Purpose: Regeneration of the cornea due to trauma or disease is a complex biological process, involving a delicate balance between cell differentiation, migration and a multitude of secreted factors and signalling molecules. We hereby present a tissue‐engineering technique as a possible first step towards getting a bench‐to‐bedside approach to assist corneal regeneration and ultimately, corneal transplantation. Methods: 3D extrusion‐based bioprinting and femtosecond laser‐assisted intrastromal keratoplasty (FLISK) were used in the study. Ex vivo cultured mesenchymal stem cells (MSCs) derived from adipose tissue, bone marrow and corneal stroma were bioprinted in a complex, collagen I‐containing hydrogel. The transparent, cell‐loaded nanocellulose and alginate‐based 3D constructs were then implanted into a pig cornea organ culture model. The 3D printed intrastromal constructs were first cultivated ex vivo, while the de novo extracellular matrix (ECM) synthesis and migration of cells was followed by optical coherence tomography and immunohistochemistry. Results: The ex vivo cultured, MSC‐loaded 3D bioprinted constructs supported cell survival and propagation for all three types of MSCs used. The transparency of the corneal stroma substitute was not compromised by the presence of MSCs in the bioprinted matrix. At day 14, the 3D‐bioprinted scaffolds appeared intact, as analysed by optical coherence tomography, while immunofluorescent staining using ECM and MSCs markers demonstrated the cellular presence in the constructs. Conclusions: Optimization of the bioink composition, cellular content and properties of the bioprinted material to be used in corneal transplantation and regeneration studies are essential in fine‐tuning the technique towards its clinical use.
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