Original article Experimental study of manual and femtosecond laser-assisted methods for cutting limbal mini-transplants B.E. Malyugin, S.A. Borzenok, O.N. Nefedova, D.S. Ostrovskii, M.Yu. Gerasymov, A.V. Shatskikh S. Fyodorov Eye Microsurger y Federal State Institution, Moscow, Russian Federation A. Yevdokimov Moscow State University of Medicine and Dentistr y, Moscow, Russian Federation Purpose. To define optimal parameters of femtosecond laser (FSL) (Femto LDV Z8, Ziemer, Switzerland) for cutting limbal mini-transplants to be further used for limbal stem cells (LSC) transplantation and compare the results with manual cutting technique using microsurgical blade. Material and methods. At first step, performed on 3 porcine and 3 cadaveric eyes, the optimal energy parameters of FSL were selected, which ensured the excision of limbal mini-transplants. At the second step, 8 eyes from 4 postmortem donors were used. In the lower and upper parts of the limbus, limbal fragments were cut out into 8 mini-transplants with a FSL (experimental group). Mini-grafts were also formed manually in symmetrical areas of each eye using dosed diamond blade (control group). The structure of the limbal bed after removing mini-transplants was studied by light and scanning electron microscopy. The effect of FSL energy on the viability of LSC was determined by intravital staining using Live & Dead dye. Finally, on the third step, the assessment of reversible and irreversible apoptosis was performed by culturing corneoscleral discs containing mini-transplants not separated from the limbal bed. The obtained samples were cultivated in a medium DMEM/F12 for 7 days followed by immunohistochemical examination. Results. The optimal parameters of FSL operation are determined by energy levels- 100, 110 and 120%. Histological analysis of the limbal residual bed zone showed that the smoothest surface was obtained at energies 110 and 120%. This fact was also confirmed by scanning electron microscopy. When stained with the Live & Dead dye, damage on the lateral and inner sides of limbal mini-transplants was revealed, mainly in the experimental group. After 7 days of cultivation in the cut area in both groups, no expression of apoptosis markers was observed. Conclusion. Cutting out the limbal zone containing LSCs using FSL is safe and allows obtaining limbal mini-grafts. The most optimal parameters of laser operation in the limbal zone were selected – a planar cut at depth of 250 µm using 110% laser energy. Key words: femtosecond laser, limbal stem cells, apoptosis, limbal stem cell deficiency syndrome, scanning electron microscopy, light microscopy, cultivation, histology

This review presents well-known surgical techniques aimed at the treatment of unilateral limbal insufficiency syndrome. Among the many available techniques, one should especially highlight the so-called “simple limbal epithelial transplantation”, which has proven to be an effective, safe and generally available method of treating unilateral limbal insufficiency syndrome (SLN). It should be noted that the risks of recurrence of SLN remain significant, and the mechanisms of engraftment of transplanted limbal epithelial stem cells on the recipient’s eye are not fully understood. The review also considers options for new promising approaches to the possible solution of existing problems in the transplantation of limbal epithelial stem cells.

Objective: to study in vitro survival and preservation of the proliferative activity of limbal stem cells (LSCs) in femtosecond laser-cut limbal tissue fragments. Materials and methods. Limbal fragments were formed from donor cadaver eyes (n = 8) in the upper and lower limbus containing the highest number of limbal stem cells, using a Z8 femtosecond laser (FSL) (Ziemer, Switzerland). The limbal fragments were fragmented into 4 mini-grafts using different energy levels (100, 110, 120%). Mini-grafts from symmetrical sections of the cadaver eyes, which were manually isolated using a microsurgical blade, served as controls. The mini-grafts were cultured for two weeks in culture media intended for limbal epithelial stem cells (LESCs) (Epilife (0.06 mM Ca++) and for multipotent mesenchymal stem cells (MMSCs) (DMEM/F12), with the addition of specific growth factors to selectively stimulate LESCs or MMSCs, respectively. The phenotype of the obtained cultured cells in the «laser» and «knife» groups was determined by flow cytometry using a set of markers (CD166, CD105, CD90, CD29, CD34) for the membrane proteins of LESCs and MMSCs. The ability of cultured cells to adhesion and proliferation in the «laser» and «knife» groups was determined by seeding the third passage of the resulting cultures on Bowman’s membrane of acellular corneas.Results. Primary cell culture was obtained from mini-grafts of all donors in both groups. Cell morphology was consistent with the phenotype of corneal epithelial cells (cobblestone pattern). When cultured in the EpiLife medium (0.06 mM Ca++), we determined the presence of LSCs proliferation from 38.6% of minigrafts; in the DMEM/F12 medium (1 : 1) the presence was determined from 31.8%. Two weeks later, cell yield from mini-grafts in the «laser» and «knife» groups was 77.2% and 63.6%, respectively. Cell growth by the end of week 2 of culturing of mini-grafts obtained by FSL at 120, 110 and 100% energies was 87.5, 71.4 and 71.4%, respectively. It was found that the resulting cell cultures in the «laser» and «knife» groups and in the «120%», «110%» and «100%» subgroups were not different phenotypically. Cytofluorimetric analysis showed that cell cultures in the groups had a mixed pattern of marker expression of both LESCs (CD29+) and MMSCs (CD90+, CD105+). Seeding of the third passage of cell culture in the test groups in all cases demonstrated adhesion and formation of a cell monolayer on the Bowman’s membrane of model corneas.Conclusion. The use of FSL for cutting out limbal grafts seems to be effective and safe in comparison with the traditional mechanical (knife) technique. Cell cultures obtained from FSL-cut mini-grafts were able to grow and migrate for at least 21 days.

Актуальность. Самым эффективным и малотравматичным методом хирургического лечения синдрома лимбальной недостаточности(СЛН) является «простая трансплантация лимбального эпителия» («simple limbal epithelialtransplantation»(SLET)), в ходе которой выделенный на 12 ч участок лимба здорового глаза протяженностью 2 мм, фрагментируется на 6-8 лоскутов, с равномерным размещением их в области стромы роговицы пораженного глаза на фибриновый клей, располагая два слоя амниона под и над фиксированными лоскутами. Цель. Оптимизировать методику SLET, исключив использование фибринового клея и одного слоя амниона. Материал и методы. Пациентке В., 59 лет, с диагнозом исход ожога роговицы, полный СЛН, состояние после сквозной кератопластики, частичное помутнение трансплантата, выполнена опeрация SLET, при которой 8 лоскутов, полученных путем фрагментации выделенного участка лимба здорового глаза, размещались в радиально сформированных туннелях на периферии роговицы. Операцию завершали наложением одного слоя амниона на поверхность роговицы пораженного глаза, наложением мягкой контактной линзы (МКЛ) и временной блефарорафией. Результаты. Спустя 3 недели МКЛ была снята, наблюдали полную эпителизацию роговицы. Спустя 12 месяцев отмечали уменьшение поверхностной неоваскуляризации, образование стойкого эпителиального покрова и увеличение прозрачности трансплантата. На здоровом глазу место выделения лимба визуализировалось слабо, было покрыто конъюнктивой. Выводы. Исключение применения фибринового клея и одного слоя амниона значительно уменьшает себестоимость операции, делая ее более доступной. Радиально сформированные туннели в строме на периферии роговицы максимально физиологичны и обеспечивают стабильное положение лоскутов без применения фибринового клея, выполняют роль своеобразных депо лимбальных стволовых клеток, тем самым способствуя их равномерной миграции по всей поверхности роговицы.