Biomimetic modelling of the lamina cribrosa region using tissue engineered scaffolds - a novel 3D model for glaucoma research

Abstract

Purpose : Fibrotic remodeling and loss of compliance (increased stiffness) are characteristic hallmarks of glaucomatous tissue change in the lamina cribrosa (LC) region of the optic nerve head. Mediating these glaucoma-related changes at the cellular level, are resident fibroblast-like LC cells. To accurately study the pro-fibrotic activity of LC cells, requires an appropriate cell culture model system to replicate the complex glaucoma tissue microenvironment. Thus, the purpose of this study was to create a novel, 3D in-vitro model of the stiffened glaucomatous LC utilizing bioengineered scaffolds.

Methods : A suspension of 84% collagen type I, 7% chondroitin-6-sulphate and 9% elastin in 0.05M acetic acid was freeze-dried to fabricate LC scaffolds (porosity 80-100µm). Scaffolds were EDAC chemically crosslinked to produce ‘normal’ and glaucomatous ‘stiff’ scaffold compliance reference values. Normal primary human LC cells were seeded onto scaffolds at a density of 3500cells/mm² and cultured for 5 days (n=2 biological replicates, n=11 scaffolds/group) and processed for histological evaluation.

Results : The microporous 3D appearance of fabricated LC scaffolds was comparable in both ‘normal’ and ‘stiff’ groups. (Fig.1) Micro-CT scanning provided a representative overview of the mesh-like structure of the bioengineered scaffolds (1A). The collagen type I composition (gold), confirmed using picrosirius red staining with birefringence, was abundantly found throughout the scaffold structure (1B). The elastin content (green, arrows) was validated with immunofluorescence (1C), and was localized to the scaffold beams using Verhoff-van Gieson stain (black, arrows, 1D). Histological analysis using eosin staining (1E) and scanning electron microscopy (1F) confirmed fabricated scaffolds had a porous ultrastructure. Analysis of cell-seeded scaffolds showed similar successful infiltration and adherence of primary LC cells within both ‘normal’ and ‘stiff’ scaffolds groups (arrows, Fig.2).

Conclusions : Novel 3D biomimetic scaffolds were developed to have similar structure and composition to the native LC. Primary LC cells have successfully been grown for the first time within these glaucomatous ‘stiff’ and ‘normal’ 3D scaffolds. This new in-vitro model of glaucoma has significant potential for deciphering the underlying fibrotic response of LC cells to stiffened glaucomatous conditions.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Fig.1

 

Fig.2