RU2779361C1 - Homogeneous transparent collagen membrane, method for production thereof, and application thereof for reconstructing the cornea - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely, to ophthalmology. Proposed is a collagen membrane for reconstructing the cornea, constituting a collagen hydrogel containing a native type I collagen, wherein the content of native type I collagen is from 5 to 20% wt. of the total weight of the collagen hydrogel, and wherein said artificial cornea is characterised by a maximum tensile load of above 20 N.

EFFECT: collagen membrane for reconstructing the cornea according to the invention is biocompatible, non-immunogenic, optically transparent and strong, is made of a highly purified biological material without applying chemical modification, and is suitable for implantation into the human body for the purpose of improving and/or restoring the anatomical and functional characteristics of the cornea of the eye.

24 cl, 3 dwg, 6 ex, 3 tbl

Description

The field of technology to which the invention belongs

The present invention relates to the field of biodegradable implants for human administration, in particular to materials for the restoration of a damaged cornea of the eye.

State of the art

The cornea, the cornea is the anterior most convex transparent part of the eyeball, one of the light-refracting media of the eye. It has the form of a convex-concave lens with its concave part facing back. The corneal diameter is 10±0.56 mm. The thickness of the cornea in the central part is 0.56 - 0.6 mm, along the edges - 1.0 - 1.2 mm. The maximum rupture load of a normal human cornea is 10-12 Newtons.

The problem of restoration of damaged corneal tissues, their integrity and transparency is relevant for ophthalmology. Belmo (leukoma) is a pathological process accompanied by partial or complete clouding of the cornea. The most common causes of the formation of a walleye are: keratitis of various etiologies (viral, tuberculosis and others), injuries (wounds, chemical and thermal burns), surgical interventions on the eye. There are also congenital corneal opacities due to intrauterine inflammation.

To restore the transparency of the damaged cornea, transplantation of a donor cornea obtained from a donor is used in clinical practice. Currently, more than 10 million people around the world are in need of a donor cornea transplant. However, the clinical use of allogeneic material is complicated due to the lack of the required number of donor corneas. Moreover, in some cases, the reaction of the patient's immune system to the antigens of the donor cornea is observed, which leads to opacity of the graft. One possible solution to this problem is the development of a material for keratoplasty that is biocompatible with corneal tissues and has minimal immunogenicity. In this case, we are talking about the rejection of the use of a donor cornea and the use of biocomposite polymers.

Close to the claimed invention in properties is a homogeneous collagen hydrogel based on recombinant human collagen type III, described in document WO 2006/020859 A3. The authors of this invention propose to use together with type III collagen proteins that promote adhesion and proliferation of corneal cells. The main disadvantage of such a product is the fact that in order to increase the strength characteristics, the product is subjected to chemical modification (including treatment with glutaraldehyde), which causes increased immunogenicity, limited biocompatibility, and impaired optical transparency after implantation.

From Xiao X., Pan S., Liu X., Zhu X., Connon C.J., Wu J., Mi S. "In vivo study of the biocompatibility of a novel compressed collagen hydrogel scaffold for artificial corneas", J. Biomed . mater. Res., 2013, 102 (6), 1782–1787 and articles by Mi S., Chen B., Wright B., Connon C.J. "Plastic compression of a collagen gel forms a much improved scaffold for ocular surface tissue engineering over conventional collagen gels", J. Biomed. mater. Res. A, 2010, 95 (2), 447–453, a homogeneous collagen hydrogel based on type I rat collagen is known at a concentration of less than 5 mg/ml. The method of preparing such a membrane consists of several stages. At the first stage, the acidic collagen solution is neutralized by mixing with the cell culture medium and 1.0 M NaOH solution. Further, to form a collagen hydrogel, the resulting neutral collagen solution is poured into special rectangular molds and incubated at +37°C for 30 minutes. Next, the obtained collagen hydrogels are subjected to plastic compression by applying a constant load of 120 g to the larger base of the hydrogel for 5 minutes, avoiding drying of the material. The material obtained in this way is hydrated, translucent and mechanically weak, does not hold a surgical suture, which limits its use for implantation.

Another technical solution close to the claimed invention in terms of the manufacturing method is a dry homogeneous collagen membrane based on type I porcine atelocollagen at a concentration of less than 5 mg / ml in the form of a Vitrigel collagen membrane, described in the article Calderón-Colón X., Xia Z., Breidenich J.L. , Mulreany D.G., Guo Q., Uy O.M., Tiffany J.E., Freund D.E., McCally R.L., Schein O.D., Elisseeff J.H., Trexler M.M. "Structure and properties of collagen vitrigel membranes for ocular repair and regeneration applications", Biomaterials, 2012, 33(33), 8286–8295 and Chae J.J., Ambrose W.M., Espinoza F.A., Mulreany D.G., Ng S., Takezawa T., Trexler M.M., Schein O.D., Chuck R.S., Elisseeff J.H. “Regeneration of corneal epithelium utilizing a collagen Vitrigel membrane in rabbit models for corneal stromal wound and limbal stem cell deficiency”, Acta Ophthalmol., 2015, 93 (1), e57–e66. The method of preparing such a membrane consists of several stages. At the first stage, the acid solution of collagen is neutralized. Next, the neutral collagen solution is converted into a hydrogel form by incubation at +37°C. At the third stage, the resulting hydrogel is converted into the form of a rigid glass-like membrane by drying in air. The dry collagen membrane thus obtained is biocompatible and transparent under in vivo conditions. The membrane is stored dry and must be rehydrated before use. However, the described dry collagen membrane based on porcine atelocollagen has a significant drawback inherent in the nature of such a product and the technology for its production - too weak strength characteristics (does not hold a surgical suture), narrowing the scope of this product only to the treatment of superficial defects in the human corneal epithelium.

Another technical solution close to the invention in terms of the manufacturing method is a homogeneous collagen hydrogel prepared from a concentrated collagen solution (90 mg/ml), described in the article Tidu A., Ghoubay-Benallaoua D., Lynch B., Haye B., Illoul C ., Allain J.M., Borderie V.M., Mosser G. "Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration", Acta Biomater., 2015, 22, 50–58. The authors of this work propose to improve the mechanical characteristics of the product not due to its chemical modification, but by increasing the concentration of collagen in the final product. The method of preparing such a material consists of concentrating the collagen solution by ultracentrifugation, and then transferring the collagen solution to the final form of collagen hydrogel, in ammonia vapor. As a result, the resulting artificial cornea is durable, transparent in distilled water (87% light transmission in the wavelength range of 380-780 nm) and compatible with corneal cells. On such an artificial cornea, human corneal epithelial cells are successfully cultivated, repeating their natural behavior in in vitro conditions. However, it should be noted that the described artificial cornea based on a concentrated collagen solution has a significant drawback inherent in its production technology - this is a significant clouding of collagen hydrogels under conditions as close as possible to in vivo (see the article by Achilli M, Mantovani D. "Tailoring Mechanical Properties of Collagen-Based Scaffolds for Vascular Tissue Engineering: The Effects of pH, Temperature and Ionic Strength on Gelation”, Polymers, 2010, 2(4), 664-680).

Closest to the invention in technical essence, method of manufacture and use is a dry collagen membrane of heterogeneous density based on native type I collagen (preferably native type I pig collagen), described in patent RU 2714943. This patent proposes a collagen membrane, which in a hydrated state is a collagen hydrogel of heterogeneous density (rigidity), having the shape of a flat cylinder, the main component of which is type I collagen. In addition to type I collagen, the composition of the proposed membrane also includes type V collagen. According to the patent, the addition of type V collagen makes it possible to control the size of the fibrils in the collagen hydrogel and, as a result, the transparency of the finished product.

Patent RU 2714943 also proposes a method for manufacturing a collagen membrane. The method of preparation is divided into two stages: obtaining a hydrogel from a sterile transparent aqueous solution of collagen and vitrifying the hydrogel with constant plastic compression. To prepare the gel, the authors used a sterile clear aqueous solution containing type I collagen and type V collagen.

Patent RU 2714943 also proposes a method for using a collagen membrane to enhance the biomechanical characteristics of the human cornea without losing its transparency; to enhance the biomechanical characteristics of human corneal leukomas in order to prepare them for keratoprosthetics; for the treatment of nonpermeable corneal leukomas using the DALK method, in which the Descemet's membrane is not damaged and the endothelial cells located on it are preserved; and/or to create a tissue-engineered construct of the cornea analog by forming a layer of human corneal epithelial cells on one of the membrane surfaces, and forming a layer of human corneal endothelial cells on the opposite membrane surface.

One of the disadvantages of the artificial cornea described in patent RU 2714943 is its complex composition (a mixture of type I collagen and type V collagen), which significantly increases the cost of production of such a material. Another disadvantage is the limited shelf life of the artificial cornea, since when stored in a dry state (more than 3 weeks), this material becomes excessively rigid and very brittle, and, as a result, it becomes impossible to use it for its intended purpose.

Thus, there remains an urgent need in the art for an artificial cornea that is biocompatible, non-immunogenic, optically transparent and durable, made from a highly purified biomaterial without the use of chemical modification.

Disclosure of the essence of the invention

The task solved in the framework of the present invention was to improve the technology for obtaining an artificial cornea, described in patent RU 2714943, in order to eliminate the above disadvantages in order to create a transparent, preferably homogeneous, collagen hydrogel for the restoration of the human cornea, obtained using a sterile transparent solution of collagen obtained by extraction from human or animal tissues without chemical modification.

In the first embodiment of the invention, a collagen membrane for corneal repair is provided, which is a collagen hydrogel containing native type I collagen, where the content of native type I collagen is from 5 to 20% wt. The collagen membrane of the invention may have a maximum tensile strength of more than 20N.

The second embodiment of the invention is a method for obtaining the collagen membrane described above. The process of preparing a collagen membrane is divided into three stages: a) obtaining a hydrogel from a sterile transparent aqueous solution of collagen, b) vitrifying the hydrogel under constant plastic compression, and c) a stage of rehydration of the collagen membrane obtained in stage b).

To prepare the gel, a sterile, clear aqueous solution containing type I collagen is used. For example, in the process of the invention, a collagen solution can be centrifuged in a cylindrical vessel, then incubated in ammonia vapor and then incubated in sterile purified water to form a gel. At the second stage, compression vitrification of the hydrogel is carried out. At this stage, the collagen hydrogel is dried. Simultaneously with drying, the cylindrical collagen hydrogel is subjected to plastic compression by applying a constant load in the range from 1 to 5 kg to the bases of the cylindrical hydrogel. As a result of vitrification, a homogeneous glass-like membrane is obtained, which, upon further soaking in sterile purified water, takes the form of a collagen membrane according to the invention.

In a third embodiment, the present invention relates to the use of the collagen membrane described above for improving/restoring the anatomical and functional characteristics of the cornea of the eye. This product can be used for anterior layering and strengthening keratoplasty for the treatment of leukomas and corneal ulcers of various etiologies, pterygium, keratoconus, as well as for the surgical treatment of traumatic injuries of the eyeball and for creating a tissue-engineered design of the cornea analogue by forming a layer of human corneal epithelial cells on one of the surfaces of collagen hydrogel and the formation of a layer of endothelial cells of the human cornea on its opposite surface.

Brief description of the drawings

In FIG. 1 is a photograph of a packaged product sample.

In FIG. 2A and FIG. 2B is a photograph of a product sample that is a uniform collagen membrane 21 mm in diameter and 500 µm thick.

In FIG. 3A and FIG. 3B is a photograph of an operated rabbit eye, 6 months postoperatively, before suture removal (FIG. 3A) and after suture removal (FIG. 3B).

Implementation of the invention

In the present invention, the terms "comprises" and "includes" and their derivatives are used interchangeably and are understood to be non-limiting, i. allowing the presence/use of other components, steps, conditions, etc., in addition to those listed explicitly. On the contrary, the expressions "consists of" and "composed of" and their derivatives are intended to indicate that the following listed components, steps, conditions, etc. are exhaustive.

The term "membrane" is used in the present invention in a broad sense as a designation of a thin transparent layer intended to be placed on the outer surface of eye structures in order to restore the cornea of the eye.

The term "corneal repair" is used in the present invention in a broad sense to refer to the elimination of any defects/damages of the cornea of the eye, including to improve and/or restore the anatomical and functional characteristics of the cornea of the eye.

In the present invention, when a range of possible values is given for any quantity, it is understood that the end points of this range are also included in the scope of the present invention. It is to be understood that all sub-ranges within the ranges given are also included within the scope of the present invention as if they were expressly stated. In the event that multiple ranges of possible values are given for any value, all ranges obtained by combining different endpoints from the indicated ranges are also included in the present invention as if they were explicitly stated.

In the event that any features of the invention are disclosed herein for one embodiment, those features may also be used in all other embodiments of the invention, provided that this does not contradict the letter and spirit of the invention.

Within the scope of the present invention, terms in the singular also cover the corresponding terms in the plural, and vice versa, unless the context clearly implies otherwise.

In this document, the expression "or" should be understood as "and/or", provided that this does not contradict the context, even though in some cases the expression "and/or" may be used explicitly.

As used herein, the expression "made essentially from" should be understood to indicate that although impurities and additional components known to those skilled in the art may be present in the product, these impurities and additional components may be present only in such an amount that does not significantly affect the phenomena and properties discussed in the present invention. In particular (but not limited to), the expression "made essentially from" includes, but is not limited to, the option "made exclusively from".

With the exception of the experimental part of the description, all numerical values expressing any quantities and conditions in the present invention are approximate and should be read as preceded by the term "approximately" or "about", even if these terms are not explicitly given. . On the contrary, in the experimental part of the description, all numerical values are given as accurately as possible, however, it should be understood that any experimentally determined value by its nature carries some error. Thus, all numerical values given in the experimental part of the description should be taken into account the existence of the specified experimental error and, at least, taking into account the number of significant figures given and standard rounding techniques.

In this document, all percentages are by weight, unless expressly stated otherwise.

As stated above, the present invention is based on a homogeneous collagen hydrogel having the shape of a flat rounded disk, which contains type I collagen and thus provides the necessary stiffness of the artificial cornea.

Preferably, the thickness of the collagen membrane according to the invention is 0.3 to 1.2 mm, preferably 400-800 µm, more preferably 410-710 µm, and its diameter is in the range of 15 to 25 mm, preferably 18-23 mm , more preferably 20-23 mm.

In a preferred embodiment, the collagen membrane is made essentially from type I collagen hydrogel, in some embodiments, exclusively from type I collagen hydrogel.

Preferably the type I collagen in the present invention is native type I collagen. Under the nativeness of collagen in the present invention is understood that this protein was extracted into solution from the tissue without enzymatic treatment with pepsin or other proteolytic drugs.

Type I collagen for use in the present invention can be obtained by known methods from various animals, including pigs, rats, cows, humans, and the like. In the most preferred embodiment within the scope of the present invention, the collagen is porcine collagen.

In a preferred embodiment, in the collagen membrane of the invention, type I collagen accounts for 5 to 20% by weight, most preferably 8 to 15% by weight. from the total mass of the hydrogel.

The light transmittance in the wavelength range from 400 to 780 nm (transparency) of the collagen membrane according to the invention, as a rule, is not less than 80% at a collagen hydrogel thickness of 0.5 mm, in the case when the membrane is made from a collagen solution with a concentration 20 mg/ml.

One of the distinguishing features of the collagen membrane according to the invention is that it is obtained and stored in a hydrated state, and such a collagen membrane for a long time retains strength characteristics sufficient for its fixation using a surgical suture. This allows you to increase the shelf life of at least 1 year.

The collagen membrane of the invention can be obtained by the method according to the second embodiment of the invention, wherein said method includes a gel formation step, a vitrification step under constant plastic compression, and a rehydration step.

Gel formation step

To carry out the gel formation stage, it is most preferable to use a sterile transparent aqueous solution containing native type I porcine collagen, obtained in accordance with the description given in patent RU 2715715 and/or in patent RU 2714943. For clarity, below is a brief description of the method for obtaining a sterile transparent concentrated solution of biocompatible collagen according to patent RU 2715715.

This method of obtaining a collagen solution includes several stages performed in a given sequence:

1) extraction of collagen-containing tissue, such as tendons, placenta of mammalian animals, such as bovine, pig, rat or human, into a solution of dilute (not more than 0.5 M) organic acid after thorough washing from protein non-collagen impurities using alkaline or neutral water-salt solutions (for example, 0.5M Na ₂ HPO ₄ ) and from impurity proteins, glycoproteins, fat-containing aggregates using organic solvents (for example, acetone, ethanol, acetonitrile);

2) repeated (at least 3 times) purification of the acidic water-salt extract from potentially immunogenic macromolecules by selective precipitation of collagen with high concentrations of sodium chloride (up to 3M) at a low temperature of the solution (less than 10°C);

3) Purification of the acidic water-salt extract from the remaining impurities of potentially immunogenic macromolecules by adsorption of the remaining impurities on DEAE-cellulose;

4) Purification of pyrogenic impurities by adsorption of liposaccharides on an affinity sorbent (for example, with immobilized polymyxin (Affi-Prep®) or LPS-specific antibodies);

5) Purification from impurities of ordinary and spore-forming microorganisms, as well as supramolecular aggregates of collagen by microfiltration of acidic solutions of collagen through membranes with a pore diameter of 0.22-0.45 microns;

6) Bringing the collagen solution to the required concentration of collagen protein in it, the molecules of which retain the triple helix structure and have a molecular weight of at least 300 kDa, by one of the known methods: ultrafiltration, evaporation, lyophilization and dissolution, or absorption of water by insoluble hydrophilic polymers.

The total concentration of collagen in the aqueous collagen solution used in the present invention can be adjusted by one skilled in the art, but is typically in the range of 5 to 35 mg/mL, most preferably in the range of 20 to 30 mg/mL.

To form a collagen hydrogel, the necessary volume of a sterile transparent collagen solution is carefully added to a cylindrical bowl, avoiding the appearance of air bubbles. Next, the bowl with the solution is centrifuged, for example at a rotor speed of 3000-5000 rpm, most preferably 4000 rpm. The centrifugation is preferably carried out at +5° C. for 30 minutes. In one embodiment, centrifugation may be performed in a plate (eg, in a 24 well plate).

After centrifugation is completed, the form with a collagen solution is incubated in ammonia vapor. In a preferred embodiment, the incubation in ammonia vapor is carried out at +4°C for 12 hours. The resulting collagen hydrogel is removed from the mold and incubated in sterile purified water. In a preferred embodiment, incubation in sterile purified water is carried out for 24 hours at +20°C. As sterile purified water, water purified by methods known to the person skilled in the art, such as distillation, reverse osmosis, and the like, can be used.

The next step is compression vitrification. At this stage, the collagen hydrogel is dried. In a preferred embodiment, drying is carried out under conditions of controlled temperature and humidity, for example at 20°C and at a relative humidity of 60-70% for 1 week. Simultaneously with drying, the cylindrical collagen hydrogel is subjected to plastic compression by applying a constant load to the bases of the hydrogel throughout the entire drying time.

A distinctive feature of the method for producing a collagen membrane according to the invention is that, in contrast to the load of 15-50 g used in patent RU 2714943 at the stage of compression vitrification, the present invention uses a higher load in the range from 1 to 5 kg, preferably about 3 kg. Applying such a load during drying and compression vitrification allows the formation of a homogeneous transparent and strong collagen membrane without the use of type V collagen.

The next step is the rehydration of the dried collagen membrane. To do this, the resulting membrane is soaked in sterile purified water. In a preferred embodiment, the soaking is carried out for a short period of time, such as 5 minutes. Then, as will be understood by those skilled in the art, the product can be placed in a pre-sterilized primary packaging that meets the requirements of GOST ISO 11607-1-2018, for example, by laying the product on a flat substrate, for example, on a fluoroplast substrate. After that, the product is packed under vacuum, for example in a sterile metallized vacuum bag. Such packaging of the product makes it possible to preserve its properties (thickness, diameter, transparency, strength, biocompatibility, sterility) for at least one year when stored at a temperature of 4°C to 10°C.

When using the required size of the collagen membrane, the surgeon can cut directly during the operation.

The collagen membrane according to the invention is advantageously characterized by a maximum tensile strength of more than 20N, and preferably more than 23N. The maximum tensile strength can be measured by any method known to those skilled in the art, for example according to the scaled analog of the ASTM D5748 test.

Further in the description, the implementation of the invention, the method of manufacturing a collagen membrane and its use are described in more detail. The examples given are illustrative and should not be taken as a basis for limiting the claims of the invention.

Example 1 Manufacturing method

For the preparation of the product, a sterile transparent aqueous solution was used containing 30 mg/ml of native porcine type I collagen, obtained according to patent RU 2715715, as disclosed above in the present invention. To form a collagen hydrogel, 0.5 ml of a sterile, clear collagen solution is carefully added to a cylindrical bowl with a diameter of 15.4 mm, avoiding the appearance of air bubbles. Next, the bowl with the solution is centrifuged at a speed of 4000 rpm at a temperature of +5°C for 30 minutes, after which the form with the collagen solution is incubated in ammonia vapor at +4°C for 12 hours. The resulting collagen hydrogel is removed from the mold and incubated in 20 ml of sterile distilled water for 24 hours at +20°C.

Then the collagen hydrogel was dried under controlled temperature (20°C) and humidity (60-70%) for 1 week. At the same time, the cylindrical collagen hydrogel was subjected to plastic compression by applying a constant load of 3 kg to the bases of the hydrogel throughout the entire drying time. At the end of the vitrification process, the compressed collagen hydrogel took the form of a homogeneous glass-like membrane.

The resulting membrane was soaked in sterile purified water for 5 minutes, after which it was laid out on a flat PTFE substrate, placed in a pre-sterilized primary package that meets the requirements of GOST ISO 11607-1-2018, and then packed under vacuum in a sterile metallized vacuum bag.

The membrane thickness after rehydration was 0.52±0.03 mm, and the diameter was 21.0±0.3 mm (see Fig. 1). A product packed in this way can retain its properties (thickness, diameter, transparency, strength, biocompatibility, sterility) for at least one year when stored at a temperature of 4°C to 10°C.

Measurement of maximum breaking load

The resulting product was clamped between two metal plates with a 3 mm hole in the center, so that the hole was in the central region. The finished structure was immersed in physiological saline to prevent the membrane from drying out during the study. Biomechanical properties were evaluated using a scaled analogue of the ASTM D5748 test. The study consists in biaxial loading of the collagen membrane at a constant rate until puncture. The tests were carried out on an Instron universal testing machine model 5982 using a ±50 N load cell of the 2530 series. Based on the test results, the maximum tensile load characterizing the strength of the samples was determined, which for this membrane was 23.2 ± 0.5 N.

Example 2. Effect of the load applied during plastic compression on the transparency and strength of the finished product

In accordance with the method described in example 1, and the method described in patent RU 2714943, varying the load applied during plastic compression, various samples of a collagen membrane with a thickness of about 500 μm were prepared from a sterile transparent collagen solution with a concentration of 25 mg/ml. The transparency of products was controlled using a PE-5400UF spectrophotometer (Ekroskhim, Russia) in the wavelength range from 400 to 780 nm. From the results presented in Table 1, it can be seen that with a load of 1 to 5 kg, samples of collagen hydrogels, both with and without type V collagen, have comparable transparency. At the same time, samples of collagen hydrogels without type V collagen had better mechanical characteristics. Thus, the load applied during plastic compression in the range from 1 to 5 kg, preferably 3 kg, makes it possible to produce a collagen hydrogel, which, in its optical characteristics, is comparable to a collagen hydrogel prepared according to the method described in patent RU 2714943. However, a higher load allows to simplify the composition of the hydrogel and increase the strength of the collagen hydrogel compared to the collagen hydrogel prepared according to the method described in the patent RU 2714943.

Table 1

Product Load applied during plastic compression Coefficient of light transmission through a product with a thickness of 500 microns,% Maximum breaking load, N According to patent RU 2714943 (using type V collagen) 30 g 85.1±1.5 (λ=400)
95.3±1.1 (λ=780) 17.1±0.6 500 g 85.5±1.1 (λ=400)
95.6±1.6 (λ=780) 17.7±0.8 1000 g 85.8±1.7 (λ=400)
95.9±1.4 (λ=780) 17.6±0.3 3000 g 85.8±1.5 (λ=400)
95.9±1.6 (λ=780) 17.8±0.7 5000 g 85.8±1.3 (λ=400)
95.9±1.7 (λ=780) 17.6±0.3 10000 g Material destruction According to example 1 (according to the invention) 30 g 75.1±1.2 (λ=400)
83.3±1.1 (λ=780) 23.2±0.5 500 g 78.1±1.2 (λ=400)
85.3±0.9 (λ=780) 23.5±0.3 1000 g 80.1±1.0 (λ=400)
90.3±1.3 (λ=780) 23.5±0.6 3000 g 81.2±0.5 (λ=400)
92.3±1.0 (λ=780) 23.4±0.1 5000 g 81.3±0.8 (λ=400)
92.4±1.1 (λ=780) 23.5±0.2 10000 g Material destruction

Example 3 Application to enhance the biomechanical characteristics of the eye

According to the method described in Example 1, collagen membranes about 500 µm thick and 18.5 mm in diameter were prepared from a sterile, clear collagen solution at a concentration of 30 mg/ml.

Twelve male chinchilla rabbits weighing 3.5 kg were operated on the left eye. Performed retrobulbar blockade with 0.5% novocaine. A corneal incision was made with a length of 5 mm for 2/3 of its depth concentric to the limbus. In this plane, a corneal intrastromal pocket "from limbus to limbus" was formed using a stratifier. Using forceps for tying and a spatula, the implant was inserted into the cavity of the formed pocket. The incision was sutured. Then, for one week, okomistin was instilled 1 drop 2 times a day.

The clinical assessment of the condition of the eyes of animals was carried out according to the degree of inflammatory reaction, corneal vascularization, and the intensity of corneal opacity.

The experimental results were evaluated morphologically. The cornea was excised along the circumference of the limbus, and then it was fixed in formalin for 24 hours. The resulting disc of the cornea was cut in half. Both fragments were dehydrated in ascending alcohols and embedded in paraffin. Sections 8–10 µm thick were made on a microtome and stained with hematoxylin and eosin. Corneal sections were examined under a light microscope at 16- and 40-fold magnification.

To study the biomechanical characteristics, the cornea was cut out along the circumference, while capturing a portion of the sclera, retreating 3 mm from the limbus. For the purity of the experiment, freshly cut animal tissues obtained immediately before the study were used. The obtained disk of the cornea was clamped between two metal plates with a hole in the center corresponding to the diameter of the cornea, so that the fixation took place behind the sclera tissue, and the cornea was intact. The finished structure was immersed in physiological saline to prevent the tissues from drying out during the study. The assessment of biomechanical properties was carried out by examining tissues for puncture using Instron tensile testing machines.

In the early postoperative period, there was a slight corneal edema. The edema was mild and apparently due to the presence of an operative injury. Clinical signs of acute infectious lesions of surrounding tissues were not found during examination. Further, the clinical picture was stable. After 2 months, there were no clinically significant changes. By the end of the observation period, after 6 months, a histological examination of the graft area showed its partial disintegration and integration with the surrounding stroma. The cornea is slightly thickened. The study of biomechanical properties showed an increase in the strength characteristics of tissues by 2.5 times.

Example 4 Corneal Integrity Application

According to the method described in Example 1, collagen membranes about 410 µm thick and 18.5 mm in diameter were prepared from a sterile, clear collagen solution at a concentration of 30 mg/ml.

Six male chinchilla rabbits weighing 3.5 kg were operated on the left eye. Performed retrobulbar blockade with 0.5% novocaine. Non-through trepanation of the superficial layers of the cornea was performed at 2/3 of its depth. Then, using a delaminator, the surface layers were excised to form a trepanation bed in the area of the operation, in which a collagen implant was then placed in the form of a disc with a diameter 1 mm larger than the diameter of the burr hole and fixed with a continuous suture. Excess material was tucked under the edge of the trepanation and provided additional mechanical fixation. Then, for one week, okomistin was instilled 1 drop 2 times a day.

Clinical assessment of the condition of the eyes of animals, morphological was carried out according to example 3.

In the early postoperative period, there was a slight corneal edema. The edema was mild and apparently due to the presence of an operative injury. Clinical signs of acute infectious lesions of surrounding tissues were not found during examination. On the 7th day, the appearance of newly formed vessels in the eccentric limbus was noted, which became empty by the end of the 2nd week. By the end of the first month, there was a stable stabilization of the clinical picture. After 2 months, there were no clinically significant changes. By the end of the observation period (6 months), the rabbit showed clear graft engraftment and no signs of inflammation (Fig. 3). Histological examination of the graft area showed its partial defibration and integration with the surrounding stroma, especially at the point of contact with the edge of the burr hole; in some places, a small amount of connective tissue was formed under the graft and signs of scarring were observed. The cornea is well epithelized, smooth and shiny.

Example 5. Illustration of the need to use vacuum packaging to preserve the main properties of the product

According to the method described in Example 1, collagen membranes about 500 µm thick and 18.5 mm in diameter were prepared from a sterile, clear collagen solution at a concentration of 30 mg/ml. The membranes were divided into 3 groups according to the method of storage. The first is in sealed vacuum packaging; the second - in a sealed package that meets the requirements of GOST ISO 11607-1-2018; the third - in a sealed glass with distilled water. Five membranes in each group were placed for experimental storage in the temperature range from +4°C to +10°C. A year later, the membranes were removed to evaluate their main properties (thickness, diameter, transparency, strength, biocompatibility, sterility) in comparison with freshly prepared material. The results are shown in table 2. As can be seen from the results presented in table 2, only membranes from group 1 (in sealed vacuum packaging) can maintain all their properties (thickness, diameter, transparency, strength, biocompatibility, sterility), at least within one year when stored at temperatures from +4°С to +10°.

table 2

Thickness, microns Diameter, mm light transmittance,
% Stress on
gap, N Biocompatibility
bridge* Sterility Group 1 500; 490; 510; 505; 502 20.1; 20.3; 20.2; 20.1; 20.0 λ=400: 81.1; 82.0; 80.5; 80.8; 81.4 λ=780: 92.3; 93.1; 92.0; 92.5;92.8 23.4; 23.3; 23.5; 23.6; 23.4 Yes, for everyone Yes, for everyone Group 2 300; 310; 305; 300; 306 20.1; 20.3; 20.2; 20.1; 20.0 λ=400: 82.1; 81.0; 81.5; 81.8; 80.4 λ=780: 91.3; 92.1; 92.0; 91.5;92.8 Unsuitable for assessment, collapsed during manipulation Yes, for everyone Yes, for everyone Group 3 1100; 1050; 1070; 1060; 1005 20.1; 20.3; 20.2; 20.1; 20.0 λ=400: 80.1; 80.0; 79.5; 79.8; 80.4λ=780: 90.3; 91.1; 90.0; 90.5;90.8 10.4; 10.3; 9.7; 10.3; 8.4 Yes, for everyone Yes, for everyone Control 500; 495; 505; 510; 508 20.1; 20.3; 20.2; 20.1; 20.0 λ=400: 81.1; 82.0; 80.5; 80.8; 81.4 λ=780: 92.3; 93.1; 92.0; 92.5;92.8 23.3; 23.4; 23.3; 23.5; 23.5 Yes, for everyone Yes, for everyone

*Biocompatibility tested in vitro using rabbit corneal stromal cells.

Example 6. Effect of the content of native type I collagen in the finished product on transparency and strength

In accordance with the method described in example 1, collagen membranes were prepared with a thickness of about 500 μm and a diameter of 20 mm, in which the content of native type I collagen varied in the range from 2% to 30% based on the total weight of the product.

The transparency of products was controlled using a PE-5400UF spectrophotometer (Ekroskhim, Russia) in the wavelength range from 400 to 780 nm. The tensile load was measured according to the method described in example 1. In accordance with the results obtained, presented in table 3, the optimal transparency-strength content of native type I collagen in the product is from 5 to 20% wt., preferably from 8 to 15% based on the total weight of the finished product.

Table 3

Content of native type I collagen, % Light transmittance, % Breaking load, N 2 87.5±0.3 (λ=400); 98.3±0.2 (λ=780) 18.2 5 85.2±0.6 (λ=400); 95.3±0.8 (λ=780) 20.4 eight 83.2±0.8 (λ=400); 93.2±0.5 (λ=780) 21.4 ten 81.0±0.8 (λ=400); 91.5±1.0 (λ=780) 23.4 fifteen 80.5±0.6 (λ=400); 90.6±1.2 (λ=780) 25.4 twenty 80.0±0.2 (λ=400); 90.1±1.1 (λ=780) 26.4 thirty 73.0±0.2 (λ=400); 84.1±1.1 (λ=780) 35.1

Claims (27)

1. Collagen membrane for corneal repair, which is a collagen hydrogel containing native type I collagen, where the content of native type I collagen is from 5 to 20 wt.% of the total mass of collagen hydrogel, and where the specified collagen membrane is characterized by a maximum rupture load of more than 20 N. 2. The collagen membrane of claim 1, wherein the collagen membrane is made essentially from a type I collagen hydrogel. 3. Collagen membrane according to claim 1, wherein the collagen membrane is exclusively made from type I collagen hydrogel. 4. Collagen membrane according to any one of paragraphs. 1-3, the thickness of which is in the range of 0.3 to 1.2 mm and the diameter of which is in the range of 18 to 25 mm. 5. Collagen membrane according to any one of paragraphs. 1-4, the thickness of which is in the range of 0.4 to 0.8 mm and the diameter of which is in the range of 20 to 23 mm. 6. Collagen membrane according to any one of paragraphs. 1-5, in which the content of native type I collagen is from 8 to 15 wt.% based on the total weight of the collagen hydrogel. 7. Collagen membrane according to any one of paragraphs. 1-6, wherein the native type I collagen is native porcine type I collagen. 8. Collagen membrane according to any one of paragraphs. 1-7, where the maximum breaking load is more than 23 N. 9. Collagen membrane according to any one of paragraphs. 1-8 which is biocompatible, sterile and optically clear. 10. Collagen membrane according to any one of paragraphs. 1-9, the transparency of which is characterized by a light transmittance in the wavelength range from 400 to 780 nm of at least 80% with an artificial cornea thickness of 0.5 mm, in the case when it is made from a collagen solution with a concentration of 20 mg / ml. 11. Collagen membrane according to any one of paragraphs. 1-10, vacuum-packed and with a shelf life of at least 1 year. 12. The method of obtaining a collagen membrane for the restoration of the cornea according to any one of paragraphs. 1-11, including the following steps: a) the step of obtaining a cylindrical hydrogel from a sterile transparent aqueous solution of type I collagen; b) the stage of vitrification of the hydrogel obtained in stage a), with constant plastic compression to obtain a homogeneous glassy membrane, and plastic compression is carried out by applying a constant load in the range from 1 to 5 kg to the bases of the collagen hydrogel; c) a step of rehydrating the collagen membrane obtained in step b). 13. The method according to claim 12, in which plastic compression is carried out by applying a constant load of 3 kg to the bases of the collagen hydrogel. 14. The method of claim 13 wherein a constant load is applied throughout the duration of the vitrification. 15. The method according to any one of paragraphs. 12-14, in which the total concentration of collagen in the collagen solution is from 5 to 35 mg/ml. 16. The method of claim 15 wherein the total concentration of collagen in the collagen solution is 20 to 30 mg/mL. 17. The method according to any one of paragraphs. 12-16, in which the preparation of the hydrogel in step a) is carried out by centrifuging the collagen solution in a cylindrical vessel, incubating in ammonia vapor, followed by incubating in sterile purified water. 18. The method according to any one of paragraphs. 12-17, wherein the vitrification step is carried out under controlled temperature and humidity conditions. 19. The method according to claim 18, wherein the vitrification step is carried out at a temperature of 20°C and at a relative humidity of 60-70%. 20. The method according to any one of paragraphs. 12-19, in which, at the stage of rehydration, the resulting membrane is soaked in sterile purified water for 5 minutes. 21. The method according to any one of paragraphs. 12-20, in which the rehydrated collagen membrane is further laid out on a flat PTFE substrate, placed in a pre-sterilized primary packaging, and then vacuum-packed in a sterile metallized vacuum bag. 22. Collagen membrane for the restoration of the cornea, obtained by the method according to any one of paragraphs. 12-21. 23. The use of a collagen membrane for the restoration of the cornea according to any one of paragraphs. 1-11 or 22 to improve and/or restore the anatomical and functional characteristics of the cornea of the eye. 24. The use according to claim 23, where the collagen membrane for corneal repair is used for anterior layering and strengthening keratoplasty for the treatment of leukomas and / or corneal ulcers of various etiologies, pterygium or keratoconus, or in the surgical treatment of traumatic injuries of the eyeball, or to create a tissue engineering structure analogue of the cornea by forming a layer of human corneal epithelial cells on one of the surfaces of the collagen hydrogel and forming a layer of human corneal endothelial cells on its opposite surface.

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