A.V. BEZUSHKO¹, A.S. DUBOVIKOV¹, A.N. KULIKOV¹, S.V. CHURASHOV¹, V.F. CHERNYSH¹, M.I. BLINOVA², O.I. ALEKSANDROVA², A.A. SUETOV¹, I.O. GAVRILYUK¹

¹Military Medical Academy named after S.M. Kirov, 6 Akademika Lebedeva Str., Saint Petersburg, Russian Federation, 194044

²Institute of Cytology of the Russian Academy of Science, 4 Tikhoretskiy Ave., Saint Petersburg, Russian Federation, 194064

 Bezushko A.V. — ophthalmologist, e-mail: konkievaav@yandex.ru

Dubovikov A.S. — сlinical resident, e-mail: dubovikovanatolyi@gmail.com

Kulikov A.N. — Head of the Department of Ophthalmology, D. Med. Sc., e-mail: alexey.kulikov@mail.ru

Churashov S.V. — Professor of the Department of Ophthalmology, D. Med. Sc., e-mail: churashoff@mail.ru

Chernysh V.F. — Associate Professor of the Department of Ophthalmology, Cand. Med. Sc., e-mail: vfchernysh@gmail.com

Blinova M.I. — Cand. Biol. Sc., senior research scientist, Laboratory of cell biology in culture, e-mail: mira.blinova@mail.ru

Aleksandrova O.I. — junior research associate, Laboratory of cell biology in culture, e-mail: elga.aleks@gmail.com

Suetov A.A. — ophthalmologist, Cand. Med. Sc., e-mail: ophtalm@mail.ru

Gavrilyuk I.O. — ophthalmologist, e-mail: iluaayaks@gmail.com.

 A promising treatment method for patients with limbal stem cell deficiency (LSCD) is the transplantation of cultured limbal epithelial stem cells (LESC) obtained from minimal biopsy samples in various carriers. To investigate the possibility of using cultured autologous LESC on the collagen scaffold (CS) to eliminate LSCD in the experiment. In 30 rabbits (60 eyes) the mechanical LSCD model was created. Animals were divided into 3 groups. The 1^st and the 2^nd were treatment groups, the 3^rd — the control group. In the 1^st group collagen scaffold (CS) with cultivated LESC was placed on denuded cornea twice with the 3-days interval, in the 2^nd group it was placed once. In the control group CS with no LESC was applied once. On the 90^th day transparency noticeably increased in the 1^st group in contradistinction to the 2^nd and control groups, where vascularization and opacification were sufficiently noticeable. According to impression cytology, epithelium with no goblet cells was found in the 1^st group in 90% of cases. Histological examination confirmed it. In the 2^nd group the presence of goblet cells was indicated in 50% of cases, in the control group goblet cells were indicated in all the animals. Double application of CS with cultured LESC proved to be effective in LSCD treatment in the experiment and can be recommended for limited clinical trials.

Key words: limbal epithelial stem cells, collagen scaffold, limbal deficiency.

References

1. Pascolini D., Mariotti SPM. Global estimates of visual impairment, 2010, British Journal Ophthalmology, 2012, no. 96, pp. 614–618.
2. Chae J.J., Ambrose W.M., Espinoza F.A., et al. Regeneration of corneal epithelium utilizing a collagen vitrigel membrane in rabbit models for corneal stromal wound and limbal stem cell deficiency. Acta Ophthalmologica, 2015, no. 93, pp. 57-66.
3. Shortt A.J., Secker G.A., Notara M.D., et al. Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol, 2007, no. 52, pp. 483–502.
4. Sefat F., McKean R., Deshpande P., et al. Production, sterilisation and storage of biodegradable electrospun PLGA membranes for delivery of limbal stem cells to the cornea. Procedia Engineering, 2013, no. 59, pp. 101 – 116.
5. Kolobov K.A., Dubovikov A.S., Konkieva A.V., et al. About the successful cultivation of corneal epithelium limbal epithelial cells in the experiment. Materials of the scientific conference of ophthalmologists «Neva Horizons 2016». St. Petersburg, Polytechnica-service Publ., 2016. pp. 497-499. (in Russ.).
6. Chandrakasan, G., Torchia, D.A. and Piez, K.A. Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution. Journal of Biological Chemistry, 1976, no. 251, pp. 6062–6067.
7. Bezushko A.V., Dubovikov A.S., Suetov A.A., et al. Modification of limbal stem cell deficiency experimental mechanical model. Modern technologiesin ophthalmology, 2017, no. 17, pp. 26-28. (in Russ.).
8. Inatomi T., Nakamura T., Koizumi N. et al. Midterm results on ocular surface reconstruction using cultivated autologous oral mucosal epithelial transplantation. American Journal of Ophthalmology, 2006, no. 141, pp. 267–275.
9. Voyno-Yasenetsky V.V. Expansion and the variability of the eye tissue during its diseases and injuries, 1979. 224 p. (in Russ.).
10. Shimazaki J., Aiba M., Goto E., et al. Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders. Ophthalmology, 2002, no. 109, pp. 1285–90.
11. Bobba S., Chow S., Watson S., Di Girolamo N. Clinical outcomes of xeno-free expansion and transplantation of autologous ocular surface epithelial stem cells via contact lens delivery: a prospective case series. Stem Cell Res Ther, 2015, no. 6, pp. 23.
12. Rama P., Bonini S., Lambiase A., et al. Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation, 2001, no. 72, pp. 1478–85.
13. Schwab I.R., Reyes M., Isseroff R.R. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea, 2000, no. 19, pp. 421–6.