Method for replacing bulbar conjunctiva defect

Abstract

FIELD: medicine; ophthalmology.SUBSTANCE: using a collagen membrane based on highly purified native chemically unmodified collagen type I - Viscoll at a concentration of 10 or 30 mg / ml and a thickness of 100 mcm, at a collagen concentration of 10 mg / ml, the membrane is subjected to crosslinking and fixed with interrupted or continuous sutures. In a particular case, the collagen membrane at a concentration of 10 mg / ml is subjected to ultraviolet crosslinking. Wherein, for 10 minutes, 0.1% riboflavin-mononucleotide solution with dextran is instilled onto the surface of the cut-out collagen membrane at a frequency of 1 drop per minute for 30 minutes, then ultraviolet irradiation is carried out for 30 minutes at a wavelength of 370 nm, a beam diameter of 8.0 mm, a power density of 3.0 mW / cm2.EFFECT: creating favourable conditions for the regeneration of the patient's own conjunctiva.2 cl, 2 ex

Description

[0001] The invention relates to ophthalmology and is intended to replace a defect in the boulevard conjunctiva.

[0002] The conjunctiva is the largest component of the ocular surface in area, consists of vascularized connective tissue and covering it multilayered nonkeratinizing epithelium interspersed with goblet cells, which provide the mucin component of the tear fluid (Paulsen F. Functional anatomy and immunological interactions of ocular surface and adnexa. Dev Ophthalmol. 2008;41:21-35. https: / / doi.org / 10.1159 / 000131068). When the conjunctiva is damaged, wound healing occurs with reepithelialization and the formation of fibrous scar tissue (Hatton MP, Rubin PA. Conjunctival regeneration. Adv Biochem Eng Biotechnol. 2005;94:125-40. https: / / doi.org / 10.1007 / b100002). Currently, conjunctival reconstruction involves excision of scar tissue and its replacement with various materials, including autologous conjunctival tissue, autologous mucous membranes, and amniotic membrane grafting.

[0003] A method for replacing conjunctival defects with an autograft of the labial mucosa is known (Gushchina MB, Tereshchenko AV, Nerobeev AI. Method for replacing conjunctival defects (variants). Patent RU 2762768; published 12 / 22 / 2021; Bulletin No. 36; Bochkareva AN, Egorov VV, Smolyakova GP, Banshchikov PA. Method for surgical treatment of pterygium. Patent RU 2611939, published 03 / 01 / 2017, Bulletin No. 7; Mai C, Bertelmann E. Oral mucosal grafts: old technique in new light. Ophthalmic Res. 2013;50(2):91-98). The disadvantages of this method include possible infection of the conjunctiva of the eye with oral microflora, stricture of the transplanted tissue, as well as obvious differences in volume, color, and texture, which differ from the recipient tissue, which may subsequently be regarded by the patient as a cosmetic defect.

[0004] A method of removing the altered conjunctiva and subsequent covering of the defect with amniotic membrane is known (Zolotarev A.V., Milyudin E.S. Surgical treatment of recurrent pterygium with plastic surgery using silica-dried amniotic membrane / / Bulletin of Ophthalmology. - 2007. - No. 7. - P. 39-42). Despite the fact that AM is a frequently used native material for plastic surgery, this type of transplant carries a risk of transmitting blood-borne diseases.

[0005] A known method for conjunctiva restoration is using allogeneic cultured limbal epithelial cells (CLET) (Cheng J, Zhai H, Wang J, Duan H, Zhou Q. Long-term outcome of allogeneic cultivated limbal epithelial transplantation for symblepharon caused by severe ocular burns. BMC Ophtalmol. 2017; 31; 17(1): 8), which consists of covering the bulbar and palpebral wound surface with an allogeneic cell culture. However, the authors themselves note that in some cases, a contraction of the transplanted tissue occurs, which subsequently leads to trichiasis, a contraction of the conjunctival fornices of the eyelids and the formation of entropion and symblepharon. This method also involves cell culturing, which is associated with expensive equipment and consumables.

[0006] Another well-known method of conjunctival defect plastic surgery is reconstruction using bulbar autoconjunctiva (Herbert E Kaufman HE, Thomas EL, Prevention and treatment of symblepharon. Am J Ophthalmol. 1979; 88(3 Pt 1):419-423; Tikhonov AB, Safonova MK, Method of surgical removal of corneal pterygium with defect plastic surgery using intact bulbar conjunctiva, Patent RU 2736914, published 11 / 23 / 2020, Bulletin No. 33). This method ensures complete closure of the postoperative wound defect. Despite its effectiveness, the use of the method is limited in bilateral diseases. Excision of the donor tissue site requires healthy intact conjunctiva in at least one eye. Therefore, conjunctival autotransplantation is impossible in cases of bilateral lesions. Disadvantages of this method also include the inability to harvest a large graft and the presence of a wound at the donor site, which requires covering or additional suturing.

[0007] The closest method for the same purpose is the method involving covering the conjunctival defect with a collagen membrane, described in the article by Witt, J., Borrelli, M., Schrader, S, et al. Evaluation of Plastic-Compressed Collagen for Conjunctival Repair in a Rabbit Model. Tissue engineering Part A, 2019, 25(15-16), 1084-1095. The authors conducted an experimental study and evaluated the surgical use of plastic-compressed collagen (Drechsler C, Kunze A, Qureshi A, Grobe G, Reichl S, Geerling G, et al. Development of a conjunctival tissue substitute based on plastic compressed collagen. Journal of tissue engineering and regenerative medicine 2015). The researchers concluded that a membrane made from collagen pressed into plastic could be used in conjunctival reconstruction without the risk of scar formation during the healing phase.However, this study did not conduct in vitro studies. It is unknown how biocompatible the material is with healthy tissue, and how this chemically modified collagen affects the survival and metabolic activity of conjunctival cells, to draw conclusions about the viability of this approach. Also, on days 10 and 28 of the study, granulocytic infiltration was noted in the histological specimen, which may be related to an immune reaction to the implanted material. Furthermore, postoperative follow-up of 3 months or more was not conducted.

[0008] The objective of the invention is to develop a method for restoring conjunctival defects using a biopolymer material based on native collagen.

[0009] The technical result of the proposed method is the creation of favorable conditions for the regeneration of one’s own conjunctiva.

[0010] The technical result is achieved through the use of a biopolymer material based on native type I collagen of optimal thickness with an increased collagen concentration and possible preliminary treatment of the membrane using crosslinking to replace the defect of the bulbar conjunctiva.

[0011] Native chemically unmodified collagen type I (http: / / imtek.ru / catalog / products / cell-culture / viscoll / Viscoll_Kit_for_3D_bioprinting_and_cell_culture / ) is an analogue of the extracellular matrix of conjunctival tissue, providing cell migration and regeneration of the altered ocular surface on its basis due to the mechanisms of its own cellular regeneration for its restoration. Thus, native chemically unmodified collagen type I has biocompatibility and biomechanical properties that are as similar as possible to healthy tissue and functions as a matrix for the regeneration of native conjunctiva. The collagen membrane (CM) was manufactured in accordance with a previously published method [Andreev AY, Osidak EO, Grigoriev TE, et al. A new collagen scaffold for the improvement of corneal biomechanical properties in a rabbit model. Exp Eye Res. 2021;207:108580. doi:10.1016 / j.exer.2021.108580].However, during the in vivo stage, using a material with a collagen concentration of 10 mg / ml, technical difficulties arose with fixation during CM implantation, namely: cutting through the applied interrupted sutures after CM hydration; CM implantation into a pre-formed intrascleral pocket and its subsequent dislocation in the postoperative period, as well as scleral perforation during the formation of intrascleral pockets due to the thinner structure of the fibrous membrane in the study animals; CM fixation using sulfacrylate glue with its polymerization and formation of a rigid film (which did not ensure the elastic properties of the conjunctiva). Also, to improve the mechanical properties of CM, a session of ultraviolet crosslinking was performed in some experimental rabbits before fixation.Compared to the intact membrane before crosslinking, biomechanical tests revealed increased elasticity, stiffness, and viscosity moduli, making the resulting sample even more resistant to external influences. The collagen crosslinking procedure was as follows: After hydration for 10 minutes, the CM was cut to obtain the material using a 4 mm diameter trephine. Next, a 0.1% solution of riboflavin mononucleotide and dextran (Khalimov AR The role of dextran in ophthalmic riboflavin solution for UV corneal crosslinking. Point of view. East - West. No. 1 2018 DOI: https: / / doi.org / 10.25276 / 2410-1257-2018-1-136-138) was installed on the surface of the CM with an instillation rate of 1 time per minute for 30 minutes, then the material was irradiated with ultraviolet light for 30 minutes using the following parameters: wavelength 370 nm, beam diameter 8.0 mm, power density 3.0 mW / cm. 2 .

[0012] To simulate real-life ophthalmic surgical conditions, a suture fixation test was performed on CM with collagen concentrations of 10, 20, and 30 mg / ml and a thickness of 100 μm, corresponding to the thickness of the bulbar conjunctiva. The test was performed by an experienced ophthalmic surgeon using standard instrumentation: microsurgical tweezers, a Castroviejo needle holder, and suture material (10 / 0 nylon). The objective was to evaluate tactile feedback, membrane resistance to deformation during suturing, and the risk of membrane eruption under clinically realistic conditions. The surgeon used two types of sutures: 12 interrupted sutures and a continuous suture along the edge of the CM. After fixing the sutures, the surgeon simulated dynamic conditions (pulling the nodes, moving the membrane with tweezers) and visually assessed the material's resistance to deformation under tension, the presence of micro-tears in the suture area, and the preservation of the integrity of the CM after 5 tension cycles.Testing was also carried out on a Nanovea indentation machine to evaluate the biomechanical properties of the samples.

[0013] When evaluating the CM with a collagen concentration of 10 mg / ml, when attempting to tighten the knot, the material deformed already at the first stitch, requiring minimal force. The suture easily cut through the material, leaving ragged edges around the puncture site, making the membrane unsuitable for fixation. At a concentration of 10 mg / ml, the CM showed a low Young's modulus (elasticity) of 0.1254 GPa under a load of 0.25 mN, which explains the rapid cut-through already during the intraoperative period. At a concentration of 20 mg / ml, after 3 cycles of interrupted suture tension, linear micro-tears 1-2 mm long appeared along the suture, which in the postoperative period led to cut-through of the suture and, consequently, dislocation of the CM. When applying a continuous suture, the CM was deformed, which is also unacceptable in the reconstruction of conjunctival defects. However, with preliminary crosslinking of the collagen type I membrane - Viscoll at a concentration of 10 mg / ml, it was possible to influence the biomechanical properties of collagen membranes.Compared to the membrane before and after ultraviolet crosslinking, Young's modulus (0.00897 GPa and 0.0511 GPa, respectively), stiffness (0.00000761 GPa and 0.0000165 GPa), and viscosity (0.3211 MPa and 0.4015 MPa) increased—the resulting sample became less viscous and more resistant to external influences. A series of suture fixation tests using interrupted sutures (Nylon 10 / 0) ​​were also performed to evaluate the potential use of the modified collagen membrane in ophthalmic surgery. The samples retained their shape and integrity during these tests.

[0014] At a concentration of 30 mg / ml, there was no suture cutting through, and the membrane demonstrated optimal rigidity: interrupted sutures fixed the material without cutting through even under maximum tension, and a continuous circumferential suture maintained the integrity of the membrane edge, with no signs of rupture or stretching. Young's modulus, which characterizes the elasticity of the CM, is comparable to that of the bulbar conjunctiva and amounted to 0.00384±0.0052 GPa and 0.00324±0.0041 GPa, respectively, under a load of 0.25 mN. These data confirm that a concentration of 30 mg / ml creates the structural integrity necessary for clinical use, while lower concentrations (10, 20 mg / ml) do not provide sufficient resistance to mechanical loads in conjunctival reconstruction.

[0015] In view of the above, in order to minimize the multi-stage preparation of CM and increase its resistance to deformation, it was decided to carry out the following. 0.2 ml of a sterile solution of highly purified native type I collagen with an increasing concentration of 30 mg / ml, isolated by acid extraction from porcine tendons (Viscoll; Imtek LLC, Moscow, Russia) was added to one well of a 24-well plate. To remove air bubbles from the collagen solution, the plates were centrifuged in an Avanti J-26XP centrifuge (JS-5.2 oscillating rotor; Beckman Coulter, Inc) at 3200 rpm and 4°C for 30 minutes. To induce collagen gelation, the plates were incubated in ammonia vapor for 12 hours at 20°C. After incubation, collagen hydrogels were collected, immersed in water for injection (Solopharm, Russia) and incubated for 24 hours at 20°C.In the next step, the collagen hydrogels were placed between two Teflon plates, one of which was subjected to a constant load of 3 kg until complete vitrification. For long-term storage, the resulting material was rehydrated in water for injection (Solopharm, Russia) for 5 minutes. The CM was then placed on a square Teflon plate and hermetically sealed in sterile primary packaging that complies with ISO 11607-1 (Clinipak, Russia). The material in the primary packaging was hermetically sealed under vacuum in a secondary vacuum packaging, allowing the CM to be stored at temperatures between 4°C and 10°C for 12 months from the packaging date. All manipulations were performed under aseptic conditions.

[0016] To evaluate the collagen membrane used in vitro, a colorimetric formazan MTS test, staining with calcein-AM (Abeam, USA), Hext nuclear stain (PanEco, Russia), and immunocytochemical staining for conjunctival epithelial cell markers CK7 and MUC5AC were performed to study the metabolic activity, survival, and phenotyping of conjunctival epithelial cells. Cell morphology was assessed at all stages of culture using phase-contrast microscopy.

[0017] The biomechanical parameters of the collagen membrane (CM) and conjunctival tissue were studied using a test on a Nanovea (California, USA) indenter testing machine under a load of 0.75 mN.

[0018] Cell migration from the explants was observed on the third day of cultivation. The cells gradually filled the surface of the culture dish, forming a continuous monolayer. On the seventh day, the formation of a subconfluent monolayer was observed, consisting of small cuboidal epithelial cells characterized by a high nuclear-to-cytoplasmic ratio.

[0019] On day 3 of cultivation on the CM, migration of conjunctival epithelial cells into the inner layers of the collagen monolayer was observed, indicating high cellular adhesion and the material's ability to create a favorable microenvironment for conjunctival epithelialization. Human conjunctival epithelial cells maintained a 64.7% survival rate on the membrane. The collagen membrane was metabolically comparable to the control (101.3%), suggesting that the material studied in this experiment does not reduce cellular metabolic activity and is biocompatible with conjunctival epithelial cells. Phenotyping of the cultured cells revealed that almost all cells expressed CK7, and MUC5AC was present in some cells.

[0020] A comparative analysis of the biomechanical characteristics revealed that Young's modulus, which characterizes the elasticity of the CM, is comparable to that of the bulbar conjunctiva and amounts to 0.0008739±0.0004332 GPa and 0.0009472±0.001323 GPa, respectively. No statistically significant differences were found between them (p=0.0549), suggesting that the elastic properties of both materials are similar.

[0021] When assessing hardness, significantly higher values ​​for this parameter were recorded for the collagen membrane compared to the bulbar conjunctiva (p<0.0001). Conversely, the viscosity of the CM was significantly lower than that of natural tissue (p<0.0001).

[0022] Based on the obtained nanoindentation results, the studied CM has the most similar elasticity and resilience, greater rigidity and lower viscosity relative to the bulbar conjunctiva, which in the future will allow the CM to resist deformation and maintain a given shape.

[0023] To obtain morphological results, the following was performed. After enucleation of the eyeball, the sclera was incised with a sharp blade along the posterior wall, 1 / 3 of the way around the semicircle, to introduce a 10% formalin solution into the vitreous for tissue fixation for 24 hours. The bulbar conjunctiva, replaced by CM, was excised, and the resulting fragment was dehydrated in ascending alcohol concentrations and embedded in paraffin. Sections 8-10 µm thick were prepared on a microtome and stained with hematoxylin and eosin. Conjunctival sections were examined under a light microscope at 16x and 40x magnification.

[0024] The experiment was conducted on five male Chinchilla rabbits, 6 months old and weighing 3.0 kg. All procedures were carried out in accordance with the Rules for the Conduct of Work Using Experimental Animals and the international principles of the Helsinki Declaration on the humane treatment of animals, as set out in the European Community Directive (86 / 609 / EC).

[0025] The method is carried out as follows.

[0026] The bulbar conjunctival defect is closed with a collagen membrane based on highly purified native chemically unmodified collagen type I - Viscoll at a concentration of 10 or 30 mg / ml and a thickness of 100 μm. The membrane is fixed with interrupted or continuous sutures. It is possible to subject the collagen membrane at a concentration of 10 mg / ml to ultraviolet crosslinking, while a 0.1% solution of riboflavin mononucleotide with dextran is installed on the surface of the cut collagen membrane for 10 minutes at a frequency of 1 instillation per minute for 30 minutes, then ultraviolet irradiation is carried out for 30 minutes at a wavelength of 370 nm, a beam diameter of 8.0 mm, a power density of 3.0 mW / cm 2 .

[0027] The proposed method is presented in the examples below.

[0028] Example 1. Under experimental conditions, a model of a conjunctival defect of the bulbar conjunctiva was created in a rabbit 1 week after its excision. Following epibulbar instillation with inocaine and subconjunctival administration of 2% lidocaine, a 4-mm diameter trephine was used to mark the tissue excision zone in the superolateral quadrant at a distance of 2 mm from the limbus. To completely remove the bulbar conjunctiva down to the exposed sclera, conjunctival scissors were used in addition to the trephine.

[0029] An intramuscular injection of Tiletamine and Zolazepam (Zoletil, 0.4 ml) and Xylazine 2% (Rometar, 0.7 ml) was performed. Preoperative preparation included surgical site preparation according to the Pirogov-Grossikh-Filonchikov technique, placement of an eyelid retractor, local anesthesia in the form of an epibulbar instillation of inocaine, and a subconjunctival injection of 2% lidocaine solution (0.3 ml). The surgery was performed under a Leica microscope. During the surgery, after determining the location and volume of the conjunctival defect, a collagen membrane flap was excised with a trephine to completely cover the defect area. Next, the wound defect of the bulbar conjunctiva measuring 4x4 mm was replaced with a CM implant based on highly purified native chemically unmodified collagen type I - Viscoll at a concentration of 30 mg / ml and a thickness of 100 μm and fixed with interrupted sutures (Nylon 10 / 0, Mani, Japan).Completion of the surgical intervention was carried out by subconjunctival administration of 0.3 ml of 0.4% dexamethasone solution in the area intact from the operation and a single instillation of 0.5% levofloxacin solution.

[0030] The clinical picture was noted by photographic recording using a slit lamp from Torsop (Japan).

[0031] On the first day postoperatively, the eye was moderately irritated. Conjunctival injection and slight mucous discharge were noted. Postoperatively, the animals were given eye drops of 0.5% levofloxacin (Oftaquix) 3 times daily for 14 days and 0.4% dexamethasone (Oftan-dexamethasone) 3 times daily, with subsequent reduction in the frequency of instillation over 3 weeks. During the postoperative period, the CM retained its transparency, interrupted sutures were clean, and the wound edges were adapted. The anterior chamber of the eye was of medium depth, and the anterior chamber fluid was transparent. After 21 days, the eye was calm, hyperemia, lacrimation, and mucous discharge were not observed, the surface was not stained with fluorescein. Ophthalmoscopic examination revealed adherence of the CM to the underlying sclera and the edges of the conjunctival defect without scar contracture. After one month of observation, the interrupted sutures were removed.

[0032] Example 2. During the preliminary preparation of the implanted material, ultraviolet collagen crosslinking was performed to increase its strength properties. The material was cut out using a 5 mm diameter trephine from a CM based on highly purified native chemically unmodified type I collagen - Viscoll with a collagen concentration of 10 mg / ml and a thickness of 100 μm. A solution of riboflavin mononucleotide and dextran was then applied to the surface of the collagen membrane with an installation frequency of 1 time per minute at 0.1% for 30 minutes, after which the material was irradiated with ultraviolet light for 30 minutes using the following parameters: wavelength 370 nm, beam diameter 8.0 mm, power density 3.0 mW / cm 2 .

[0033] In this experiment, a model of bulbar conjunctival defect similar to Example 1 was created in a rabbit. Next, intramuscular injections of Tiletamine and Zolazepam (Zoletil, 0.4 ml) and Xylazine 2% (Rometar, 0.7 ml) were performed. All manipulations were performed using a Leica microscope. Subsequently, after preoperative preparation of the surgical field according to the Pirogov-Grossikh-Filonchikov technique, placement of a blepharostat, epibulbar instillation of inocaine, and subconjunctival injection of 2% lidocaine solution (0.3 ml), after determining the location and area of ​​the conjunctival defect, a collagen membrane implant was formed with a trephine to cover the entire defect. Next, the bulbar conjunctiva defect measuring 5x5 mm was replaced with a prepared CM implant after ultraviolet crosslinking and fixed with a continuous suture (Nylon 10 / 0, Mani, Japan).At the end, a subconjunctival injection of 0.3 ml of 0.4% dexamethasone solution was performed in the area intact from the operation and a single instillation of 0.5% levofloxacin solution was performed.

[0034] Registration of the clinical picture was carried out using a slit lamp from the Torsop company (Japan).

[0035] Postoperative treatment included instillation of 0.5% levofloxacin (Oftaquix) eye drops three times daily for 14 days and 0.4% dexamethasone (Oftan-dexamethasone) three times daily, followed by a reduction in the frequency of instillations over 3 weeks. On the first day after CM implantation, conjunctival injection and mucous discharge from the eye were observed. During the postoperative period, the CM was fixed with a continuous suture, which adapted the wound edges. The anterior chamber of the eye of the experimental animals was of medium depth, and the aqueous humor was transparent. After 15 days, the eye was calm, with no hyperemia, lacrimation, or mucous discharge. Three weeks after surgery, the implantation area was not stained with fluorescein. Furthermore, optical coherence tomography data showed that the CM adhered tightly to the underlying sclera and was aligned with the edges of the conjunctival defect. After one month of observation, the continuous suture was removed.

[0036] The experimental results were assessed morphologically. By the end of the observation period, after 6 months, histological examination of the transplanted area revealed its complete replacement with newly formed autologous tissue, covered by stratified squamous nonkeratinized epithelium of varying thickness, with conjunctival cells migrating into the newly formed tissue, as well as the absence of fibrous tissue. Thus, the collagen carrier integrated into the iatrogenic conjunctival defect in rabbits and supported the formation of an organized monolayer of regenerated epithelial cells on its surface.

[0037] The expected effect of the claimed method appears to be quite significant and is associated with increased treatment effectiveness and a reduction in the volume and trauma of surgical intervention.

[0038] Thus, the use of the claimed method allows: the use of an analogue of the extracellular matrix of conjunctival tissue based on native type I collagen, facilitating the process of cell migration into the structure of the material while maintaining the epitheliomorphic structure and intercellular contacts, which indicates the biocompatibility of the studied material and creates the prerequisites for its further use in clinical practice for the elimination of conjunctival defects; avoidance of conjunctival tissue scarring; in the long term, complete elimination of excision of autologous and allogeneic tissues and the need for additional trauma.

[0039] The data obtained during the development of this method certainly allows the extrapolation of the method’s techniques to clinical conditions.

Claims

1. A method for replacing a defect of the bulbar conjunctiva, including closing the defect with a collagen membrane, characterized in that a collagen membrane based on highly purified native chemically unmodified collagen type I - Viscoll at a concentration of 10 or 30 mg / ml and a thickness of 100 μm is used; at a collagen concentration of 10 mg / ml, the membrane is subjected to crosslinking and fixed with interrupted or continuous sutures.

2. The method according to paragraph 1, characterized in that the collagen membrane at a concentration of 10 mg / ml is subjected to ultraviolet crosslinking, while for 10 minutes a 0.1% solution of riboflavin mononucleotide with dextran is installed on the surface of the cut collagen membrane with an instillation rate of 1 time per minute for 30 minutes, then ultraviolet irradiation is carried out for 30 minutes at a wavelength of 370 nm, a beam diameter of 8.0 mm, a power density of 3.0 mW / cm 2 .