Confocal Microscopy Study Of Corneal Stroma Regeneration With Cellular Therapy: Stromal Cell Density And Immune Cell Infiltration

This study investigated the regenerative potential of autologous adipose-derived adult stem cells (ADASCs) and decellularized human donor corneal stromal lamina (dCL), or ADASCs-recellularized dCL (ADASCs-rCL) corneal tissue in advanced keratoconus patients. Using in vivo confocal microscopy (IVCM), we evaluated the in vivo evolution of injected ADASCs into keratocytes over a one-year follow-up period and the corresponding changes in corneal dCL or ADASCs-rCL. Additionally, we assessed immune cell (ICs) infiltration in these patients, providing a comprehensive understanding of the therapeutic effects and cellular dynamics involved in this novel surgical approach.

Division of Ophthalmology, Universidad Miguel Hernández, Elche, Spain. Lebanese University, Doctoral School of Sciences and Technology, Lebanon. Lebanese University, Genomic Surveillance and Biotherapy GSBT, Faculty of Sciences, RasMaska, Lebanon. Research in Ophthalmology, Optica General, Saida, Lebanon.

We conducted an experimental, prospective clinical trial involving fourteen patients divided into three groups: G-1 received autologous ADASCs, G-2 received dCL, and G-3 received ADASCs-rCL. Femtosecond-assisted implantation was performed. We used an original method of quantitative IVCM cell counting to study cell density, the morphological evolution of the implanted ADASCs, the dCL, ADASCs-rCL, and infiltrated ICs. A morphological study differentiated host cells from ICs, and cell area calculations of ICs were performed using ImageJ analysis. Assessments were conducted at preoperative and at 1, 3, 6, and 12 months post-implant, providing comprehensive insights into therapeutic effects and cellular dynamics.

A significant increase (P<0.001) was observed in cellularity in the anterior and posterior stroma in G-1, G-2, and G-3 at one year/preoperative density level. The mid corneal stroma in G-1 and the anterior, posterior surfaces, and within the laminas in G-2 and G-3 showed similar results. Cell density in patients with recellularized laminas was significantly higher than in those with decellularized laminas. Infiltrated ICs, including granulocytes and agranulocytes, were seen across all groups. Stromal ICs infiltration ranged from 1.19% to 6.62%, with significant group-related increases (F=10.68, P<.0001). G-3 had notable variations at 6 and 12 months (2.0 and 1.87-fold). IC size shifts occurred in G-3, indicating a role in tissue repair.

Confocal microscopy has proven to be an essential tool for the assessment and "in vivo" follow-up of corneas implanted with ADASCs for corneal regeneration purposes. A significant increase in the cellularity of the corneal stroma was observed following the implantation of ADASCs alone, as well as with patients implanted with  dCL, and ADASCs-rCL. ADASCs-recellularized lamina therapy led to increased ICs infiltration compared to ADASCs alone, impacting cell distribution and size due to the presence of the lamina. These findings reveal intricate immune patterns shaped by the corneal microenvironment and underscore the importance of understanding immune responses for the development of future therapeutic strategies.