Citation: González-Andrades M, Carriel V, Rivera-Izquierdo M, et al. Effects of detergent-based protocols on decelluarization of corneas with sclerocorneal limbus. Evaluation of regional differences. Tran Vis Sci Tech. 2014;4(2):13, http://tvstjournal. org/doi/full/10.1167/tvst. 4.2.13, doi: 10.1167/tvst.4.2.13 Purpose: In this work, we decellularized whole porcine corneas including the sclerocorneal limbus (SCL) and we evaluated regional differences in order to identify an efficient method to decellularize whole corneas for future clinical use. Methods:We analyzed the efficiency of four decellularization protocols based on benzalkonium chloride (BAK), Igepal, sodium dodecyl sulfate (SDS), and Triton X-100 detergents on whole porcine corneas.Results: Results showed that the decellularization efficiency of most protocols was low, with the specific protocol resulting in more efficient levels of decellularization being 0.1% SDS for 48 hours, especially in the medium and posterior cornea regions. A significant correlation was found between the decellularization efficiency and the concentration of agent used (P ¼ 0.0174; r ¼ 0.1540), but not for the incubation time (P . 0.05). The analysis of cornea components preservation demonstrated that all protocols were able to preserve the integrity of the Bowman's layer and Descemet's membrane. Although the collagen structure was partially altered, the global decellularization groups showing highest preservation of the ECM collagen contents and orientation were Igepal and SDS, with differences among the three regions of the cornea. All global groups showed high levels of proteoglycan and glycoprotein preservation after decellularization, with the best results were found in the SDS group followed by the Igepal group.Conclusions: These results suggest that very powerful protocols are necessary for whole-cornea decellularization. For the generation of lamelar corneas for clinical use, decellularization regional differences should be taken into account.Translational Relevance: Decellularized whole corneas may be potential therapeutic agents for lamelar keratoplasty.

Ideally, biomaterials designed to play specific physical and physiological roles in vivo should comprise components and microarchitectures analogous to those of the native tissues they intend to replace. For that, implantable biomaterials need to be carefully designed to have the correct structural and compositional properties, which consequently impart their bio-function. In this study, we showed that the control of such properties can be defined from the bottom-up, using smart surface templates to modulate the structure, composition, and bio-mechanics of human transplantable tissues. Using multi-functional peptide amphiphile-coated surfaces with different anisotropies, we were able to control the phenotype of corneal stromal cells and instruct them to fabricate self-lifting tissues that closely emulated the native stromal lamellae of the human cornea. The type and arrangement of the extracellular matrix comprising these corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs) were then evaluated in detail, and was shown to correlate with tissue function. Specifically, SLATEs comprising aligned collagen fibrils were shown to be significantly thicker, denser, and more resistant to proteolytic degradation compared to SLATEs formed with randomly-oriented constituents. In addition, SLATEs were highly transparent while providing increased absorption to near-UV radiation. Importantly, corneal stromal SLATEs were capable of constituting tissues with a higher-order complexity, either by creating thicker tissues through stacking or by serving as substrate to support a fully-differentiated, stratified corneal epithelium. SLATEs were also deemed safe as implants in a rabbit corneal model, being capable of integrating with the surrounding host tissue without provoking inflammation, neo-vascularization, or any other signs of rejection after a 9-months follow-up. This work thus paves the way for the de novo bio-fabrication of easy-retrievable, scaffold-free human tissues with controlled structural, compositional, and functional properties to replace corneal, as well as other, tissues.

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