3D Cell Printed Corneal Tissue Analogues with Shear-aligned Fibers of Enhanced Tissue-specific Bioinks

Abstract

The cornea is the outermost transparent tissue of the eye, with the most cases for transplantation. Nevertheless, due to the lack of donor cornea, domestic corneal graft waiting time is reported to be at an average of 7 years or more. The inadequate supply of donated cornea is becoming further intensified due to the worldwide trends in population aging and the increasing popularity of refractive surgery such as laser-assisted in situ keratomileusis (LASIK). Finally, the average transplant waiting time is expected to be elongated. So far, clinically available artificial corneas based on synthetic polymers were developed, but various postoperative complications have been reported due to the limitation of cornea-specific properties and their ability to bio-integrate with surrounding host tissues. To overcome this problem, tissue-engineered corneas have been proposed as donor corneal replacements. However, these substitutes have different corneal specific properties (such as transparency) from that of native corneal tissue, or also hardly recruit from host cells to be integrated with the surrounding tissue. In this study, corneal bioink was developed using cornea-derived decellularized extracellular matrix and was aimed at increasing the possibility of bio-integration with surrounding tissues. The microstructural and biochemical tissue-specific environment was recapitulated in the 3D cell-printed structure using the bioink. Furthermore, physical properties including suturability increased by the additional crosslinking in the bioink, demonstrating the potential as an actual corneal substitute. The newly developed tissue engineered cornea was then implanted into a beagle dog model to evaluate its regeneration and efficacy as corneal tissue. The details of this study are as follows. To recapitulate the tissue-specific microenvironment, we first developed corneal tissue-derived decellularized extracellular matrix bioink. Compared with the collagen hydrogels commonly used in corneal tissue engineering research, the bioinks exhibited similar protein composition to corneal tissue, high transparency, and high differentiation rate from stem cells to corneal cells (cornea specific properties). In addition, when transplanted in vivo, the rate of immune response was at a similar level to that of clinical grade collagen hydrogel. The internal structure of the corneal tissue is organized in a lattice pattern consisting of collagen fibrils. To mimic this collagenous architecture, the collagen fibrils were arranged using the shear stress generated during 3D cell-printing process. The level of shear stress, controlled by the various size of the printing nozzle, manipulates the arrangement of the fibrillar structure. With proper selection of parameters, the printed cornea exhibited a high cellular alignment capability, indicating a tissue-specific structural organization of collagen fibrils. Printing conditions have been established according to the cellular performances. The collagen fibrils that remodeled along with the printing path created a lattice pattern similar to the structure of human cornea after 4 weeks in vivo, with transparency higher than that of native corneal tissue. Thereafter, the corneal grafts were transplanted to rabbit model and high transparency along with tissue integration were verified. Based on the bio-inks developed previously and the fabrication methods to recapitulate shear-aligned collagen fibril patterns upon 3D cell printing, tissue-engineered corneas were developed with improved mechanical properties. In order to improve the physical properties, the methacrylated collagen was incorporated with the bio-ink to induce further crosslinking. After additional bonding, the physical properties of the corneal equivalents improved by approximately 80 times, while no significant difference in cellular behaviour was observed. These tissue-engineered corneas with improved properties were implanted into the beagle dog model using the Deep Anterior Lamellar Keratoplasty, which is widely used in corneal transplantation. Epithelialization occurred above the graft surface at the first week of transplantation. At postoperative sixth months, the tissue-engineered corneas were integrated with the surrounding corneal tissue, replacing newly reconstructed corneal matrix. Moreover, beagles that underwent transplantation recovered with visual acuity similar to that of the normal cornea. The present dissertation demonstrates the practical use of the corneal equivalents based on 3D cell printing technologies incorporating with enhanced cornea-specific bioink. In addition, the printing technology aligning fibers can be applied not only to develop corneal equivalent, but also to mimic various tissue analogues such as muscle, skin, and cartilage, enabling the construction of micropatterned constructs. This technology will provide a blueprint for developing human scale tissue replacing donor tissues/organs.