Optimal fixation period of a combined bioconstruction with buccal epithelial cells in limbal cell deficiency in experiment

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Abstract

BACKGROUND: The search for an effective method for the treatment of limbal stem cell deficiency of various etiologies, leading to intense clouding and vascularization of the cornea, followed by a significant decrease in visual acuity, remains an important and relevant topic in ophthalmology. The results of the studies showed that transplantation of buccal epithelial cells could significantly improve the prognosis of treatment in this category of patients.

AIM: To determine the optimal fixation period of a combined bioconstruction with buccal epithelial cells for the treatment of limbal cell deficiency in experiment.

MATERIALS AND METHODS: At the first stage, the corneal epithelium was removed with a scraper in the eyes of experimental animals (12 eyes), and the limb was excised along the entire circumference. Next, to isolate epithelial buccal cells and manufacture a combined bioconstruction consisting of buccal cells, a collagen carrier and a soft contact lens, a flap of the mucous membrane of 5 × 5 mm was taken from the cheeks of rabbits. At the second stage, after the formation of a fibrovascular pannus on the cornea, it was excised to transparent layers of the cornea, a combined bioconstruction was placed on top. Further, one U-shaped suture was applied at the border of the inner and outer third of the eyelids. Temporary blepharorraphia persisted for 3 days (6 eyes) and 5 days (6 eyes), after the specified time, stitches were removed from the eyelids, and bioconstructions were removed. The bioconstructions removed from the eyes were stained with a vital (lifetime) fluorochrome dye based on tripaflavin and acridine orange, followed by analysis in a fluorescent microscope. The structure of cell nuclei, the overall integrity of the cytoplasm, and the presence of secretory vesicles were evaluated. After 7 and 14 days after transplantation of the bioconstruction, the area of erosion, the extent of new vessels and the transparency of the cornea were evaluated. When the observation was completed, the rabbits were removed from the experiment, and the eyes were enucleated and subjected to histological examination.

RESULTS: On the surface of all bioconstructions removed after 5 days, only leukocytes were detected. At the same time, on collagen matrix of bioconstructions removed after 3 days, in addition to leukocytes, there were also buccal epithelial cells with normal nuclear structure and chromatin topography in its composition, as well as with secretory vesicles. On days 7–14, both groups showed a decrease in the area of erosion, while the dynamics of ocular surface recovery processes was more pronounced in the group with fixation of the bioconstruction for 5 days.

CONCLUSIONS: According to the results of in vivo staining with fluorochrome dye of combined bioconstructions removed from the cornea of experimental animals 3 and 5 days after superficial keratectomy, as well as the data of clinical and histological examination, the optimal period for bioconstruction fixation was established to be 5 days.

About the authors

Ekaterina V. Сhentsova

Helmholtz National Medical Research Center of Eye Diseases

Email: chentsova27@yandex.ru
ORCID iD: 0000-0002-8394-1038
SPIN-code: 8191-8338

MD, Dr. Sci. (Med.), professor, head of the traumatology and reconstructive surgery Department

Russian Federation, Moscow

Natalia V. Borovkova

N.V. Sklifosovsky Research Institute for Emergency Medicine

Email: borovkovanv@yandex.ru
ORCID iD: 0000-0002-8897-7523
SPIN-code: 9339-2800

MD, Dr. Sci. (Med.), head of the biotechnology and transfusiology Department

Russian Federation, Moscow

Tatyana V. Tselaya

Helmholtz National Medical Research Center of Eye Diseases

Author for correspondence.
Email: tatyana.tselaya@yandex.ru
ORCID iD: 0000-0003-3013-685X

postgraduate student of the traumatology and reconstructive surgery Department

Russian Federation, Moscow

Maya V. Storozheva

N.V. Sklifosovsky Research Institute for Emergency Medicine

Email: mayya.storozheva@yandex.ru
ORCID iD: 0000-0003-1927-2404
SPIN-code: 7789-3277

research associate, the biotechnology and transfusiology Department

Russian Federation, Moscow

Maksim S. Makarov

N.V. Sklifosovsky Research Institute for Emergency Medicine

Email: mcsimmc@yandex.ru
ORCID iD: 0000-0002-2184-2982
SPIN-code: 3543-5800

Cand. Sci. (Med.), senior research associate, Biotechnology and Transfusiology Department

Russian Federation, Moscow

Aleksey A. Churilov

Helmholtz National Medical Research Center of Eye Diseases

Email: churilov_aa@mail.ru
ORCID iD: 0000-0003-1018-8257
SPIN-code: 8648-0654

junior research associate, pathological anatomy and histology Department

Russian Federation, Moscow

Ivan N. Ponomarev

N.V. Sklifosovsky Research Institute for Emergency Medicine

Email: rzam@yandex.ru
ORCID iD: 0000-0002-2523-6939
SPIN-code: 4705-9314

Cand. Sci. (Med.), senior research associate, biotechnology and transfusiology Department

Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Slit lamp photograph of the eye immediately after limbectomy and deepithelization (vital fluorescein staining)

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3. Fig. 2. Fibrovascular pannus — a model of limbal cell deficiency (1 month after deepithelization and limbectomy)

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4. Fig. 3. Eye of a rabbit after keratectomy and combined bioconstruction placed over the cornea (indicated by the arrow)

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5. Fig. 4. Combined bioconstruction: a — removed 3 days after keratectomy; b — removed 5 days after keratectomy. Vital staining with tripaflavin acridine-orange. Magnification ×200

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6. Fig. 5. Slit lamp photographs of the eye on the 7th day after keratectomy and transplantation of combined bioconstruction (vital fluorescein staining): a — the 1st group (the bioconstruction was removed 3 days after surgery); b — the 2nd group (the bioconstruction was removed 5 days after surgery)

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7. Fig. 6. Slit lamp photographs of the eye on the 14th day after keratectomy and transplantation of combined bioconstruction (vital fluorescein staining): a — the 1st group (the bioconstruction was removed 3 days after surgery); b — the 2nd group (the bioconstruction was removed 5 days after surgery)

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8. Fig. 7. Histological analysis of the cornea 14 days after keratectomy and transplantation of the combined bioconstruction: a — 1st group, the cornea is covered with stratified squamous epithelium with stratification of its own epithelial substance. There are thin fibers visible subepithelially with a violation of parallelism, with the formation of empty cracks and edema at 1/4 of the corneal thickness in the stroma; b — 2nd group, the cornea is covered with 2–3-layered squamous epithelium with changes in the stroma, proliferation of capillaries and fibroblasts. Hematoxylin-eosin staining. Magnification ×200

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