Advanced Bioprinting And Bioengineering Approaches For Corneal Injury Repair: Emphasis On Limbal Niche And Organoid Construction

The limbal stem cell niche (LSCN) plays a vital role as a reservoir for limbal stem cells (LSCs), essential for the regeneration of the corneal epithelium. Pathological alterations within the LSCN can obstruct LSCs from effectively renewing the corneal epithelium, resulting in limbal stem cell deficiency (LSCD) and progressing to corneal blindness. Here, we produced a collagen membrane with LSCN microstructure and developed a microphysiological system (MPS) platform consisting of Matrigel, ultra-low attachment U-shaped plates, and PDMS grid-like biochips to form LOs. This study aims to innovate bioprinting and bioengineering methodologies to reconstruct the LSCN and facilitate the repair of corneal injuries, potentially reversing LSCD.

In a pioneering study conducted at a leading research institution's state-of-the-art laboratory, scientists embarked on a groundbreaking investigation into the restoration of corneal injuries. Leveraging cutting-edge bioprinting and bioengineering techniques, the team focused on the intricate construction of the limbal stem cell niche (LSCN) and the development of limbal organoids (LOs) to address limbal stem cell deficiency (LSCD), a critical factor leading to corneal blindness.

Initially, the microstructure of the limbal niche was precisely sculpted using ultraviolet (UV) lithography, followed by the creation of collagen membranes imprinted with this microstructure to mimic the corneal limbus's physiological traits. Limbal organoids (LOs), derived from LSCs, were cultured in a microphysiological system (MPS) for one month, leading to their bio-printing onto the collagen membranes with a microstructure ring of the limbal niche. These engineered constructs were then transplanted onto the corneal surfaces of New Zealand white rabbits afflicted with LSCD to evaluate the reparative effects of this bioengineered limbal niche microenvironment on corneal injuries.

The study demonstrated that the MPS fostered the formation of homogeneous LOs. LSCs in LOs adhered to and expanded on the culture plate or collagen membrane, showing near confluence after 24 hours. Immunofluorescence staining revealed key marker expressions, indicating successful cellular differentiation and proliferation. The LOs bio-printed on collagen membranes exhibited enhanced gene and protein marker expressions related to stemness and cellular junctions, outperforming traditional LSC sheets. Transplantation of these LOs-sheets significantly accelerated corneal epithelial healing compared to untreated groups, with a notable restoration of the corneal epithelial layer and reduction in inflammatory cells.

This study underscores the potential of bioprinted LOs-sheets, enriched with a limbal niche microstructure, in promoting corneal injury repair and regeneration. By simulating the limbal stem cell niche's environment and utilizing bioengineered LOs, this approach presents a promising avenue for treating LSCD and restoring vision, marking a significant advancement in the field of regenerative medicine and bioengineering.