Human corneal lenticule bio-engineered with rhNGF and Mesenchymal Stromal Cells and its effect on pre-clinical models of glaucoma and diabetic retinopathy.

Abstract

Purpose : Glaucoma and diabetic retinopathy (DR) stand as two of the leading causes of blindness. Among therapeutic strategies, attention was gained to Nerve Growth Factor (NGF) and mesenchymal stromal cells (MSCs). However, the delivery of these factors into the eye remains a challenge. Hence, employing preclinical models of glaucoma and DR, this study assesed the feasibility of using human corneal lenticule (hCL), a thin and disc-shaped part of the cornea obtained during refractive surgery, as a natural delivery system of neurotrophic and regenerative factors.

Methods : hCLs (thickness 100-120 µm), collected from patients’ cornea, were decellularized (0.1% SDS) and engineered with recombinant human NGF microparticles (rhNGF-MPs) and/or with MSCs from human amniotic fluid (hAFSCs). The glaucoma model was established treating Human Trabecular Meshwork Cells (HTMCs) with TGF-β2 (5 ng/mL for 4 days) to induce an increased extracellular matrix (ECM) deposition associate to higher intraocular pressure. Porcine neuroretina explants were exposed to high glucose (HG; 25 mM for 4 days) to mimic DR. In both models the effect of engineered hCLs was tested.

Results : Data obtained from the glaucoma model revealed that TGF-β2 increased the deposition of ECM in HTMCs as demonstrated by cells permeability reduction and higher COL4a1, COL1a1 and ACTA1 expression. Notably, these effects were ameliorated by the incubation with engineered hCLs.
In the DR model, HG increased the expression of TNF-α, NFkB, NRF2, BAX/Bcl2 ratio, p21promoting inflammation and apoptosis. Increased inflammation was also confirmed by cytokine array experiments performed on porcine neuroretina explants conditioned media. Of note, incubation with engineered hCLs reduced the expression of both inflammatory/apoptosis markers and the level of pro-inflammatory cytokines (GM-CSF, IL-1β and PDGF-BB), while increased the anti-inflammatory IL-10 level. Interestingly, engineered hCLs was able also to restore the structural changes induced by HG as revealed by TUBB3 and Rho reduction.

Conclusions : Our findings indicate that this advanced bioengineered scaffold, through the delivery of neurotrophic and regenerative factors, was able to ameliorate glaucoma and DR-induced damage in preclinical models representing a potential therapeutic approach for such disorders.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.