Optimal fixation period of a combined bioconstruction with buccal epithelial cells in limbal cell deficiency in experiment
- Authors: Сhentsova E.V.1, Borovkova N.V.2, Tselaya T.V.1, Storozheva M.V.2, Makarov M.S.2, Churilov A.A.1, Ponomarev I.N.2
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Affiliations:
- Helmholtz National Medical Research Center of Eye Diseases
- N.V. Sklifosovsky Research Institute for Emergency Medicine
- Issue: Vol 16, No 2 (2023)
- Pages: 63-72
- Section: Experimental trials
- URL: https://journals.rcsi.science/ov/article/view/253837
- DOI: https://doi.org/10.17816/OV321988
- ID: 253837
Cite item
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.
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##article.viewOnOriginalSite##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, MoscowNatalia 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, MoscowTatyana 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, MoscowMaya 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, MoscowMaksim 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, MoscowAleksey 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, MoscowIvan 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, MoscowReferences
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