Generation of a Bioengineered Substitute of the Human Sclero-Corneal Limbus Using a Novel Decellularization Method

<b>Background</b>: Severe dysfunction of the human limbus associated with limbal stem cell deficiency is a therapeutic challenge, especially when a structural alteration of the limbal niche is associated. <b>Methods:</b> We have evaluated seven decellularization protocols applied to 20 human sclero-corneal limbus, based on the use of SDS (protocol P1), SDS + NaCl (P2), SDS + triton X-100 + SDC + enzymatic treatment (P3), SDS + triton X-100 + SDC + enzymatic treatment + trypsin (P4), sulfobetains + DNAse (P5), sulfobetains + SDC + DNAse (P6) and SDC + DNAse (P7). The decellularization efficiency of each protocol, biocompatibility and safety, as well as their capability to support cell attachment and differentiation, were evaluated. <b>Results:</b> Results showed that the use of protocols P1 to P4, based on strong ionic detergents such as SDS, was not efficient for decellularizing the human limbus. Conversely, protocols P5, P6 and P7 removed more than 95% of DNA while preserving 60-100% of the extracellular matrix components. These protocols were biocompatible, as macrophages cultured with decellularized scaffolds were viable and differentiated to the pro-regenerative M2 phenotype (CD163/CD86 ratio > 2) without inducing a significant increase in reactive oxygen species (ROS). Protocols P6 and P7 supported cell attachment, survival and differentiation of corneal epithelial cells and four types of mesenchymal stem cells cultured on the surface of these scaffolds. Cellularized limbi showed positive expression of several limbal cell markers, especially in scaffolds decellularized with protocol P6. <b>Conclusions:</b> These results support the use of protocol P6 for the generation of human limbal substitutes by tissue engineering using decellularized human limbi. Future studies should determine the clinical potential of the regenerative biomaterial generated in patients with structural limbal damage, particularly in patients with chemical burns and aniridia, where conventional stem cell therapies fail.