THE USE OF COLLAGEN SCAFFOLD AND AMNIOTIC MEMBRANE WITH LABORATORY-REARED STEM CELLS TO MANAGE LIMBAL DEFICIENCY: EXPERIMENTAL STUDY

Objective: comparative analysis of the use of collagen scaffold (CS) and amniotic membrane (AM) with laboratory-reared limbal stem cells (LSC) to manage limbal deficiency.
Methods: The study was performed on 40 adult rabbits (80 eyes), Chinchilla breed. All animals were divided into four groups of 10 animals each to run two set of experiments with CS and AM as LSC carriers. After creation of a model of limbal deficiency and superficial keratectomy we used CS and AM with laboratoryreared LSC in experimental groups experimental groups, and CS and AM without cells in control groups.
Results: On the 90th days after transplantation we observed a significant increase of corneal transparency in experimental groups. Epithelial surface was smooth, glossy, not stained with fluorescein. In control groups we registered pronounced corneal stroma opacity, its vascularization, persistent abrasions. According to the results impression cytology and histological study we detected normal multilayer non-squamous epithelium with basal membrane not containing goblet cells in experimental groups. In control groups epithelium contained goblet ells, had different number of ranges, abrasions were detected.
Conclusions:Transplantation of carriers with laboratory-reared LSC on the surface of cornea enables to create its repository sufficient to recover epithelium of corneal phenotype.

1. 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. P. 101–116.

2. Haagdorens M., Van Acker S.I., Van Gerwen V. [et al.]. Limbal stem cell deficiency: current treatment options and emerging therapies // Stem Cells International. 2016. Vol. 2016, No. 4. 22 p.

3. Aleksandrova O.I., Okolov I.N., Khorolskaya J.I. [et al.]. Possibilities of cellular technologies for rational pharmacotherapy of ocular pathologies // Modern Technologies in Ophthalmology. 2017. Vol. 20, No. 7. P. 5–7.

4. Kim K.H., Mian S.I. Diagnosis of corneal limbal stem cell deficiency // Current Opinion in Ophthalmology. 2017. Vol. 28, No. 4, P. 355–362.

5. Aleksandrova O.I., Khorolskaya J.V., Maychuk D.Y. et al. Study of the total cytotoxicity of aminoglycoside and fluoroquinolone antibiotics on cell cultures // The Russian Annals of Ophthalmology. 2015. Vol. 131, No. 5. P. 39–48.

6. 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. Vol. 52, No. 2. P. 483–502.

7. Meller D., Pires R.T.F., Tseng S.C.G. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures // Br. J. Ophthalmol. 2002. Vol. 86, No. 4. P. 463–471.

8. Bezushko A.V., Dubovikov A.S., Kulikov A.N et al. Modification of mechanical limbal stem cell deficiency model // Ophthalmology in Russia. 2018. Vol. 1, No. 15. P. 51–57.

9. Niknejad H., Peirovi H., Jorjani M. [et al.]. Properties of the amniotic membrane for potential use in tissue engineering // Eur. Cells Mater. 2008. Vol. 15, No. 15. P. 88–99.

10. Kolobov K.A., Dubovikov A.S., Konkieva A.V. [et al.]. About the successful cultivation of corneal epithelium limbal epithelial cells in the experiment // Neva Horizons 2016: Materials of the scientific conference of ophthalmologists. St. Petersburg, 2016. P. 497–499.

11. 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. P. 101–116.

12. Chernish V.F., Boiko E.V. Eye burns. State of the problem and new approaches. St. Petersburg: Bastion, 2008. P. 11–17.

13. 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. Vol. 52, No. 2. P. 483–502.

14. Baylis O., Figueiredo F., Henein C. [et al.]. 13 years of cultured limbal epithelial cell therapy: a review of the outcomes // J. Cell. Biochem. 2011 Vol. 112, No. 4. P. 993–1002.

15. 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. Vol. 93, No. 1. P. e57–e66.

16. Chandrakasan G., Torchia D.A., 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. Vol. 251, No. 19, P. 6062–6067.

17. Glowacki J., Mizuno S. Collagen scaffolds for tissue engineering // Biopolymers 2008. Vol. 89, No. 5. P. 338–344.

18. Haagdorens M., Van Acker S.I., Van Gerwen V. [et al.]. Limbal stem cell deficiency: current treatment options and emerging therapies // Stem Cells International. 2016. Vol. 2016, No. 4. 22 p.

19. Kim K.H., Mian S.I. Diagnosis of corneal limbal stem cell deficiency // Current Opinion in Ophthalmology. 2017. Vol. 28, No. 4, P. 355–362.

20. Meller D., Pires R.T.F., Tseng S.C.G. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures // Br. J. Ophthalmol. 2002. Vol. 86, No. 4. P. 463–471.

21. Niknejad H., Peirovi H., Jorjani M. [et al.]. Properties of the amniotic membrane for potential use in tissue engineering // Eur. Cells Mater. 2008. Vol. 15, No. 15. P. 88–99.

22. 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. P. 101–116.

23. 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. Vol. 52, No. 2. P. 483–502.