RU2776561C1 - Method for treating bullous keratopathy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely, to ophthalmology. A surgical access to the cornea and implantation of an implant into the anterior chamber of the eye are performed. An incision is made preliminarily from 12 to 4 o'clock by the limbus, followed by implanting a polycaprolactone film with a diameter of 6.0 mm, a thickness of 20 mcm, and suturing said film to the cornea with two interrupted sutures. A cellular suspension - autologous mononuclear leukocytes - is therein layered on the endothelium at the boundary between the film and the inner surface of the cornea. The corneal incision is sutured shut with a continuous suture, followed by inflating sterile air into the anterior chamber.

EFFECT: possibility of reducing the injury rate of surgical intervention, reducing the risk of developing tissue incompatibility, facilitating the production of cells, reducing the swelling of the corneal tissue, thereby contributing to the regeneration of the transparency thereof and relief of surface bullae, significantly reducing the thickness of the implant.

1 cl, 17 dwg

Description

The invention relates to medicine, in particular to ophthalmology, and can be used for the treatment of bullous keratopathy.

Bullous keratopathy is a severe, progressive disease associated with decompensation of the endothelial layer of the cornea. After damage to the endothelium and the loss of its function as a semipermeable membrane between the stroma of the cornea and the moisture of the anterior chamber, edema of the stroma of the cornea gradually develops. In the future, the moisture of the anterior chamber penetrates under the epithelium of the cornea and exfoliates it with the development of recurrent erosions, causing excruciating pain for the patient, photophobia and lacrimation.

Currently, various methods of surgical treatment of bullous keratopathy have been developed: penetrating keratoplasty with replacement of the cornea with an allograft (Kopaeva V.G., Subtotal penetrating keratoplasty in corneal dystrophy. Opticoreconstructive operations and alloplasty in ophthalmology. M. 1974), layered keratoplasty with transplantation of the posterior layers of the cornea (Volkov V.V. To develop a surgical method for the treatment of endothelial-epithelial dystrophy of the cornea. The First Congress of Transcaucasian ophthalmologists: Abstracts of the report. Tbilisi: 1976), interlamellar transplantation of the posterior lens capsule (USSR Author's certificate N 810235, class A61F 9 / 007, 1981), layered keratoplasty using a gelatin film (Method of treating bullous keratopathy, RF patent No. 2082364, 06/27/1997), posterior layered keratoplasty - Posterior lamellar keratoplasty (PLK) and Deep lamellar endothelial keratoplasty (DLEK) (Ko, W. W. Feldman , S. T. Frueh, B. E. Experimental posterior lamellar trans plantation of the rabbit cornea / W. W. Ko, S. T. Feldman, B. E. Frueh //Invest. Ophthalmol. - 1993. - No. 34. - P. 1102.; Terry, M. A. Ousley, P. J. Deep lamellar endothelial keratoplasty in the first United States patients: early clinical results /M. A. Terry, P. J. Ousley, // Cornea. 2001. - No. 20. - P. 239 - 243.), keratoplasty with transplantation of Descemet's membrane (Mamikonyan, V.R. Trufanov, S.V. Automated endothelial keratoplasty with transplantation of Descemet's membrane (DMAEK) / V.R. Mamikonyan, S.V. Trufanov / / East-West: collection of scientific papers of scientific - practical conferences - Ufa, 2011. - P. 89 Gorovoy, M. Descemet stripping automated endothelial keratoplasty / M. Gorovoy // Cornea. - 2006. - No. 25: 8. - P. 886 - 889. Melles, G. R. Ong, T. S. Ververs, B. van der Wees, J. Descemet membrane endothelial keratoplasty (DMEK) / G. R. Melles, T. S. Ong, B. Ververs, J. van der Wees // Cornea. - 2006. - Vol. 25. - No. 8. - P. 987 - 990), keratoplasty using a hydrogel (Method for the treatment of bullous keratopathy, RF patent No. sheet for the transplantation of corneal endothelium patent US 20140377326, 2018), keratoplasty, by intralamellar transplantation of biological membranes (Method of treating edematous corneal degeneration, 1979, USSR patent No. 810235), keratoplasty using pseudoendothelial implants (Implantes pseudoendoteliales hidrofobos para tratar un edema corneal, Pat. 2 442 768, 2010, Sergio RIZZO). However, these methods have a number of significant drawbacks: the technical complexity of the operation, the high incidence of complications, the likelihood of an immune conflict with subsequent rejection of the donor material, and relapse of the disease.

A known method of keratoplasty for the treatment of bullous keratopathy using a hydrogel disc acting as a biological membrane, which is inserted at a distance of 1.5-2 mm from the limbus at 12 hours transconjunctivally in the direction of the deep layers of the corneal stroma, a tunnel incision 2.5-3 wide is formed, 0 mm and 2.5-3.0 mm long. The corneal stroma is exfoliated at a depth of 2/3 of its thickness, followed by the formation of a rounded intrastromal pocket with a diameter of 8.5-9.0 mm. A folded hydrogel disc 8.0 mm in diameter and 0.1 mm thick is implanted into the formed intrastromal pocket using a standard injector for implanting intraocular lenses. The disadvantages of this method are: an extended incision of the cornea and the formation of a pocket, which is associated with a rupture of collagen fibers and the absence of a smooth contact surface of the stroma of the stratified cornea with a hydrogel disk, as well as with a deterioration in the trophism of the cornea and the preservation of an incompetent edematous endothelial layer (Method of treating bullous keratopathy, RU 2405513 , 2010).

A known method for the same purpose, including implantation between the stroma and Descemet's membrane of the cornea of the hydrogel disc. In this case, a trepanation hole of the cornea is first formed so that its bottom does not reach the Descemet's membrane by 80-120 μm. The trepanation disk of the cornea is removed. At the bottom of the burr hole, a channel is created up to the Descemet's membrane and the colored viscomaterial is injected through it until the Descemet's membrane is peeled off. The viscomaterial is washed out and a hydrogel disk is implanted into the space formed between the stroma and the Descemet's membrane. The removed burr disc of the cornea is placed into the burr hole of the cornea, after which air is introduced into the anterior chamber of the eye. The main disadvantage of this method is the complete preservation of the Descemet's membrane, which can also be involved in the pathological process and causes a decrease in the transparency of the cornea and, consequently, low visual acuity (Method of intralamellar keratoplasty, patent RU 2463025, 10.10.2012).

A known method of keratoplasty using implants ranging in size from 1-2 mm to 6 mm, thickness from 1 μm to 100 μm based on biologically compatible materials: polymethyl methacrylate (PMMA), silicone, silicone rubber, collagen, hyaluronic acid, acrylic or methacrylic hydrogels, hydroxyethyl methacrylate or partially hydrolyzed poly (2-hydroxyethyl methacrylate / methacrylic acid / copolymer), polysulfone (Fabrication of gelatin hydrogel sheet for the transplantation of corneal endothelium, patent US 100523506, 2018). According to this method, an implant based on one of these polymers with formed paracentral holes is attached to the posterior surface of the cornea using the method of posterior lamellar keratoplasty (PLK) or endothelial keratoplasty with Descemet's membrane replacement (DSEK) by preparing the corneal bed (preliminary removal of a part of the endothelium and Descemet's membrane ) followed by the introduction of a binder. As a binder, poly-L-lysine, poly-D-lysine, fibronectin, laminin, type I, II, III and IV collagen, thrombospondin, polystyrene, vitronectin, polyarginine and platelet factor IV can act. In addition, this method indicates the need for the use of cytotoxic agents: proteins that inhibit ribosomes (saporin and ricin), as well as antimitotic drugs such as methotrexate, 5-fluorouracil, daunomycin, doxorubicin, mitoxantrone, vinca alkaloids, vinblastine, colchicine and cytochalasins and ionophores such as monensin and ouabain. Despite a number of advantages of this method (the possibility of varying the material for creating an implant, a wide range of its dimensions and thickness), the main disadvantages are: the need to prepare the corneal bed with the removal of the endothelium and Descemet's membrane, the mandatory use of a binding agent, the need to use cytotoxic drugs.

In addition, cellular technologies are used as an alternative to existing surgical methods for the treatment of bullous keratopathy. A known method of intracameral local express autocytokine therapy (LEACCT) (Method for the treatment of corneal edema and other manifestations of early bullous keratopathy, RF patent No. 2357743, 13.10.2009). According to this method, the patient's autologous blood mononuclear cells activated by Poludan are introduced into the anterior chamber of the diseased eye. Blood sampling from the patient is carried out in compliance with the rules of asepsis and antiseptics in the conditions of the treatment room. Blood is taken from the patient's cubital vein in an amount of 5.0 ml. The contents of 2 vials of Poludan are dissolved in 2.0 ml of water for injection, and the aqueous solution of Poludan is transferred into a sterile tube with 5.0 ml of blood. The contents of the tube are thoroughly mixed by inverting. Next, the test tube is placed in a thermostat for 2-4 hours. After the end of incubation, the test tube with clotted blood is centrifuged for 5-10 minutes at 500-1000 rpm. Further, at the border of the serum and the coagulated blood clot, a cell suspension is collected with a pipette. In the conditions of the operating room, after processing the surgical field and retrobulbar anesthesia of the diseased eye of the patient near the limbus, a corneal paracentesis is performed with a blade. A suspension of autologous blood mononuclear cells is gradually infused into the anterior chamber in an amount of 1.0-2.0 ml until it completely replaces the anterior chamber moisture. In the zone of paracentesis of the cornea, a supramid suture is applied. Immediately after the operation, the patient should take a position face down and lie down for 1 hour so that the injected cells come into contact with the corneal endothelium. LEACCT sessions are carried out with an interval of 3-6 days in an amount of 1 to 4 times. The disadvantages of this method are: the duration and complexity of obtaining autologous blood mononuclear cells activated with Poludan, the high probability of cell loss due to the lack of a substrate for attachment and a directed flow of intraocular fluid to the inner corner of the eye.

Closest to the proposed method is a method of replacing the damaged endothelial layer of the cornea by transplantation using a gelatin hydrogel disk (Fabrication of gelatin hydrogel sheet for the transplantation of corneal endothelium Patent US 20140377326, 2012). According to this method, a gelatin-hydrogel scaffold (scaffold) 100-300 μm thick, with pores 20-30 μm in size, modified with heparin, containing one layer of corneal endothelial cells pre-cultured for 7-21 days, is implanted on a previously prepared - after removal endothelium and Descemet's membrane of the recipient - corneal bed. The graft introduced into the anterior chamber of the eye is fixed to a previously prepared bed with interrupted sutures, fibrin glue or fibronectin is used to immobilize the frame, and sterile air is introduced into the anterior chamber.

The disadvantages of this method are: the technical complexity of the surgical intervention (preparation of the corneal bed with the removal of the endothelial layer and Descemet's membrane), the need for cultivation of corneal endothelial cells, a high risk of tissue incompatibility between the donor's endotheliocytes and the recipient's corneal tissues, the relatively large thickness of the implanted disc (100-300 micron), the use of fibrin glue or fibronectin as a binding agent, modification with heparin.

A new technical objective of the invention is to reduce the trauma of surgical intervention, the use of biodegradable material for implantation as a temporary cell substrate, the use of autologous cells, which reduces the risk of tissue incompatibility, simplification of cell production, a significant reduction in the thickness of the implant (20-30 microns).

To solve the problem in the method of surgical treatment of bullous keratopathy, which includes the implantation of a polymeric material into the anterior chamber with its subsequent suturing to the cornea with interrupted sutures, autologous mononuclear leukocytes are layered on the damaged surface of the cornea at the border between the film and the cornea. The implant is a film of polycaprolactone with a diameter of 6.0 mm, a thickness of 20-30 microns.

Thanks to this method of using autologous mononuclear leukocytes and an implanted polymer film as a cell substrate, the swelling of the corneal tissue is significantly reduced, which contributes to the restoration of its transparency and the relief of bullae on the surface.

The essence of the invention is illustrated by drawings. Below is a brief description of the attached drawings used to describe the implementation of the invention.

Figure 1 shows the side of the polycaprolactone film;

figure 2 shows a photograph of the contact angle (60.1°) of the film of polycaprolactone after treatment of the sides with plasma;

in Fig.3. the transmission spectrum of the polycaprolactone film is shown, where 1 is the initial sample; 2 - after plasma treatment;

in Fig.4. shows the injection of conjunctival vessels, severe edema, edema of the cornea in an experimental animal 2 weeks after the modeling of bullous keratopathy;

in Fig.5. shows the injection of conjunctival vessels, lacrimation, corneal edema of the experimental animal of the second group on the 3rd day after implantation of the polycaprolactone film and layering of mononuclear leukocytes;

in Fig.6. a moderately pronounced corneal edema of the experimental animal of the second group was shown on the 14th day after the implantation of the polycaprolactone film and the layering of mononuclear leukocytes;

in Fig.7. shows a mild swelling of the surface layers of the cornea of the experimental animal of the second group 4 weeks after the implantation of the polycaprolactone film and the layering of mononuclear leukocytes;

in Fig.8. shows a moderately pronounced injection of conjunctival vessels and swelling of the cornea of the experimental animal of the first group after 6 weeks from the start of the experiment;

in Fig.9. presents optical coherence tomography (OCT) of the cornea of an experimental animal 2 weeks after the modeling of bullous keratopathy;

in Fig.10. OCT of the cornea of the experimental animal of the second group is shown 4 weeks after the implantation of the polycaprolactone film and the layering of mononuclear leukocytes;

in Fig.11. presented OCT of the cornea of the experimental animal of the first group after 6 weeks from the start of the experiment;

in Fig.12. shows the anterior epithelium, Bowman's membrane and corneal proper substance of the experimental animal of the second group. Hematoxylin-eosin stain, x200;

in Fig.13. the own substance of the cornea of the experimental animal of the second group is shown. Hematoxylin-eosin stain, x200;

in Fig.14. adhesion of mononuclear leukocytes to the Descemet's membrane of the cornea of an experimental animal of the second group is shown. Hematoxylin-eosin stain, x400;

in Fig.15. layers of process cells of the cornea of an experimental animal of the second group are shown. Hematoxylin-eosin stain, x200;

in Fig.16. shows the anterior epithelium, Bowman's membrane and corneal proper substance of the experimental animal of the first group. Hematoxylin-eosin stain, x200;

in Fig.17. shows the anterior epithelium, Bowman's membrane and corneal proper substance of the experimental animal of the first group. Hematoxylin-eosin stain, x200.

Film polycaprolactone (figure 1) is obtained from a 1% solution of polycaprolactone with a molecular weight, Mw=80000 g/mol (Sigma-Aldrich, England). The finished solution in an amount of 12 g is poured into Petri dishes and kept in a fume hood until the solvent has completely evaporated (48 hours). The formed polymer films are removed from the Petri dish and placed in a vacuum chamber for 24 hours (pressure 10-3 Torr, temperature 25°C) to completely eliminate the residual solvent. The thickness of the films obtained by this method is (25±5) µm. In order to increase the wettability of the polymer and, as a result, improve the adhesion of cells introduced into the anterior chamber of the eye between the film and the inner surface of the cornea, the sides of the material are treated with low-temperature atmospheric pressure plasma with a voltage of 25 kV and a frequency of 5 kHz for 30 seconds. The contact angle of wettability (in water) of polycaprolactone films after treatment of the sides with plasma is 60.6°±2.3° (figure 2). The transmittance of the visible spectrum of polycaprolactone films is 90%-95% (figure 3). The method is carried out as follows.

Autologous mononuclear cells from the blood of an experimental animal are isolated by density gradient fractionation on a ficoll-verografin separating solution. Blood taken in the amount of 4.0-5.0 ml is placed in a sterile test tube containing 1.0 ml of heparin solution. Heparinized blood is diluted 2 times with isotonic sodium chloride solution. The resulting suspension is layered on 3.0 ml of a mixture of ficoll-verografin. Samples are centrifuged at room temperature for 15 min at 800 g (2000 rpm). After centrifugation, the interphase layer containing mononuclear cells and located between the plasma and the gradient is taken with a Pasteur pipette. 1.0 ml of isotonic sodium chloride solution is added and the resulting suspension is again centrifuged for 7 minutes at 400 g (1550 rpm). The purity of mononuclear cells obtained on a ficoll-verografin gradient is up to 96-98%. The viability of the cell material is assessed in the Triplan blue test in the following way: a 0.1% eosin solution is preliminarily prepared on the basis of Ringer's solution. Next, a 0.1% solution of trypan blue is prepared on the basis of distilled water. After that, 1-2 drops of a fresh mixture of solutions of previously prepared dyes, taken in equal volumes, are added to a drop of the cell suspension. The resulting mixture is placed in the Goryaev chamber. When counting cells, the percentage of stained (dead) elements should be 1.5-2%, which would not exceed the allowable (no more than 3%) amount. The viability of cellular material in the test with trypan blue is 97-98%.

In the conditions of the operating room after anesthesia and processing of the surgical field in compliance with the rules of asepsis and antisepsis, the animals undergo surgery. In the cornea with pre-induced bullous keratopathy, an incision is made from 12 to 4 hours at the limbus, through which a polycaprolactone film is implanted into the anterior chamber of the eye. The film is sutured with two interrupted sutures to the cornea. On the border between the film and the inner surface of the cornea, a cell suspension is layered on the endothelium, the corneal incision is sutured with a continuous suture. Sterile air is introduced into the anterior chamber.

The method was tested on 12 Sylvilagus bachmani rabbits weighing 2.5-3.0 kg.

Review of the experimental material: the total duration of the experiment was 6 weeks. At stage I, bullous keratopathy was simulated in each animal under operating conditions by mechanical damage and removal of the corneal endothelium of one of the eyes. At stage II, 2 weeks after the development of the pathological process, the animals were divided into the following groups:

group 1 - (n=6) disease model group;

group 2 - (n=6) a group of animals in which the anterior chamber of the eye was implanted with a film of polycaprolactone and layered with autologous mononuclear leukocytes by the described method.

During the experiment, an external examination, photographic recording, and optical coherence tomography (OCT) of the cornea were performed. The material was taken 6 weeks after the start of the experiment.

Enucleated eyes were fixed in 12% neutral formalin. Fixation was carried out at room temperature for 24 hours. Then the objects, after 24-hour washing in running water, were subjected to dehydration in alcohols of increasing concentration, clarified in O-xylene, and embedded in paraffin. The resulting sections were stained with hematoxylin and eosin, as well as by the Van Gieson method.

Various structural components and cell infiltration were counted using a LOMO Biolam AU-12 light microscope (approx. x7, x40, x90, native magnification of the microscope x1.5), an Avtandilov 50-point eyepiece grid, and an eyepiece insert with a known area.

The obtained data were processed using the IBM SPSS Statistics 20 software package.

As a result of external examination 2 weeks after the modeling of bullous keratopathy in all animals, lacrimation, photophobia, injection of conjunctival vessels, severe swelling of the cornea (figure 4) were noted. On the third day after implantation in animals of the second group, the injection of conjunctival vessels, lacrimation, corneal edema (figure 5) remained, on the 14th day the corneal edema decreased markedly, lacrimation and injection of conjunctival vessels were completely stopped (figure 6). 4 weeks after the implantation of polycaprolactone films and layering of mononuclear leukocytes in animals of the second group, a mild swelling of the surface layers of the cornea was noticeable (Fig.7). In animals of the first group throughout the experiment, lacrimation, moderately pronounced injection of conjunctival vessels and swelling of the cornea remained (Fig.8).

During OCT of the cornea, an increase in the thickness of the cornea was revealed in all animals 2 weeks after the modeling of bullous keratopathy, which averaged 745 μm (Fig.9). 2 weeks after implantation of polycaprolactone films and layering of mononuclear leukocytes in animals of the second group, a decrease in the thickness of the cornea to 703 μm was observed, after 4 weeks - to 633 μm, p<0.05 (compared to the first group at the same time according to the Mann criterion -Whitney) (Fig.10). In animals of the first group, the thickness of the cornea at 6 weeks from the start of the experiment was 702 μm (Fig.11).

In the course of morphological studies in the second group of animals, the following was revealed.

The anterior corneal epithelium is represented by 4-5 layers of squamous epithelium with normochromic nuclei and is preserved throughout (Fig.12). Bowman's membrane was visualized as a homogeneous eosinophilic strip. In its own substance of the cornea showed uneven moderate changes (Fig.12, Fig. 13). Own substance of the cornea contained predominantly compact collagen fibers (Fig.12, Fig. 13). The specific volume of gaps between them was 9.9%. The posterior limiting membrane was not changed and was well visualized throughout. In places, adhesion of mononuclear leukocytes, layered on the damaged surface of the cornea, to the Descemet's membrane was found (Fig.14). In many places, the endothelium was represented by layers of process cells (Fig.15).

In the course of morphological studies in the first group of animals, the following was revealed.

The anterior corneal epithelium is represented by layers of squamous epithelium and is preserved throughout (Fig.16). Bowman's membrane was visualized as a homogeneous eosinophilic strip, unevenly thickened in places (Fig. 16). The intrinsic substance of the cornea contained predominantly tortuous collagen fibers, the specific volume of gaps between which was 16%, p<0.05 (compared to the second group according to the Mann-Whitney test) (Fig. 17). The posterior limiting membrane was visualized throughout. The endothelium was absent (Fig.17).

Analysis of the data obtained during the experiment indicates a decrease in corneal edema, and, as a result, stabilization of the pathological process.

Thus, the results of an experimental study showed that the implantation of polycaprolactone films in the anterior chamber of the eye and the layering of mononuclear leukocytes at the border between the material and the inner surface of the cornea in bullous keratopathy can compensate for the impaired function of the endothelial layer of the cornea and stabilize the course of the pathological process, thereby reducing tissue edema. and the transparency of the cornea is restored.

Claims (1)

A method for the treatment of bullous keratopathy, which includes performing a surgical access to the cornea and implanting an implant in the anterior chamber of the eye, characterized in that an incision is made from 12 to 4 o'clock at the limbus, followed by implantation of a polycaprolactone film with a diameter of 6.0 mm, a thickness of 20 μm and by suturing it with two interrupted sutures to the cornea, while at the border between the film and the inner surface of the cornea, a cell suspension is layered on the endothelium - autologous mononuclear leukocytes, the corneal incision is sutured with a continuous suture, after which sterile air is introduced into the anterior chamber.

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