Corneal Stroma Cell Density Evolution in Keratoconus Corneas Following the Implantation of Adipose Mesenchymal Stem Cells and Corneal Laminas: An In Vivo Confocal Microscopy Study

Abstract

Purpose: To report the corneal stroma cell density evolution identified by in vivo corneal confocal microscopy in humans using injected autologous adipose-derived adult stem cells (ADASCs) and corneal decellularized laminas in corneas with advanced keratoconus.

Methods: Interventional prospective, consecutive, randomized, comparative series of cases. A total of 14 keratoconic patients were randomly distributed into three groups for three types of surgical interventions: group 1 (G-1), autologous ADASC implantation (n = 5); group 2 (G-2), decellularized human corneal stroma (n = 5); and group 3 (G-3), autologous ADASCs + decellularized human corneal stroma (n = 4).

Results: A gradual and significant increase (P < 0.001) was observed in the cellularity in the anterior and posterior stroma of patients in G-1, G-2, and G-3 a year after the surgery in comparison with the preoperative density level. The same result was observed at the mid-corneal stroma in G-1 and at the anterior and posterior surfaces and within the laminas in G-2 and G-3. The cell density of patients receiving ADASC recellularized laminas (G-3) was statistically significantly higher (P = 0.011) at the anterior surface and within the lamina (P = 0.029) and at the posterior surface than in those implanted only with decellularized laminas (G-2).

Conclusions: A significant increase in cell density occurred up to 1 postoperative year at the corneal stroma following the implantation of ADASCs alone, as well as in those cases implanted with decellularized and recellularized laminas at the different levels of the analysis. However, this increase was significantly higher in the ADASC recellularized laminas.

Conflict of interest statement

Disclosure: M. El Zarif, None; K.A. Jawad, None; J.L. Alió Del Barrio, CSO (C); Z.A. Jawad, None; A. Palazón-Bru, None; M.P. de Miguel, Roche Farma SA (F); P. Saba, None; N. Makdissy, None; J.L. Alió, AkkoLens (C, R), Blue Green Company (F), Carl Zeiss Meditec (C, R), CSO (R), Dompé (R), Hanita Lenses (C, R), Jaypee Brothers (P), Maghrabi Hospital (C), Mediphacos (R), Oftalcare Nutravision (F), Omeros (C), Ophtec (F), Santen Pharmaceutical (C, F, R), Oculentis (C, R), Presbia (C), Schwind eye-tech-solutions (F, R), Slack Incorporated (C), Topcon Medical Systems (C), VisiDome (F)

Figures

Figure 1.

Cornea Visante OCT images and pachymetric maps for case 2 from G-1. (A, B) Preoperative examination, and (C, D) 12 months after surgery. Observe the patchy hyperreflective areas (red arrows) at the level of the stromal pocket compatible with areas of new collagen production. The distribution of these areas was not homogeneous along the surgical plane.

Figure 2.

Cornea OCT images and pachymetric maps for case 5 from G-2: (A) Preoperative OCT examination, (B) preoperative pachymetric map, (C) 12-month postoperative OCT (red arrows represent the graft edges), and (D) significant improvement in the pachymetric map 12 months after surgery. Cornea OCT images and pachymetric maps for case 10 from G-3: (E) preoperative OCT examination, (F) preoperative pachymetric map, (G) 12-month postoperative OCT (red arrows represent the graft edges), and (H) significant improvement in the pachymetric map 12 months after surgery.

Figure 3.

(A) Cell count performed with medium brightness and contrast. The more illuminated and more refringent cells were counted, and the keratocytes are marked in blue. (B) Elimination of keratocytes that do not belong to the plane under observation is possible using low brightness and high contrast settings.

Figure 4.

ADASC counting in G-1. (A) Count of ADASCs (red arrows) and keratocytes 1 month following surgery of a keratoconic patient; the cells are marked in blue. The ADASCs have a rounded shape and are larger, more luminous, and more refringent than the normal keratocytes. (B) Cell count of a keratoconic patient 1 year after implantation of ADASCs; all corneal stroma cells demonstrate a similar shape.

Figure 5.

Cell count on decellularized laminas for case 9 from G-2. (A) Anterior surface of a decellularized lamina that appears without cells 1 month after surgery. (B) Cell count on the posterior surface of a lamina 3 months after surgery. Cells show different morphology from the host corneal stromal cells. These cells are smaller. (C) Cell count on the posterior surface of a lamina 1 year after surgery. All cells demonstrate a morphology identical to that of normal corneal stromal keratocytes.

Figure 6.

Cell count on recellularized lamina for case 13 from G-3. (A) Anterior surface of a recellularized lamina 1 month after the operation; few ADASCs can be seen (marked in blue). (B) Posterior surface of the recellularized lamina 1 month after the operation; note the presence of a few ADASCs similar in morphology to keratocytes. (C) Anterior surface of the recellularized lamina 12 months after surgery showing an abundant number of stromal cells. (D) Mid-stroma of the lamina 12 months after surgery showing a high number of stromal cells. (E) Posterior surface of the recellularized lamina 12 months after surgery showing a high number of stromal cells. (F) OCT image where the red arrows represent the anterior and posterior surfaces, as well as the mid-stroma, of the recellularized lamina 12 months after surgery.

Figure 7.

Cellular density change in the corneal stroma of G-1, G-2, and G-3. (A) Increase in cell density over time in the anterior stroma of the cornea in G-1, G-2, and G-3 from the preoperative period until 12 months after surgery. A significant statistical difference (P = 0.025) among the groups was detected. (B) Statistically significant increase in cell density (P < 0.001) over time in the mid-stroma of the cornea in G-1 from the preoperative period until 12 months after surgery. (C) Increase in cell density over time in the posterior stroma of the cornea in G-1, G-2, and G-3 from the preoperative period until 12 months after surgery. There was no significant difference among the three groups (P = 0.311).

Figure 8.

Cellular density change in the anterior and posterior surfaces of the lamina, as well as the mid-stroma of the lamina. (A) Increase in cell density over time in the anterior surface of the lamina in G-2 and G-3 from the preoperative period until 12 months after surgery. A significant statistical difference (P = 0.011) exists between G-2 and G-3. (B) Increase in cell density over time in the mid-stroma of the lamina in G-2 and G-3 from the preoperative period until 12 months after surgery. A significant statistical difference (P = 0.011) between G-2 and G-3 was detected. (C) Increase in cell density over time in the posterior surface of the lamina in G-2 and G-3 from the preoperative period until 12 months after surgery. A significant statistical difference (P = 0.029) between G-2 and G-3 was detected.

Figure 9.

Cell count of the anterior and posterior corneal stroma for case 10 from G-3 12 months after the operation. (A) Anterior corneal stroma with abundant corneal stroma cells in a female patient 12 months after surgery. The number and morphology of corneal stroma cells are very similar to those of a normal stroma. (B) Posterior corneal stroma with a high number of corneal stroma cells for the same patient 12 months after surgery. The image shows that corneal stroma cells of the posterior stroma are similar in number and morphology to stromal cells of a normal cornea. (C) Presence of hyperreflective deposits (red arrow) corresponded with the preoperative paracentral corneal scarring observed in preoperative case 2 from G-1. (D) Improvement of the fibrotic tissue (red arrow) at the same level with the same case 2 from G-1 is observed. Note the presence of fibroblast (yellow arrow). Part of the superficial corneal nerve can be noted (blue arrow).

Figure 10.

(A) The presence of highly reflective fibrotic tissue (red arrow) of keratoconic case 11 corresponds with the preoperative paracentral anterior stroma scar observed at 3 months after surgery. (B) The presence of fibrotic tissue (red arrow) is highly reflective on the periphery of the posterior surface of the lamina at 3 months for the same case. (C) Improvement of the paracentral preoperative fibrotic tissue (red arrow) on the anterior stroma of the same keratoconic case 6 months after the operation. (D) Improvement of the fibrotic tissue (red arrow) on the periphery of the posterior surface of the recellularized lamina of the same keratoconic case 12 months after surgery. (E) Presence of fibroblast or myofibroblast (yellow arrow) and fibrotic tissue (blue arrow) on the posterior surface of the decellularized lamina of a keratoconic patient 3 months after surgery. (F) Transition zone between the posterior surface of the decellularized lamina and the host stroma showing a number of migrating corneal stroma cells (possibly keratocytes?) with dendritical shapes (marked with red arrows) in a keratoconic patient 12 months after surgery. (G) Presence of highly reflective fibrotic tissue (red arrow) on the periphery in the mid-stroma of the decellularized lamina of a keratoconic patient 6 months after surgery. (H) Slit-lamp image of a keratoconic patient demonstrating peripheral scar tissue 6 months after surgery.