Decellularized porcine cornea in-situ cross-linked molded lens implant for keratoconus operation and preparation method thereof

Abstract

The invention relates to the technical field of ophthalmic material preparation, and particularly discloses a decellularized porcine cornea in-situ cross-linked molded lens implant for keratoconus surgery and a preparation method thereof. The preparation method comprises the following steps: (1) cleaning a fresh porcine cornea, removing epithelium, and then performing decellularization treatment to obtain a decellularized porcine cornea; (2) soaking the decellularized porcine cornea in a mixed solution containing riboflavin and dextran; after soaking is finished, the decellularized porcine cornea is taken out and subjected to in-situ cross-linking through ultraviolet irradiation, and after cross-linking is finished, the decellularized porcine cornea in-situ cross-linking forming lens implant for the keratoconus operation is obtained. According to the method, a brand new implant is prepared, and the donor source of corneal transplantation is expanded; besides, the inventor also finds that the lens implant prepared by carrying out in-situ crosslinking on the decellularized porcine cornea and riboflavin also has an antibacterial effect and an important application value in research.

Description

Technical Field

[0001] The present invention relates to the technical field of ophthalmic material preparation, and particularly relates to a decellularized porcine cornea in-situ crosslinked and molded lens implant for keratoconus surgery and a preparation method thereof. Background Art

[0002] Keratoconus is a non-inflammatory, progressive corneal ectasia disease with an incidence of about 1 / 2000. It mostly occurs during adolescence and gradually stabilizes around the age of 30. Its characteristic is that the central or paracentral part of the cornea thins and expands, protruding forward in a conical shape, often causing highly irregular astigmatism. In the late stage, due to corneal scar formation, vision significantly decreases or even leads to blindness.

[0003] Before the emergence of corneal collagen crosslinking surgery, in the early stage of keratoconus, vision was mainly corrected by wearing frame glasses or corneal contact lenses, or corneal stromal rings were implanted to improve vision; in the late stage, patients with corneal scar formation needed corneal transplantation, including penetrating keratoplasty or deep anterior lamellar keratoplasty. However, the source of corneal materials is limited, only some patients can receive corneal transplantation surgery, and the surgery cost is high, the surgical skills of doctors are required to be high, and there are also complications after the surgery, making the surgical effect unsatisfactory.

[0004] To break through the donor limitation, bioderived corneal substitute materials have become a research hotspot, mainly including two directions: decellularized corneal matrix (DCM) and synthetic polymer composite materials. Decellularized corneal matrix refers to removing the cellular components in xenogeneic or allogeneic corneas through chemical or enzymatic treatment, and retaining the natural collagen fiber structure and bioactive components. Porcine corneas have become the main raw materials because of their highly similar anatomical structure to humans (the difference in curvature radius <5%) and rich sources. In 2003, Wollensak et al. proposed corneal collagen crosslinking surgery. This surgery uses riboflavin to generate singlet oxygen and reactive oxygen under ultraviolet light irradiation at a wavelength of 370 nm, inducing chemical crosslinking between corneal stromal collagen fiber molecules and increasing the biomechanical strength of the cornea. The emergence of this surgery has significantly reduced the need for corneal transplantation. After the surgery, the corneal refractive power of some patients has decreased, the corneal topographic parameters have improved, and the vision has been improved to a certain extent, and the clinical effect is good. However, there is no research report in the prior art on in-situ crosslinking using decellularized porcine corneas and riboflavin. Summary of the Invention

[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a decellularized porcine cornea in-situ crosslinked and molded lens implant for keratoconus surgery and a preparation method thereof.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention first provides a method for preparing an acellular porcine corneal in-situ crosslinked formed lens implant for keratoconus surgery, which comprises the following steps:

[0008] (1) After taking a fresh porcine cornea and cleaning it, the epithelium is removed, and then acellular treatment is carried out to obtain an acellular porcine cornea;

[0009] (2) The acellular porcine cornea is immersed in a mixed solution containing riboflavin and dextran; after the immersion ends, the acellular porcine cornea is taken out and irradiated with ultraviolet light for in-situ crosslinking, and after the crosslinking is completed, the acellular porcine corneal in-situ crosslinked formed lens implant for keratoconus surgery is obtained.

[0010] The present invention provides a completely new method for in-situ crosslinking an acellular porcine cornea with riboflavin to prepare a lens implant, and a completely new implant is prepared, which expands the donor source for corneal transplantation; it has important application value.

[0011] In addition, the inventors also found in the research that the lens implant prepared by in-situ crosslinking an acellular porcine cornea with riboflavin also has an antibacterial effect, especially has a certain effect against methicillin-resistant Staphylococcus aureus (MRSA).

[0012] Preferably, the cleaning in step (1) specifically refers to cleaning with physiological saline.

[0013] Preferably, the specific method for carrying out acellular treatment in step (1) is: putting the cleaned fresh porcine cornea into a solution containing a detergent and incubating it at 1-5°C for 8-24 h.

[0014] Preferably, the solution containing a detergent refers to an aqueous solution containing a detergent; the detergent is Triton X-100.

[0015] Preferably, the mass concentration of the detergent in the solution containing a detergent is 0.1%-1%.

[0016] Preferably, the mass concentration of riboflavin in the mixed solution containing riboflavin and dextran is 0.1-1%, and the mass concentration of dextran is 3-6%.

[0017] Most preferably, the mass concentration of riboflavin in the mixed solution containing riboflavin and dextran is 0.5%, and the mass concentration of dextran is 5%.

[0018] Preferably, the mixed solution refers to a mixed aqueous solution.

[0019] Preferably, the mixed solution containing riboflavin and dextran also contains saffron extract.

[0020] In further research, the inventors found that after adding saffron extract to a mixed solution containing riboflavin and dextran, the lens implant obtained by cross-linking with acellular porcine cornea had a significantly improved anti-Methicillin-resistant Staphylococcus aureus (MRSA) effect compared to the lens implant obtained by cross-linking acellular porcine cornea without adding saffron extract.

[0021] The saffron extract described above is prepared by the following method:

[0022] Take traditional Chinese medicine saffron, heat it to boiling with ether and reflux for 1 h. After the extraction is completed, take the extract and concentrate and dry it to obtain the saffron extract.

[0023] In further research, the inventors found that the selection of the extraction solvent for saffron extract is crucial; only the saffron extract obtained by using ether as an organic solvent can further significantly improve the anti-MRSA effect of the lens implant obtained by cross-linking; however, the saffron extract obtained by using other organic solvents cannot further significantly improve the anti-MRSA effect of the lens implant obtained by cross-linking.

[0024] Among them, the dosage ratio of traditional Chinese medicine saffron to ether is 1 kg: 8-20 L.

[0025] Most preferably, the dosage ratio of traditional Chinese medicine saffron to ether is 1 kg: 12 L.

[0026] Preferably, the mass concentration of saffron extract in the mixed solution containing riboflavin and dextran is 1-3%.

[0027] Most preferably, the mass concentration of saffron extract in the mixed solution containing riboflavin and dextran is 2%.

[0028] Preferably, the specific conditions for ultraviolet light irradiation are: wavelength is 360-380 nm, intensity is 4-6 mW / cm 2 , total energy is 4-8 J / cm 2 , irradiation time is 20-40 min.

[0029] Preferably, the specific conditions for ultraviolet light irradiation are: wavelength is 370 nm, intensity is 5 mW / cm 2 , total energy is 6 J / cm 2 , irradiation time is 30 min.

[0030] The present invention also provides a lens implant for in-situ cross-linking molding of acellular porcine cornea for keratoconus surgery prepared by the above preparation method.

[0031] Beneficial effects: The present invention provides a novel preparation method for an acellular porcine cornea in-situ crosslinked molded lens implant for keratoconus surgery; the method prepares a novel implant, expanding the donor source for corneal transplantation; it has important application value. In addition, the inventors also found in the research that the lens implant prepared by in-situ crosslinking of acellular porcine cornea and riboflavin also has antibacterial effects, especially has a certain effect against methicillin-resistant Staphylococcus aureus (MRSA). Detailed implementation manners

[0032] The following specific examples are used to further explain the present invention, but the examples do not limit the present invention in any form.

[0033] Example 1 Preparation method for an acellular porcine cornea in-situ crosslinked molded lens implant for keratoconus surgery

[0034] (1) After taking a fresh porcine cornea and washing it with physiological saline, the epithelium is removed, and then acellular treatment is carried out to obtain an acellular porcine cornea; the specific method for carrying out acellular treatment is: putting the washed fresh porcine cornea into an aqueous solution containing 0.5% (mass concentration) of Triton X-100 and incubating it at 4°C for 12 h;

[0035] (2) The acellular porcine cornea is put into a mixed aqueous solution containing riboflavin and dextran and soaked for 30 min; after the soaking is completed, the acellular porcine cornea is taken out and irradiated with ultraviolet light for in-situ crosslinking, and the acellular porcine cornea in-situ crosslinked molded lens implant for keratoconus surgery is obtained after the crosslinking is completed;

[0036] The specific conditions for the ultraviolet light irradiation in step (2) are: wavelength is 370 nm, intensity is 5 mW / cm 2 , total energy is 6 J / cm 2 , irradiation time is 30 min; the mass concentration of riboflavin in the mixed aqueous solution containing riboflavin and dextran is 0.5%, and the mass concentration of dextran is 5%.

[0037] Example 2 Preparation method for an acellular porcine cornea in-situ crosslinked molded lens implant for keratoconus surgery

[0038] (1) After taking a fresh porcine cornea and washing it with physiological saline, the epithelium is removed, and then acellular treatment is carried out to obtain an acellular porcine cornea; the specific method for carrying out acellular treatment is: putting the washed fresh porcine cornea into an aqueous solution containing 0.5% (mass concentration) of Triton X-100 and incubating it at 4°C for 12 h;

[0039] (2) Immerse the decellularized porcine cornea in a mixed aqueous solution containing riboflavin, dextran, and saffron extract for 30 minutes; after the immersion, take out the decellularized porcine cornea and perform in-situ crosslinking by ultraviolet light irradiation. After the crosslinking is completed, the in-situ crosslinked and shaped lens implant for keratoconus surgery is obtained;

[0040] The specific conditions of the ultraviolet light irradiation in step (2) are: wavelength is 370 nm, intensity is 5 mW / cm 2 , total energy is 6 J / cm 2 , irradiation time is 30 minutes; in the mixed aqueous solution containing riboflavin, dextran, and saffron extract, the mass concentration of riboflavin is 0.5%, the mass concentration of dextran is 5%, and the mass concentration of saffron extract is 2%;

[0041] The saffron extract is prepared by the following method: Take traditional Chinese medicine saffron, heat it to boiling with ether and reflux extract for 1 hour. After the extraction, take the extract and concentrate and dry it to obtain the saffron extract. Among them, the dosage ratio of traditional Chinese medicine saffron to ether is 1 kg:12 L.

[0042] Preparation method of in-situ crosslinked and shaped lens implant for keratoconus surgery in Comparative Example 1

[0043] (1) After taking a fresh porcine cornea and washing it with physiological saline, remove the epithelium, and then perform decellularization treatment to obtain a decellularized porcine cornea; the specific method of the decellularization treatment is: put the washed fresh porcine cornea into an aqueous solution containing 0.5% Triton X-100 and incubate at 4°C for 12 hours;

[0044] (2) Immerse the decellularized porcine cornea in a mixed aqueous solution containing riboflavin, dextran, and saffron extract for 30 minutes; after the immersion, take out the decellularized porcine cornea and perform in-situ crosslinking by ultraviolet light irradiation. After the crosslinking is completed, the in-situ crosslinked and shaped lens implant for keratoconus surgery is obtained;

[0045] The specific conditions of the ultraviolet light irradiation in step (2) are: wavelength is 370 nm, intensity is 5 mW / cm 2 , total energy is 6 J / cm 2 , irradiation time is 30 minutes; in the mixed aqueous solution containing riboflavin, dextran, and saffron extract, the mass concentration of riboflavin is 0.5%, the mass concentration of dextran is 5%, and the mass concentration of saffron extract is 2%;

[0046] The saffron extract is prepared by the following method: Take traditional Chinese medicine saffron, heat it with methanol to boiling and then reflux for 1 h. After the extraction is completed, take the extract and concentrate and dry it to obtain the saffron extract. Among them, the dosage ratio of traditional Chinese medicine saffron to methanol is 1 kg:12 L.

[0047] The difference between Comparative Example 1 and Example 2 lies in the different selection of the extraction solvent for saffron.

[0048] Preparation method of acellular porcine corneal in-situ crosslinked molding lens implant for keratoconus surgery in Comparative Example 2

[0049] (1) Take fresh porcine cornea, wash it with normal saline, remove the epithelium, and then perform acellular treatment to obtain acellular porcine cornea; the specific method for performing acellular treatment is: put the washed fresh porcine cornea into an aqueous solution containing 0.5% (mass concentration) of Triton X-100 and incubate at 4 °C for 12 h;

[0050] (2) Immerse the acellular porcine cornea in a mixed aqueous solution containing riboflavin, dextran and saffron extract for 30 min; after the immersion is completed, take out the acellular porcine cornea and irradiate it with ultraviolet light for in-situ crosslinking. After the crosslinking is completed, the acellular porcine corneal in-situ crosslinked molding lens implant for keratoconus surgery is obtained;

[0051] The specific conditions of the ultraviolet light irradiation in step (2) are: wavelength is 370 nm, intensity is 5 mW / cm 2 , total energy is 6 J / cm 2 , irradiation time is 30 min; in the mixed aqueous solution containing riboflavin, dextran and saffron extract, the mass concentration of riboflavin is 0.5%, the mass concentration of dextran is 5%, and the mass concentration of saffron extract is 2%;

[0052] The saffron extract is prepared by the following method: Take traditional Chinese medicine saffron, heat it with acetone to boiling and then reflux for 1 h. After the extraction is completed, take the extract and concentrate and dry it to obtain the saffron extract. Among them, the dosage ratio of traditional Chinese medicine saffron to acetone is 1 kg:12 L.

[0053] The difference between Comparative Example 2 and Example 2 lies in the different selection of the extraction solvent for saffron.

[0054] The antibacterial rates of the acellular porcine corneal in-situ crosslinked molding lens implants prepared in Examples 1-2 and Comparative Examples 1-2 against methicillin-resistant Staphylococcus aureus (MRSA) are shown in Table 1.

[0055] Table 1.

[0056] MRSA antibacterial rate Lens implant prepared in Example 1 46.8％ Lens implant prepared in Example 2 95.2％ Lens implant prepared in Comparative Example 1 62.4％ Lens implant prepared in Comparative Example 2 67.9％

[0057] As can be seen from the experimental data in Table 1, the antibacterial rate of the lens implant prepared in Example 2 against methicillin-resistant Staphylococcus aureus (MRSA) reached 95.2%, which was significantly higher than that of the lens implant prepared in Example 1, and had excellent antibacterial effect against methicillin-resistant Staphylococcus aureus (MRSA). This shows that: after adding saffron extract to the mixed solution containing riboflavin and dextran, the lens implant crosslinked with acellular porcine cornea has a significantly improved antibacterial effect against methicillin-resistant Staphylococcus aureus (MRSA) compared with the lens implant crosslinked with acellular porcine cornea without adding saffron extract.

[0058] As can be seen from the experimental data in Table 1, the antibacterial rates of the lens implants prepared in Comparative Examples 1 and 2 against methicillin-resistant Staphylococcus aureus (MRSA) did not increase significantly compared with that of the lens implant prepared in Example 1. This shows that: the selection of the extraction solvent for saffron extract is crucial; only the saffron extract obtained by using ether as an organic solvent can further significantly improve the antibacterial effect of the crosslinked lens implant against methicillin-resistant Staphylococcus aureus (MRSA); however, the saffron extract obtained by using other organic solvents cannot further significantly improve the antibacterial effect of the crosslinked lens implant against methicillin-resistant Staphylococcus aureus (MRSA).

Claims

1. A method for preparing a decellularized porcine cornea in situ cross-linked lens implant for keratoconus surgery, characterized in that: The following steps are included: (1) taking a fresh porcine cornea, cleaning it, removing the epithelium, and then performing a decellularization treatment to obtain a decellularized porcine cornea; (2) soaking the decellularized porcine cornea in a mixed solution containing riboflavin and dextran; taking out the decellularized porcine cornea and irradiating it with ultraviolet light for in situ cross-linking, and obtaining the decellularized porcine cornea in situ cross-linked lens implant for keratoconus surgery after the cross-linking is completed.

2. The preparation method according to claim 1, characterized in that: The cleaning described in step (1) specifically refers to cleaning with physiological saline.

3. The preparation method according to claim 1, characterized in that: The specific method of performing the decellularization treatment in step (1) is: placing the cleaned fresh pig cornea in a solution containing a detergent and incubating it at 1 to 5° C. for 8 to 24 hours.

4. The preparation method according to claim 3, characterized in that: The solution containing detergent refers to an aqueous solution containing detergent; the detergent is Triton X-100.

5. The preparation method according to claim 4, characterized in that: The mass concentration of the detergent in the solution containing the detergent is 0.1% to 1%.

6. The preparation method according to claim 1, characterized in that: The mass concentration of riboflavin in the mixed solution containing riboflavin and dextran is 0.1-1%, and the mass concentration of dextran is 3-6%.

7. The preparation method according to claim 6, characterized in that: The mixed solution refers to a mixed aqueous solution.

8. The preparation method according to claim 1, characterized in that: The specific conditions of ultraviolet light irradiation are: wavelength 360-380nm, intensity 4-6mW / cm 2 , total energy is 4~8J / cm 2 The irradiation time is 20 to 40 minutes.

9. The preparation method according to claim 8, characterized in that: The specific conditions of UV irradiation are: wavelength 370nm, intensity 5mW / cm 2 , the total energy is 6J / cm 2 , the irradiation time is 30min.

10. A decellularized porcine cornea in situ cross-linked lens implant for keratoconus surgery prepared by the preparation method according to any one of claims 1 to 9.