Posterior lamellar techniques, such as Descemet stripping (automated) endothelial keratoplasty (DSEK/DSAEK) and Descemet membrane endothelial keratoplasty (DMEK), have been developed and became an alternative to the traditional full thickness corneal replacement known as penetrating keratoplasty in the early stage of corneal decompensation. In general, thinner graft results in shorter rehabilitation time and better visual function outcome.
The thickness of posterior cornea grafts dissected by microkeratome in DSAEK is not that predictable. While DMEK grafts preparation requires much more training and suffer a higher failure rate, McCauley et al. even reported a grafts reattachment rate up to 25% after DMEK surgery. Constructing an in vitro bioengineered graft can achieve a predictability in both sheets' thickness and cell density. As for CC, this biomaterial has easy production progress and is able to be manipulated to achieve the ideal thickness, which may reach the balance point between decreasing recovery time and reducing grafts dislocation rate. Alarcon et al. even successfully added antibiotic properties to collagen implants foreboding the broad application prospect of collagen-constructing grafts. Laminin is coated onto CC to form effective connection and increase grafts cell density, for CECs express laminin receptor and previous experimental study have proved that laminin can promote adhesion, migration, and moderate proliferation of cultured CECs. Levis et al. seeded CECs onto CC without coated laminin and reached a final graft cell density <2000 cells/mm2, while in our study, laminin-coated CC can eventually got a graft cell density more than 3600 cells/mm2.
In our study, B-CECs formed a confluent monolayer on CC. The morphology of cells were similar to in vivo status, displayed a homogeneous and hexagonal shape, with the cell density higher than cadaveric graft. The ultrastructural analysis also suggested an integral endothelial layer with tight cell junction and microvilli, which are critical structure for endothelium cell to form a boundary and pump water from corneal stroma into aqueous humor. ZO-1 expression identified in this bioengineered graft further confirmed this observation. ZO-1 is a prominent protein in cell tight junction complex, is also considered to regulate paracellular permeability. Bioengineering an endothelial sheet relies on the fact that the CECs can be expanded in vitro, but this approach would fail if cells did not maintain functional phenotype. The ZO-1 labeled at B-CECs lateral cell borders indicated that transferred B-CECs still remain the key function which underpins the possibility of constructing a biomimetic endothelium sheet with viability.
To establish a bioengineered graft that can apply to real clinic require further study. Our study is a heterogenic study using bovine cells, human endothelial cells' expansion ability on CC should be tested, as well as the cell function phenotype of transferred human endothelium cell. The animal experiment also needs to be performed to verify the safety and efficiency of graft.
We successfully constructed a biomimetic endothelium graft with B-CECs expanded on CC sheet. The morphology and ultrastructure of cells on biomaterial similar to that ofendothelial cells in vivo. The graft has also been improved to possess the basic specific function of endothelium layer. This method set the cornerstone of using one donor's cornea to form multiple uniformed endothelium grafts to overcome the shortage of cadaveric cornea tissue.
This study was supported by a grant from the National Natural Science Foundation of China (No. 81700799).
There are no conflicts of interest.
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Edited by: Yi Cui