Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose Stem Cells and Corneal Decellularized Laminas in Patients with Keratoconus

Abstract

Keratoconus is described as a noninflammatory degeneration and ectasia of the cornea of the eye that progresses to a severe corneal deformation, with a thin and debilitated cornea that is associated with a sharp decrease in the amount of keratocytes.

Managing Keratoconus is performed by following different surgical approaches, one of them is the implant of stem cells. The use of confocal microscopy has allowed a better understanding of physiological mechanisms and visualization of microscopic changes to the stromal cells of the keratoconic cornea in vivo.

The aim of this chapter is to describe using corneal confocal microscopy the evolution in vivo of injected adipose autologous stem cells (ADAS) into the human cornea affected by keratoconus and their transformation along the follow up into keratocytes.

This confocal microscopy study was performed in an experimental clinical study, prospectively designed . 14 keratoconic patients were included and distributed into 3 groups and were submitted to different surgical aproximations : Group 1: Autologous ADASC implantation (5 patients), Group 2: Decellularized human corneal stroma transplantation (5 patients), Group 3: Autologous ADASC + Decellularized human corneal stroma transplantation (4 patients).

A description of the original method used with the confocal microscope for cell counting and the method of cell counting is described in detail, as well as the use of the confocal microscope to observe the viability and the evolution of the isolated transplanted cells, the evolution of the transplanted acellular laminas or including stem cells. Calculation of the corneal cell density was also described as well as the difficulties in confocal microscopy cell counting.

The method described in this study allowed to appreciate a gradual increase (P value = 0.07) in the density of cells was evident in the anterior, middle, and posterior stromas of the patients with transplanted ADASC one year after the operation, in comparison to the density level preoperative. Keratocyte density in both the anterior (P value = 0.008) and posterior (P value = 0.008) stroma of patients with decellularized lamina and recellularized lamina, was statistically significant one year after the operations were performed.

We could also demonstrate a statistically significant increase in the keratocyte density within the lamina, the cell density of the patients with recellularized laminas was higher than those treated with a decellularized lamina.

As a conclusion confocal corneal microscopy has shown to be an essential tool in the assessment and “in vivo” follow-up of the corneas implanted with mesenchymal cells for corneal regeneration purposes. Using corneal bioconfocal microscopy, we were able to observe an increase in the cellularity of the corneal stroma following the implantation of ADASC cells alone, and also an increase in keratocytes of patients with transplanted decellularized and recellularized laminas.