*4.11. Cornea*

The cornea is a transparent, avascular, and highly innervated connective tissue that acts as the primary structural barrier to infections, and is the first lens of the eye optical system. The human cornea is organized in five layers, three of which are cellular (i.e., the epithelium, stroma, and endothelium), and two are considered interphases (i.e., the Bowman membrane and the Descemet membrane). This highly organized structure contributes to the cornea's transparency and mechanical strength, while disruptions to this pattern disturb said transparency and result in loss of vision [176,177]. The cornea's ECM is composed of water, inorganic salts, proteoglycans, glycoproteins, and collagens. The stromal lamellar collagen fibrils are heterotypic hybrids of types I and V, with significant amounts of collagen types VI, XII, and XIV. A high concentration of small leucine-rich proteoglycans, including decorin, lumican, and keratocan, decorated with dermatan sulfate and keratan sulfate are present in the lamellae, credited with maintaining the interfibrillar spacing required for transparency, and contributing to the regulation of corneal hydration.

Many corneal disorders require a corneal transplant, while obviously there is limited availability of donor tissue. As an alternative to cadaveric corneas, among others, the dECM from acellular porcine and bovine cornea and decellularized amniotic membrane have been combined with di fferent cell types to form full-thickness corneas with stroma, epithelium, and endothelium layers [178–180]. This dECM replicates the structure and functional requirements of the native cornea, with the maintenance of the collagen fibril organization, transparency, biocompatibility, suitable mechanical toughness, and low immunogenicity [181,182].

Detergents such as SDS and Triton X-100 were commonly used in the pioneering cornea decellularization methods. Du et al. used a 24 h SDS (0.5% or 1%) treatment to generate a decellularized porcine cornea matrix, which was opaque and swollen after the decellularization [48]. Transparency was restored after soaking in sterile glycerol for one hour, but implantation in a rabbit model showed stromal edema and worsening of corneal opacity throughout the 28-day observation period. Another comparative study used NaCl, 0.05% SDS, or 1% Triton-X100 to decellularize

human corneas, and observed that NaCl did not affect transparency, while Triton-X-treated corneas experiencing tissue clouding; meanwhile, SDS-treated corneas appeared the most cloudy/opaque after decellularization [183,184]. Conversely, SDS treatment was combined with benzonase (nuclease) and protease inhibitors in human corneal sheets; in this case, when the dECM was recellularized and implanted in a rabbit model, the implanted tissue maintained complete transparency for three months [185]. A conceptually similar decellularization approach combines sodium N-lauroyl glutamate (SLG) surfactant with supernuclease (a nuclease homologous to benzonase), and also provides adequate transparency and good biocompatibility without degradation 28 days after transplantation [57].

Benzonase endonuclease is often used as the main decellularizing agen<sup>t</sup> in a detergent-free approach, based on its ability to quickly infiltrate the corneal stroma, combined with its easy removal by repeated washes. This approach minimizes the destruction of the ECM, with minimal loss of optical transparency and proper results in animal transplantation assays [186].
