Digital light processing bioprinting of functional keratoprosthesis

Abstract

Corneal transplantation is the main treatment option for severe corneal diseases. However, an unsteady supply of corneal donors and complications, such as possible transmission of infectious agents, necessitate the development of artificial corneas. Three-dimensional (3D) bioprinting technology has the potential to regenerate functional organs including corneas in a customised manner. Nevertheless, there is currently limited evidence of 3D bioprinted corneas achieving a level of clinical functionality and practicality on par with donated corneal tissues.

This doctoral work comprised two parts: reconstruction of functional cornea and bioprinting of corneal constructs. The first part focused on addressing two key issues in keratoprostheses research: (i) physiochemical characteristics required for corneal alternatives to repair any dysfunction of the diseased cornea, and (ii) optimisation of the biocompatibility and mechanical properties for clinical adoption. The second part centred on exploring the feasibility of 3D bioprinting technique as a new branch of tissue engineering in the field of corneal research, including key elements and procedures for successful bioprinting of keratoprostheses.

The objective of this research was to apply a digital light processing (DLP) 3D bioprinting technique with varied photo-sensitive biomimicry inks in the reconstruction of customisable keratoprostheses, providing potential therapeutic strategies in functional recovery upon cornea-related blindness. Results suggested the efficacy of DLP-bioprinted keratoprostheses with personalised physical dimensions, which is promising for achieving better clinical outcomes and addressing the current worldwide shortage of donor corneas.

First, the cornea’s desired physical dimensions were imported into the DLP printer’s software, and the ink tank was filled with either poly NAGA-GelMA (PNG) or poly PEGDA-HAMA (PEH) hybrid bio-ink. Then, suitability tests were carried out for the water content, dynamic swelling behaviors, and optical and mechanical properties of the printouts prior to implantation. The results demonstrated DLP-bioprinting could print pre-specified parameters. Material testing indicated that both PNG and PEH displayed strong water-retaining capability, excellent light transmittance and refractive index, but the structural strength and optical stability in the PNG group appeared more competitive.

Second, the cytocompatibility of PNG was evaluated with human corneal epithelial, stromal, and endothelial cell lines. The in-vitro immune response of PNG was analysed with human peripheral blood mononuclear cells (hPBMC) by Illumina RNA sequencing. The in-vivo performances of both PNG and PEH keratoprostheses were assessed using anterior lamellar keratoplasty (ALK) and intrastromal keratoplasty (ISK) models in New Zealand white rabbits. Results from in-vitro evaluation showed that PNG supported the adhesion and viability of corneal epithelial, stromal, and endothelial cells, while maintaining the phenotype and function of keratocytes. RNA sequencing results indicated that PNG activated type 2 immunity in macrophages, facilitated tissue regeneration, and suppressed inflammation. In-vivo assessment in rabbits showed although epithelialization was not achieved with the ALK model, postoperative IOP, corneal sensitivity, and tear formation remained unaffected with either PNG or PEH keratoprostheses. PNG keratoprostheses had excellent surgical handling characteristics, and no adverse effects on the host, while poor in-vivo outcomes of PEH keratoprostheses were observed. To conclude, considering its bio-safety and functional efficacy, PNG is a suitable candidate material for DLP-bioprinted keratoprosthesis.