Native-Like, Cell-Laden, Orthogonal-Multilayer and Transplantable Tissue- Engineered Corneal Stroma Induced by a Mechanical Collagen Microenvironment

Abstract

Purpose : In native corneal tissue, the highly ordered spatial arrangement of collagen fibrils in stroma is critical for optical transparency. Thus, engineering aligned corneal stroma enables the implementation of corneal functionalities. However, achieving desired alignment is challenging and the vast majority of attempts to reproduce stroma follow a scar model.

Methods : Here, we have developed a protocol for the efficient generation of a native-like, cell-laden and orthogonal-multilayer tissue-engineered (TE) corneal stroma, which is induced by the mechanical effects of compressed collagen (CC) or stretched compressed collagen (SCC).

Results : Compared with those cultured on tissue culture plates (TCP), corneal stromal cells (CSCs) in CC or SCC showed a shift from the activation (A) state of fibroblasts (A₁) or myofibroblasts (A₂) to the keratocyte quiescent state (A₀) by analysis of gene expression and protein expression. Furthermore, within SCC, long CSCs and orthogonal collagen microfibres with d-period are orderly arranged, as observed under confocal microscopy and scanning and transmission electron microscopy (Fig1). The down-regulated genes and proteins of proliferation, cytoskeletal actin and tubulin, up-regulatedgene of GJA1 (gap junction protein 1), down-regulated gene of TRPV4 and up-regulated gene of TRPA1 are associated with changes in CSCs of SCC. Orthogonal-multilayer SCC shows strong mechanical strength and transparency similar to that present in native cornea. Moreover, SCC grafts displayed good biocompatibility and integration with host corneal tissue after rabbit allogenic lamellar implantation by wk 6 under slit lamp and in vivo confocal microscopes (Fig2) and histological examination.

Conclusions : The SCC model facilitates the construction of physiological feature TE corneal stroma instead of the fibrotic scar model. This method can be applied for reconstructing other tissues where ordered alignment in a multi-modular structure is desired. And the study also helps to reverse fibrosis pathologies in general.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Figure 1 | The arrangement of collagen fibers and CSCs in CC and SCC.
In the SCC, cells are arranged orderly. The d-period is observed (arrows).

 

Figure 2 | The corneal lamellar implantation of CC, SCC and acellular collagen grafts. Slit lamp biomicroscopic photographs and In vivo confocal microscopic images in post-operative 6wk.