Supporting data for "Digital Light Processing Bioprinting of Functional Keratoprosthesis"

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 comprises two parts: reconstruction of functional cornea and bioprinting of corneal constructs. The first part focuses on addressing two key issues in keratoprosthesis 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 of the existing keratoprosthesis for clinical adoption. The second part centres on exploring the feasibility of 3D bioprinting techniques, including (i) recent advances in 3D bioprinting as a new branch of tissue engineering in the field of corneal research, and (ii) key elements and procedures for successful bioprinting of keratoprosthesis.

The objective of this research is to apply a digital light processing (DLP) 3D bioprinting technique with varied photosensitive biomimicry inks in the reconstruction of customisable keratoprosthesis, providing potential therapeutic strategies in functional recovery upon cornea-related blindness. Results suggest the efficacy of DLP bioprinted keratoprosthesis 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 bioink. Then, suitability tests were carried out for the water content, dynamic stability, and optical and mechanical properties of the printouts prior to corneal transplantation. The results demonstrated DLP-bioprinting can print pre-specified parameters. Material testing indicated that both PNG and PEH keratoprosthesis display strong wettability, high long-term stability in PBS, and excellent light transmittance and refractive index, but the structural strength in the PNG group appears more competitive.

Second, the cytocompatibility of PNG keratoprosthesis was evaluated with human corneal epithelial, stromal, and endothelial cell lines. The in-vitro immune response of PNG keratoprosthesis was analysed with human peripheral blood mononuclear cells by illumina RNA sequencing. The in-vivo performances of both PNG and PEH keratoprosthesis 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 keratoprosthesis supports 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 keratoprosthesis activates type 2 immunity in macrophages, facilitates tissue regeneration, and suppresses inflammation. In-vivo assessment in rabbits showed although epithelialisation was not achieved with the ALK model, postoperative IOP, corneal sensitivity, and tear formation remain unaffected with either PNG or PEH keratoprosthesis. PNG keratoprosthesis has excellent surgical handling characteristics, and no adverse effects on the host, while poor in vivo outcomes of PEH keratoprosthesis are observed.