.15 g/mL; the mass ratio of the niobium aluminum carbide to the hydrofluoric acid is preferably 1: (1-5), more preferably 1: (1-3), and preferably 1: 2; the reaction temperature is preferably 70-90 ℃; the reaction time is preferably 1-3 hours; the solvent used for washing is ethanol; the centrifugal rotating speed is 2000-3000 r.min^-1More preferably 2500 r.min^-1(ii) a The centrifugation time is preferably 20-40 min, and more preferably 30 min; the power of the ultrasonic stripping is preferably 100-200W, and more preferably 150W; the ultrasonic stripping time is 30-50 min, more preferably 35-45 min, and still more preferably 40 min.

The mass ratio of graphene oxide to niobium carbide in the composite material is preferably 1: (1.5-2.5), more preferably 1: 2; the composite material is preferably prepared according to the following steps: mixing graphene oxide and niobium carbide in water, and performing ultrasonic treatment to obtain a composite material; the mass ratio of the graphene oxide to the water is preferably 1: 2; the power of the ultrasonic wave is preferably 100-200W, and more preferably 150W; the ultrasonic time is preferably 30-60 min, and more preferably 40-50 min.

Filtering and molding the composite material in a mold, and calcining to obtain a skirt; the calcination temperature is preferably 150-250 ℃, and more preferably 200 ℃; the calcination time is preferably 0.5-2 h, more preferably 0.5-1.5 h, and still more preferably 1 h.

According to the invention, one surface of the skirt edge, which is opposite to the umbrella-shaped part, is coated with a nano coating; the thickness of the nano coating is preferably 100-300 nm, more preferably 150-250 nm, and further preferably 200 nm.

According to the invention, the side of the skirt edge, which is not opposite to the umbrella-shaped part, is preferably provided with a vascular endothelial growth factor coating; the thickness of the vascular endothelial growth factor coating is preferably 100-300 nm, more preferably 150-250 nm, and further preferably 200 nm.

In order to further illustrate the present invention, a keratoprosthesis provided by the present invention is described in detail below with reference to examples.

The reagents used in the following examples are all commercially available.

Example 1

1.1 preparing graphene oxide by an improved Hummers method, pre-oxidation, deep oxidation, centrifugal washing and ultrasonic stripping. The pre-oxidation adopts potassium persulfate and phosphorus pentoxide; the mass ratio of the graphite to the graphite is 2.5:2.5: 3.0; the pre-oxidation temperature is 35 ℃, and the time duration is 4 hours; the deep oxidation oxidant is potassium persulfate and phosphorus pentoxide; the mass ratio of the addition amount to the graphite is 2.5:2.5: 3; the deep oxidation is carried out at the temperature of 80 ℃ for 1 hour; the rotating speed of the centrifugation is 2500 r.min^-1Time is 30 minutes; the solvent adopted for washing is a 1:10 HCl solution; the power and time of ultrasonic stripping are 150W and 40 minutes respectively

1.2 niobium aluminum carbide with the grain diameter of 0.02mm is taken as a raw material, and the mass ratio of the niobium aluminum carbide to the niobium aluminum carbide is 1:2, reacting the mixture with hydrofluoric acid with the concentration of 1.15g/mL for 2 hours to remove aluminum atoms, washing with ethanol for 2500 r.min^-1Centrifuging for 30min, and ultrasonic stripping for 40min at 150W to prepare the two-dimensional niobium carbide.

1.3, mixing graphene oxide (50 wt%) and niobium carbide suspension (50 wt%) according to a mass ratio of 1:2, and carrying out ultrasonic treatment for 50min at 150W to obtain the composite material.

1.4 preparation parameters: injecting the mixed material into a mold, filtering, molding, and calcining at 200 ℃ for 1h to obtain discs of three specifications, wherein the discs have the thickness of 100 micrometers and the diameter of 4-8 mm; a disk with a thickness of 200 μm and a diameter of 4-8 mm; a disk with a thickness of 300 μm and a diameter of 4-8 mm.

And (3) constructing the artificial cornea skirt composite material through surface modification.

Respectively modifying a coating VEGF200nm on the front surface of the artificial cornea skirt composite material (VEGF powder of lug/mL is diluted to 20.0mL by double distilled water and prepared into a VEGF solution with the concentration of 50ng/mL to prepare a coating), and modifying a coating TA200nm (triamcinolone acetonide powder, polyglycolic acid-polyglycolic acid PLLGA, the components are ground to be in a fine particle shape, and the significance of the coating is that the ratio of 200mg of the triamcinolone acetonide powder to 800mg of the polyglycolic acid-polyglycolic acid PLLGA and the triamcinolone acetonide powder are dissolved in 20mL of Tetrahydrofuran (THF) of a common solvent): the front coating reduces cell proliferation and the back coating reduces fibrous membrane hyperplasia.

And (3) covering the disc prepared in the step (1.4) with a coating, soaking in ethanol for 24 hours, baking in vacuum, and then taking the disc as a sample to perform cell experiments to prove that the sample has low cytotoxicity and no influence on cell proliferation and apoptosis.

Cell culture conditions and grouping

1) Cell culture: human corneal stromal cells (HK) were cultured in special complete medium and 10% FBS + 1% double antibody DMEM medium at 37 deg.C and 5% CO₂And culturing in a saturated humidity incubator.

2) Sample set samples were placed in 24-well plates and log-grown HK cells were taken at 2 × 10⁵And (4) inoculating the cells.

3) Control group: HK cells were not treated at all.

Calcein/PI cell activity and cytotoxicity detection and test steps

Washing: absorbing the culture solution, and washing with PBS for 2 times;

dyeing, namely adding Calcein AM/PI detection working solution with proper volume, and incubating for 30min at 37 ℃ in the dark;

detecting: after the incubation, the cells were detected by a luciferase reader (Calcein AM: green fluorescence, Ex/Em: 494/517 nm; PI: red fluorescence, Ex/Em: 535/617 nm). By comparing the RFU (relative fluorescence values) of the control group and the treated group, the change in the number of dead cells and living cells can be found.

And fourthly, removing the dye solution, washing the dye solution for 3 times by PBS, and then taking a picture under a microscope to obtain a microscope picture as shown in figure 3.

Apoptosis detection procedure

Firstly, digesting and collecting the treated cells by using pancreatin without EDTA;

② washing the cells with PBS for 2 times, centrifuging for 5min at 1000g each time, and collecting the cells;

③ adding 195ul Annexin V-FITC binding solution to gently resuspend the cells;

adding 5ul Annexin V-FITC, mixing uniformly, adding 10ul propidium iodide staining solution, and mixing lightly;

fifthly, reacting for 10min at room temperature in a dark place;

sixthly, within 1h, observing and detecting by a flow cytometer to obtain a picture as shown in figure 4.

EdU-488 cell proliferation assay

EdU tag

1.1 culturing cells in a cell culture medium according to a ratio of 1000: the EdU solution (reagent A) was diluted at a ratio of 1 to prepare an appropriate amount of 50. mu.M EdU medium.

1.2 adding 100 μ L of 50 μ M EDU culture medium into each well, incubating overnight, and removing the culture medium;

1.3PBS washing cells 1 ~ 2 times, each time 5 minutes.

2. Immobilization of cells

2.1 adding 50 μ L of cell fixing solution (4% paraformaldehyde) into each well, incubating at room temperature for 30min, and removing the fixing solution;

2.2 adding 50 mu L of 2mg/mL glycine into each hole, and after incubating for 5 minutes by a decoloring shaker, discarding the glycine solution;

2.3 adding 100 mul PBS into each hole, washing for 5 minutes by a decoloring shaker, and discarding the PBS;

2.4 adding 100 mul of penetrant into each hole, decoloring and shaking for 10 minutes; PBS wash 1 times, 5 minutes.

Apollo staining

3.1 Add 100. mu.L of 1X per wellIncubating the dyeing reaction liquid in a dark place at room temperature for 30 minutes by a decoloring shaking bed, and then discarding the dyeing reaction liquid;

3.2 adding 100 mu L of penetrant, decoloring and shaking the mixture for 2-3 times, and removing the penetrant after 10 minutes each time;

3.3 adding 100 mu L of methanol into each hole for cleaning for 1-2 times, 5 minutes each time; PBS wash was performed 1 time for 5 minutes each.

DNA staining

4.1 adding deionized water according to the weight ratio of 100: 1, preparing a proper amount of 1x Hoechst33342 reaction solution, and storing in a dark place;

4.2 adding 100 μ L of 1x Hoechst33342 reaction solution into each hole, incubating for 30 minutes in a dark place at room temperature and a decoloring shaker, and then discarding the dyeing reaction solution;

4.3 adding 100 mu L PBS to each hole for washing for 1-3 times;

5. image acquisition and analysis: immediately after the completion of staining, the staining pattern was observed as shown in FIG. 5.

The disc obtained in 1.4 was analyzed by an electron scanning microscope, and a scanning electron micrograph thereof is shown in FIG. 6.