3D bioprinting of a corneal stroma equivalent

Abstract

Corneal transplantation constitutes one of the leading treatments for severe cases of loss of corneal function. Due to its limitations, a concerted effort has been made by tissue engineers to produce functional, synthetic corneal prostheses as an alternative recourse. However, successful translation of these therapies into the clinic has not yet been accomplished. 3D bioprinting is an emerging technology that can be harnessed for the fabrication of biological tissue for clinical applications. We applied this to the area of corneal tissue engineering in order to fabricate corneal structures that resembled the structure of the native human corneal stroma using an existing 3D digital human corneal model and a suitable support structure. These were 3D bioprinted from an in-house collagen-based bio-ink containing encapsulated corneal keratocytes. Keratocytes exhibited high cell viability both at day 1 post-printing (>90%) and at day 7 (83%). We established 3D bio-printing to be a feasible method by which artificial corneal structures can be engineered.

Keywords: 3D bioprinting; Bio-ink; Collagen; Cornea; Keratocytes; Tissue engineering.

Graphical abstract

Fig. 1

Stages of support structure generation. (A) Human cornea showing size and natural curvature across surface. (B) Original corneal model derived by (Simonini and Pandolfi, 2015) via the Finite Element Method (FEM). Corneal model is first converted to a solid to enable the execution of Boolean operations required for the generation of a support structure. Cornea is then ‘sealed’ (C) with a planar circle in order to subtract its volume from the support structure. (D) Wireframe view of cornea situated at the centre of cuboid prior to subtraction. (E) Digital support structure after subtraction. (F) The 3D printed plastic support structure.

Fig. 2

Using support structure to facilitate the printing of a corneal structure with 3% alginate (nozzle diameter = 200 μm) and optimisation of bio-inks for corneal 3D bioprinting. (A) Digital cornea is imported to the computer driving the 3D printer software slic3r and a preview of the concentric directionality of print is displayed. (B) The support structure is coated with FRESH to facilitate the 3D bioprinting of corneal structures. (C) View of the 3D bioprinting process. Corneal structures were printed with 3% alginate bio-ink stained with trypan blue to increase visibility. (D) Image of 3D bioprinted corneal structure captured prior to incubation. (E) FRESH is aspirated after 8 min of incubation and corneal structure is carefully removed from support, but begins to unravel 1 day post-printing once keratocytes were combined with the alginate bio-ink. (F) Images of corneal structures 3D bioprinted from composite bio-inks. (G) Relationship between nozzle diameter and printed thickness of corneal structures (left) and depiction of transparency of corneal structure 3D bioprinted from Coll-1 bio-ink (H) Brightfield image of 3D bioprinted corneal structure containing cells at day 1 (left) and cell viability measurements over 7 days (right). (I) Representative live/dead stain images using fluorescence microscopy at days 1 and 7 after 3D bioprinting in Coll-1.