Patent Application
20250170305
The present application claims priority to the Chinese Patent Application No. CN202211003956.8, filed with the China National Intellectual Property Administration (CNIPA) on Aug. 19, 2022, and entitled “TRANSPARENT SCLERA-BASED CORNEAL REPAIR MATERIAL, AND PREPARATION METHOD AND USE THEREOF”, which is incorporated herein by reference in its entirety.
The present disclosure relates to the technical field of medical repair materials, in particular to a transparent sclera-based corneal repair material, and a preparation method and use thereof.
Corneal blindness is the fourth leading cause of blindness worldwide. There are about 10 million corneal blindness patients in the world, but only about 18,500 patients are capable of receiving keratoplasty annually. So far, for patients with severe corneal damage, the most common and effective way to improve visual function is still keratoplasty. This can ease the economic pressure on the country and improve the quality of life for patients. However, since the number of corneas donated by donors is far less than the demand for keratoplasty, it is necessary to develop new materials for corneal defect repair.
The cornea does not contain vascular tissues inside, which make the cornea have desirable optical penetration properties. Sutures are required during keratoplasty, such that repair materials need to have a certain suture-resistant strength. In other words, the repair materials need to show excellent biomechanical properties. After the keratoplasty, donor materials need to be epithelialized in vivo to complete the repair of biological tissues. Therefore, corneal repair materials require desirable biocompatibility. Due to different cases, these corneal repair materials also need to realize size controllability of the materials during the preparation.
The current corneal repair materials mainly include three categories. The first is that porcine cornea processed by decellularization is used as a substitute material for lamellar keratoplasty. Such a material has the disadvantage of heterogeneous origin. The second is biomimetic biomaterials prepared from the materials such as collagen, silk fibroin, alginate, or chitosan. This type of material has a poor biomechanical strength. The third is the artificial cornea made of polymethacrylate. This material has a narrow scope of application and is only suitable for patients with severe corneal damage.
Sclera is a natural biological material. From the source of development, both the sclera and cornea come from mesoderm. Moreover, both the sclera and cornea are derived from neural crest cells. The sclera is a collagen-rich extracellular matrix and has a collagen type similar to that of the cornea. From an ultrastructural point of view, like the cornea, the sclera is rich in transverse and longitudinal fibers. However, the sclera has characteristic oblique fibers that render the sclera opaque. As a result, sclera cannot currently be used as a corneal repair material.
An objective of the present disclosure is to provide a transparent sclera-based corneal repair material, and a preparation method and use thereof. The corneal repair material is a type of transparent sclera with desirable transparency and excellent biocompatibility.
To achieve the above objective, the present disclosure provides the following technical solutions:
The present disclosure provides a preparation method of a transparent sclera-based corneal repair material, including the following steps:
Preferably, the pretreatment includes removal of uvea and accessory tissues, sectioning, and cleaning that are conducted in sequence.
Preferably, a sclera section obtained by the sectioning has a thickness of 350 μm to 500 μm.
Preferably, a reagent for the decellularization is a mixture II of Triton X-100 and a super nuclease; the Triton X-100 has a mass concentration of 1% to 3% in the mixture II; and the super nuclease has a concentration of 2,000 u/mL in the mixture II.
Preferably, the compressing is conducted at 10 MPa to 12 MPa for 48 h to 50 h.
Preferably, the compressing is conducted at a radian of 43° to 44°.
Preferably, the drying is conducted at 20° C. to 35° C. for 24 h to 26 h.
Preferably, a cross-linking agent in the cross-linking agent solution is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM); the cross-linking agent solution has a concentration of 30 wt %; and the cross-linking is conducted for 4.5 h to 5 h.
The present disclosure further provides a transparent sclera-based corneal repair material prepared by the preparation method.
The present disclosure further provides use of the transparent sclera-based corneal repair material in corneal repair.
The present disclosure provides a preparation method of a transparent sclera-based corneal repair material. In the present disclosure, the sclera, a natural biological material that is convenient to obtain, is used as a source of the corneal repair material. The opaque sclera is compressed into a sheet, such that the sclera has a reduced fiber spacing, a denser tissue, and a more orderly arrangement of fibers in three directions. In this way, the light transmittance is increased, and the transparency is increased, thereby realizing hyalinization of the sclera. Carboxyl groups on a scleral fibrin is activated by cross-linking, such that amino groups of the sclera combine with the carboxyl groups to form an amide bond. The amide bond belongs to a covalent bond and has a desirable stability, thereby maintaining a transparency of the sclera after the compressing. Therefore, obtained transparent sclera can be used as a transparent corneal tissue repair material.
In the present disclosure, sclera is used as a raw material. Accordingly, the prepared corneal repair material has fiber structure, as well as physical and chemical properties and biological properties similar to those of a natural corneal tissue, and shows excellent optical properties. Moreover, the sclera is a natural biological material with desirable biocompatibility and tissue compatibility. After being coated with Matrigel, corneal epithelial cells can adhere to, proliferate, and secrete extracellular matrix on this repair material, making the material epithelialized and facilitating postoperative corneal epithelial recovery. Therefore, the material can be used as an alternative of the corneal donor for the repair and replacement of damaged corneal tissues in the medical field.
In the present disclosure, the corneal tissue repair material uses natural sclera as a raw material. The mechanical properties of the natural sclera are higher than those of the cornea. Therefore, the repair material has desirable biomechanical properties, is suture-resistant, and can be made into materials with controllable size and thickness according to specific injury cases.
In the present disclosure, the preparation method has a simple process, cheap and easy-to-obtain raw materials, and a low cost, which are beneficial to large-scale production.
The present disclosure provides a preparation method of a transparent sclera-based corneal repair material, including the following steps:
In the present disclosure, unless otherwise specified, all raw materials required for preparation are commercially available products well known to persons skilled in the art.
In the present disclosure, all operations in the preparation method are conducted in a sterile ultra-clean environment. The PBS has a concentration of 0.02 M and a pH of 7.4.
In the present disclosure, pretreatment and decellularization are conducted on pig eye sclera in sequence to obtain a decellularized sclera section.
In the present disclosure, the pig eye sclera is preferably obtained from fresh eyeballs collected within 2 h, and the sclera is cleaned with sterile double distilled water. There is no special limitation on a cleaning process, which can be conducted according to the well-known process in the art.
In the present disclosure, the pretreatment includes preferably removal of uvea and accessory tissues, sectioning, and cleaning that are conducted in sequence. Preferably, the uvea and accessory tissues are surgically removed; more preferably, the uvea and accessory muscular tissues are removed from the inner and outer surfaces of the pig eye sclera. There is no special limitation on a removal process, which can be conducted according to the process well known in the art.
In the present disclosure, the inner and outer sides of the sclera are resected through the sectioning. Preferably, the sectioning is conducted using a Moria microkeratome and a trephine of a specified size, and sections of the resulting size and thickness are obtained. The sclera section has a thickness of preferably 350 μm to 500 μm and a diameter of 6.25 mm.
In the present disclosure, a cleaning solution for the cleaning is preferably a mixture I of Triton X-100 and PBS, and the Triton X-100 has a mass concentration of preferably 1% to 3%, more preferably 2% in the mixture I. The cleaning solution is preferably used after being sterilized under high temperature and high pressure. The cleanings is preferably conducted greater than or equal to 3 times for preferably 30 min each time and a total cleaning time of preferably 2 h to 2.5 h. There is no special limitation on a composition of the PBS, and commercially available products well known in the art can be used; in an example, the PBS has a concentration of 0.02 mol/L and a pH value of 7.4. There is no special limitation on a process of the sterilization under high temperature and high pressure, which can be conducted according to the process well known in the art.
In the present disclosure, after the pretreatment is completed, the sclera section is subjected to the decellularization. A reagent for the decellularization is preferably a mixture II of Triton X-100 and a super nuclease. A solvent used in the mixture II of the Triton X-100 and the super nuclease is preferably pure water. The Triton X-100 has a mass concentration of 1% to 3%, more preferably 2% in the mixture II. The super nuclease has a concentration of 2,000 u/mL in the mixture II. There is no special limitation on a dosage of the mixture II of the Triton X-100 and the super nuclease, as long as the decellularization is fully conducted.
In the present disclosure, the decellularization is conducted at preferably 37° C. for preferably 15 h to 17 h in preferably a shaker with a rotation speed of preferably 100 rpm. The scleral fibroblasts are removed through the decellularization, thereby alleviating a rejection reaction of the sclera sheet after being applied to living body transplantation.
In the present disclosure, after the decellularization is completed, the sclera section is preferably washed with PBS. There is no special limitation on a washing process, and the sclera section can be washed according to the well-known process in the art.
In the present disclosure, the decellularized sclera section is compressed, and an obtained product is dried until transparent to obtain a sclera sheet. The compressing is preferably conducted in a press. The compressing is conducted at preferably 10 MPa to 12 MPa for preferably 48 h to 50 h. The compressing is conducted at a radian of preferably 43° to 44° (same as that of the cornea), more preferably 43.5°. The fibrin arrangement of the sclera is more orderly by the compressing. After the fiber is ordered, the scattering of light after passing through the tissues is reduced, and the transmittance of light is increased.
In the present disclosure, the drying is preferably conducted under natural conditions at preferably 20° C. to 35° C. for preferably 24 h to 26 h. There is no special limitation on a degree of the drying to transparency, as long as a desired degree of transparency can be achieved.
In the present disclosure, the sclera sheet is subjected to cross-linking in a cross-linking agent solution, and immersed in water to obtain the transparent sclera-based corneal repair material.
In the present disclosure, a cross-linking agent in the cross-linking agent solution is preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM); the cross-linking agent solution has a concentration of preferably 30 wt %; and the cross-linking is conducted for preferably 4.5 h to 5 h. The cross-linking agent solution is preferably obtained by dissolving a DMTMM powder in an HCl solution with a pH value of 6.5. There is no special limitation on a dosage of the cross-linking agent solution, as long as the solution can completely infiltrate the sclera sheet.
In the present disclosure, the cross-linking agent can activate the carboxyl groups of scleral fibrin, and make the amino groups and carboxyl groups of sclera combine to form an amide bond. The amide bond is a covalent bond, which is more stable compared with the non-cross-linked amino and carboxyl groups, and can maintain the transparency of sclera after compressing. An amidation equation is: R—COOH+R′—NH2→R—CO—NH—R′ (final structure, type I collagen)+H2O.
In the present disclosure, a process of the immersing includes preferably: immersing the sclera sheet obtained by cross-linking into PBS or a 0.9 wt % sodium chloride aqueous solution until the immersing is completed. The PBS has a concentration of preferably 0.02 mol/L and a pH value of preferably 7.4.
The present disclosure further provides a transparent sclera-based corneal repair material prepared by the preparation method.
The present disclosure further provides use of the transparent sclera-based corneal repair material in corneal repair. In the present disclosure, there is no special limitation on a method of the use, and methods well known in the art can be used. In the present disclosure, preferably, the transparent sclera-based corneal repair material is coated with a Matrigel basilar membrane, so as to increase the biocompatibility of the tissues, thereby promoting the rapid repair of the corneal epithelium. There is no special limitation on a process of coating with the Matrigel basilar membrane, which can be conducted according to the process well known in the art. In an example, the process specifically includes: implanting the corneal repair material into a Matrigel basilar membrane solution with a concentration of 8 mg/mL (the Matrigel basilar membrane dissolved in a Gibco's serum-free medium), spreading, and drying in an oven at 37° C.; conducting immersing with PBS (with a concentration of 0.02 mol/L and a pH value of 7.4) to obtain a composite membrane. Preferably, the composite membrane is directly used for corneal repair.
The technical solutions of the present disclosure will be clearly and completely described below with reference to the examples of the present disclosure. Apparently, the described examples are merely a part rather than all of the examples of the present disclosure. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure.
In the following examples, the PBS has a concentration of 0.02 mol/L and a pH value of 7.4.
1) fresh pig eye sclera within 2 h was taken, and uvea and accessory muscle tissues on the inner and outer surfaces of the sclera were removed; treated sclera was sectioned with a Moria microkeratome and a trephine of a specific size, to obtain a sclera section with a thickness of 500 μm and a diameter of 6.25 mm.
the sclera section was washed in a Triton X-100-PBS solution with a mass concentration of 2% for 2 h, 4 times, 30 min for each time; a resulting washed sclera section was placed in a mixture of 2% TritonX-100 and 2,000 u/mL super nuclease, and the subjected to decellularization in a shaker at 37° C. and 100 rpm for 17 h; a resulting product was washed again with PBS to obtain a decellularized sclera sheet;
The corneal repair material prepared in Example 1 was placed in a petri dish, and 200 μL of a Matrigel basilar membrane solution with a concentration of 8 mg/mL (a Matrigel basilar membrane was dissolved in a Gibco's serum-free medium) was added dropwise into an upper surface of the material, spread with a pipette tip, dried in an oven at 37° C. for 2 h, and then immersed with PBS (with a concentration: 0.02 mol/L and a pH value: 7.4) to obtain a composite membrane. Note: since the Matrigel membrane was easy to gel at higher than 10° C., all operations were conducted on ice before drying in the oven.
1)
2)
3)
4) The corneal repair material prepared in Example 1 was stained with hematoxylin-eosin (H&E), specifically including:
1. Fixation: the corneal repair material prepared in Example 1 was fixated in a 10 wt % formaldehyde solution for 24 h.
2. Dehydration: an obtained fixated material was placed in an embedding box, rinsed in running water for 30 min, and dehydrated in an alcohol solution.
3. Transparency treatment: an obtained dehydrated material was placed in xylene to replace the alcohol to maintain the transparency of the material.
4. Wax dipping and embedding: the transparent material was placed in melted paraffin, and cooled after the paraffin completely covered the material, so as to achieve the embedding.
5. Slicing, unfolding, and baking: an obtained embedded material was cut into thin slices with a thickness of 3 μm in a microtome, the thin slices were put in water and unfolded, and placed on slides. The slides were dried in a thermostat at 60° C.
6. Dewaxing and dehydration: the slides were dewaxed in xylene, dehydrated in alcohol, and placed in distilled water.
7. Staining: the slices in distilled water were stained for 10 min in a hematoxylin aqueous solution, placed in 1 wt % hydrochloric acid for 3 s, and then placed in tap water for 5 min to conduct bluing; resulting bluish slices were dehydrated in 70% and 90% alcohol separately for 10 min, and then stained in an eosin staining solution for 5 min.
8. Dehydration and hyalinization: an obtained dyed material was dehydrated in pure alcohol, and then placed in xylene for hyalinization.
9. Sealing and observation: the slices were observed after adding resin on the glass slide and sealing with the cover glass.
The stained slices (×400) obtained through the above steps 1 to 9 were shown in
5) A TEM test was conducted on the corneal repair material prepared in Example 1, and the obtained results were shown in
The composite membrane prepared in Use Example 1 was used to repair the defect of rabbit cornea, and conduct lamellar keratoplasty in rabbits.
(1) Animal model preparation: healthy New Zealand white rabbits were anesthetized, and the corneal stroma with a diameter of 6.0 mm and a thickness of 200 μm was removed in a center of the cornea with a trephine to prepare a corneal stroma defect model.
(2) Defect repair: the corneal defect was filled with the transparent sclera material coated with the Matrigel basilar membrane prepared in Use Example 1, sutured intermittently with 10-0 nylon suture, and a thread knot was transferred to the deep corneal stroma. Note: the transplant was aligned with the planting bed.
(3) Rinsing: the cornea was rinsed with normal saline, and after binding with a corneal bandage mirror, the New Zealand rabbits were sent back to their breeding room.
(5) Postoperative care: the New Zealand white rabbits were smeared with Alcon® eye drops (tobramycin and dexamethasone eye drops) every morning and evening within seven days after the surgery.
1. General photo observation: 3 d after surgery, a repair effect of the transparent sclera material coated with the Matrigel basilar membrane prepared in Use Example 1 was observed by a slit lamp. The results were shown in
2. Observation of sodium fluorescein staining: 3 d after surgery, the cornea repaired with the transparent sclera repair material coated with the Matrigel basilar membrane prepared in Use Example 1 was stained with sodium fluorescein (the conjunctival sac at the outer ⅓ of the lower eyelid of the rabbit was lightly touched with a sodium fluorescein staining test paper to make the rabbit blink, such that the stain was evenly applied to the corneal surface of the rabbit). After the stained cornea was rinsed with normal saline, photographing and observation were conducted under cobalt blue light, and the results were shown in
The composite membrane prepared in Use Example 1 was used to treat corneal perforation in rabbits, and conduct penetrating keratoplasty in rabbits.
(1) Animal model preparation: healthy New Zealand white rabbits were anesthetized, and a full-thickness cornea with a diameter of 2.75 mm was removed by a trephine in a center of the cornea to prepare a corneal perforation model.
(2) Defect repair: the corneal perforation was filled with the transparent sclera material coated with the Matrigel basilar membrane prepared in Use Example 1, sutured intermittently with 10-0 nylon suture, and a thread knot was transferred to the deep corneal stroma. Note: the transplant was aligned with the planting bed.
(3) Rinsing: the cornea was rinsed with normal saline, and after binding with a corneal bandage mirror, the New Zealand rabbits were sent back to their breeding room.
(5) Postoperative care: the New Zealand white rabbits were smeared with Alcon® eye drops (tobramycin and dexamethasone eye drops) every morning and evening within seven days after the surgery.
1) General photo observation: 3 d after surgery, a repair effect of the transparent sclera material coated with the Matrigel basilar membrane prepared in Use Example 1 was observed by a slit lamp. The results were shown in
2) 3 d after the surgery, digital slit lamp inspection was conducted on the rabbit in vivo model, and the results were shown in
3) Observation of sodium fluorescein staining: 3 d after surgery, the cornea repaired with the transparent sclera repair material coated with the Matrigel basilar membrane prepared in Use Example 1 was stained with sodium fluorescein (the conjunctival sac at the outer ⅓ of the lower eyelid of the rabbit was lightly touched with a sodium fluorescein staining test paper to make the rabbit blink, such that the stain was evenly applied to the corneal surface of the rabbit). After the stained cornea was rinsed with normal saline, photographing and observation were conducted under cobalt blue light, and the results were shown in
The above are merely preferred implementations of the present disclosure. It should be noted that several improvements and modifications may further be made by a person of ordinary skill in the art without departing from the principle of the present disclosure, and such improvements and modifications should also be deemed as falling within the protection scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202211003956.8 | Aug 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/071153 | 1/9/2023 | WO |
