Ex Vivo Functionality of 3D Bioprinted Corneal Endothelium Engineered with Ribonuclease 5‐Overexpressing Human Corneal Endothelial Cells

Abstract

Human corneal endothelial cells (HCECs) are scarcely proliferative in vivo. The cultured HCECs engineered to overexpress ribonuclease (RNase) 5 (R5-HCECs) are prepared after transient transfection with RNase 5 plasmid vector. As candidate targets of R5-HCECs for enhancement of cellular proliferation and survival of R5-HCECs, programmed cell death protein 4 is inhibited, and cyclin D1 and cyclin E1 are activated. The cultured R5-HCECs and control HCECs on lyophilized amniotic membrane (AM) are deposited as a carrier by extrusion-based 3D bioprinting to prepare transplantable RNase 5 vector-transfected HCECs-laden AM graft (R5-Graft) and the control HCECs-laden AM graft (Ct-Graft), respectively. The ready-to-use R5-Graft shows clearer basolateral expression of Na -K ATPase pump and higher cell confluency than Ct-Graft. From 2 weeks after graft transplantation, both R5-Graft and Ct-Graft start restoring clarity of the rabbit corneas, and their central corneal edema are much less than those in the control group at 3 and 4 weeks. The ex vivo expression of corneal endothelial phenotypical markers is clear in R5-Grafs rather than in Ct-Grafts at 4 weeks. In conclusion, the fabricated corneal endothelium with cultured HCECs easily survives and functions as corneal endothelium in vivo. Furthermore, the use of the cultured HCECs engineered to overexpress RNase 5 (R5-HCECs) may be an option to obtain higher graft cellularity and to enhance the function of transplanted grafts.

Prospects and Challenges of Translational Corneal Bioprinting

“…Given that corneal stromal bioprinting has shown encouraging results up to now, one can consider expanding this knowledge to manufacturing in vitro the remaining layers of the cornea, despite their distinct characteristics relative to the stroma. Two examples of studies that experimented with epithelial and endothelial corneal bioprinting are listed in Table 1 [90,91]. In the study by Zhang et al [90], human CEpCs were embedded in 15% GelMa hydrogels and printed by extrusion.…”

“…In the study by Zhang et al [90], human CEpCs were embedded in 15% GelMa hydrogels and printed by extrusion. In the other study by Kim et al [91], human CECs were embedded in a gelatin-RGD bioink and printed in an amniotic membrane support. In both studies, cells remained round inside the matrices after printing and did not form mono/multilayers, questioning the choice of materials or the need for printing these very thin layers with an automated dispensing mechanism.…”

The progress in corneal translational medicine

“…In the study by Zhang et al, 204 human CEpCs were embedded in 15% GelMa hydrogels and printed by extrusion. In another study by Kim et al ), 205 human CECs were embedded in a gelatin-RGD bioink and printed in an amniotic membrane support. In both studies, the cells remained round inside the matrices after printing and did not form mono/multilayers, challenging the choice of materials and the need for printing these very thin layers with an automated dispensing mechanism.…”

“…After harvesting, the cell-cell junctions and the extracellular matrix were maintained. A gentle enzymatic approach was used by Kim in two human studies 205,276 (9.52% of the human studies). Engineered cell sheets were detached using dispase treatment, which cleaves collagen and fibronectin from the extracellular matrix.…”

Aktueller Stand und neue Entwicklungen des 3D-Drucks in der Unfallchirurgie

“…"Bioprinting" bezieht sich auf eine schichtbasierte Technologie, in der zellbasierte Materialien oder Zellen in bestimmter räumlicher Anordnungen auf einer Matrix aufgebracht werden. Bioprints -mit unterschiedlichen Techniken hergestellt -werden zum Ersatz von Kornea [33] oder Blutgefäßen [65] intensiv beforscht und im Bereich der Haut bereits erfolgreich eingesetzt. Bei anderen Geweben stellen Vaskularisierung und Biointegration die größten biologischen Herausforderungen, die nur inter-und multidisziplinär (Materialwissenschaftlern, Gewebeingenieure, Chirurgen) gelöst werden können [29,35,38].…”