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Introduction: Corneal transplantation is the gold standard procedure to cure corneal diseases that can lead to blindness. Fabrication of corneal tissues is a promising solution to overcome the shortage of human donors. Our future goal is to utilize a bioprinter mounted to a robotic arm to print corneal tissue directly onto the eye of the patient. One interesting cell type for in situ and in vivo corneal bioprinting is the use of bone marrow-derived mesenchymal stromal cells (BM-MSC) in their differentiated state toward the keratocyte lineage.
Objective: This study aims to improve the differentiation of BM-MSC into keratocytes using 3D culture techniques, including differentiation post-bioprinting. Specifically, we aim to characterize the expression of primary corneal stromal keratocyte (CSK) specific markers of differentiated cells for their suitability in in vivo corneal bioprinting. To further validate the applicability of the model for in vivo corneal repair, ex vivo experiments were performed using porcine corneal tissue.
Materials & Methods: BM-MSC from 3 donors were cultured in BM-MSC medium. Briefly, BM-MSC were encapsulated in collagen-based hydrogels. After 3 days, differentiation was initiated by subculturing the casted BM-MSC for 14 days in a differentiation medium based on DMEM/F12, 1 % MEM Vitamin Solution, 1 % MEM nonessential amino acids, 1 % Insulin-Transferrin-Selen, 1 mM L-Ascorbate 2-Phosphate, 10 ng/ml FGF-b, and 0.1 ng/ml TGF-b3. The expressions of the CSK-specific markers keratocan, lumican, and ALDH3A1 were investigated by immunofluorescence staining and qPCR. Porcine eyes were cleaned, and the cornea was carefully dissected from the ocular globe. A stromal defect was simulated via central trephination, followed by defect filling using an inkjet-based bioprinter. Cells spreading and differentiation were monitored by IF and qPCR.
Results: 3D differentiation of BM-MSC enhanced their differentiation towards keratocyte lineages compared to traditional 2D culture methods. IF and qPCR analyses revealed an upregulation of CSK-specific markers, indicating successful differentiation, in both 3D cast and 3D printed constructs. Moreover, differentiated cells exhibited typical phenotypes of primary CSK. The ex vivo experiments demonstrated successful filling of the corneal defect with the bioink, along with evidence of cell spreading and keratocyte differentiation as confirmed by protein analysis.
Conclusion: Our work demonstrates the efficiency of 3D culture techniques in promoting the differentiation of BM-MSC into keratocyte lineages for corneal tissue engineering applications. The enhanced expression of CSK-specific markers and functional properties of differentiated cells underscore the potential of this approach for generating bioengineered corneal substitutes. Moreover, the ex vivo tests highlight the potential use of this technique for in vivo corneal bioprinting.
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