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Introduction: Damage of the sclerocorneal limbus may lead to limbal stem cell deficiency (LSCD), loss of corneal transparency, and blindness. Keratoplasty is the standard treatment for corneal transplantation disorders, but it is contraindicated in cases with LSCD or limbal damage1, which are very difficult to manage. Generation of a biological substitute of the sclerocorneal limbus could contribute to treat these patients. However, the complex three-dimensional structure of the sclerocorneal limbus makes it difficult to generate fully functional artificial tissues2. In the present work, we describe a bioengineered sclerocorneal limbus substitute generated by decellularization techniques.
Methodology: Four different decellularization protocols were applied to the porcine sclerocorneal limbus. These protocols were based on the use, alone or in combination, of osmotic agents (distilled water and NaCl), detergents (SDS and SD) and enzymes (nucleases) in different proportions to remove native cellular components. The efficiency of the decellularization procedure was assessed by DAPI staining and DNA quantification. Morphology, structure, and ECM composition were assessed histologically. In addition, transparency of decellularized limbus were evaluated.
Results: All protocols achieved effective decellularization, with HE and DAPI analyses showing no remaining cell nuclei in any protocol. However, some differences were found for the DNA quantification analysis, with protocols based on 0.1% SDS showing the best results. Analysis of corneal transparency revealed that all decellularized tissues were able to transmit the incoming light, although the most adequate results were found in the 0.1% SDS group. Interestingly, the analysis of collagen fibers showed that the 0.1% SDS was able to more adequately preserve the amount and orientation of the stromal collagen fibers as compared to the other experimental groups.
Conclusions: Our results demonstrate that decellularized sclerocorneal limbus xenografts can be efficiently generated in the laboratory. Application of specific decellularization protocols based on 0.1% SDS showed the most optimal results in terms of remaining nuclear components, transparency and ECM composition, being a promising alternative for LSCD treatment.
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