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PURPOSE A malfunction of the corneal endothelium leading to corneal opacity is one of the main causes of impaired vision. Currently, keratoplasty is the one and only donor cornea-dependent treatment, and this calls for alternatives because of the worldwide lack of donor corneas. Recently, the topography of Descemet membrane (DM) has been discovered as a feasible stem cell differentiation tool. With this study, we further confirm this mechanotransductive system by using preinduced Wharton jelly-derived mesenchymal stem cells (WJ-EPCs). METHODS To measure the mechanotransductive potential of Descemet-like topography (DLT), WJ-EPCs were cultivated on collagen imprints with DLT. Changes in the gene and protein expressions of corneal endothelial cells (CECs), typical markers such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured. In addition, CEC functionality has been evaluated by measuring the relative potential differences in a 2-compartment system and by measuring corneal transparency in an ex vivo rabbit cornea model. To confirm the activity of WJ-EPCs, rabbit CECs were restless deleted by collagen digestion of a thin layer of rabbit Descemet membrane. RESULTS The proper CEC-typical hexagonal morphology of WJ-EPCs in combination with a significant expression of ZO-1, Na/K-ATPase, PITX2, and COL-8 could be demonstrated. In addition, the WJ-EPCs were able to build up a relative potential difference of 40 mV and to keep corneas clear and transparent. CONCLUSIONS These data indicate that a well-characterized, functional CEC monolayer was developed by using a DLT-mediated mechanotransductive differentiation of WJ-EPCs.

Supplemental Digital Content is Available in the Text., Purpose: Loss of corneal endothelial cells (CECs) bears disastrous consequences for the patient, including corneal clouding and blindness. Corneal transplantation is currently the only therapy for severe corneal disorders. However, the worldwide shortages of corneal donor material generate a strong demand for personalized stem cell–based alternative therapies. Because human mesenchymal stem cells are known to be sensitive to their mechanical environments, we investigated the mechanotransductive potential of Descemet membrane–like microtopography (DLT) to differentiate human mesenchymal stem cells into CEC-like cells. Methods: Master molds with inverted DLT were produced by 2-photon lithography (2-PL). To measure the mechanotransductive potential of DLT, mesenchymal stem cells were cultivated on silicone or collagen imprints with DLT. Changes in morphology were imaged, and changes in gene expression of CEC typical genes such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured with real-time polymerase chain reaction. At least immunofluorescence analysis has been conducted to confirm gene data on the protein level. Results: Adhesion of MSCs to DLT molded in silicone and particularly in collagen initiates polygonal morphology and monolayer formation and enhances not only transcription of CEC typical genes such as ZO-1, Na/K-ATPase, PITX2, and COL-8 but also expression of the corresponding proteins. Conclusions: Artificial reproduction of Descemet membrane with respect to topography and similar stiffness offers a potential innovative way to bioengineer a functional CEC monolayer from autologous stem cells.

Two-photon polymerization (TPP) has become an established generative fabrication technique for individual, up to three-dimensional micro- and nanostructures. Due to its high resolution beyond the diffraction limit, its writing speed is limited and in most cases, very special structures are fabricated in small quantities. With regard to the trends of the optical market towards higher efficiencies, miniaturization and higher functionalities, there is a high demand for so called intelligent light management systems, including also individual optical elements. Here, TPP could offer a fabrication technique, enabling higher complexities of structures than conventional cutting and lithographic technologies do. But how can TPP opened up for production? In the following, some approaches to establish TPP as a mastering technique for molding are presented against this background.

Two-Photon Polymerization (2PP) has become an established process for fabricating individual micro-and nanostructures nearly in the last two decades. Its high degree of freedom opened up novel possibilities for a large range of applications like functional structures for cell growth, photonic crystals, nanoantennas, diffractive optical elements and lab-on-a-chip structures (just to name a few). Since the measurement of structures written with 2PP is always very time consuming, we present a comparison between white light interferometry (WLI) and confocal microscopy (CM) which were used for measuring structures written with 2PP. By performing a GageRR analysis with both metrology devices, we calculated the process tolerance one has to accept when measuring these structures with WLI or CM.

Optical Materials Express 5(2), 456-461 (2015). doi:10.1364/OME.5.000456, Published by OSA, Washington, DC