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. 2015 Apr 10;4(2):13.
doi: 10.1167/tvst.4.2.13. eCollection 2015 Apr.

Effects of Detergent-Based Protocols on Decellularization of Corneas With Sclerocorneal Limbus. Evaluation of Regional Differences

Miguel González-Andrades  1 , Victor Carriel  2 , Mario Rivera-Izquierdo  2 , Ingrid Garzón  2 , Elena González-Andrades  2 , Santiago Medialdea  3 , Miguel Alaminos  2 , Antonio Campos  2
Affiliations

Effects of Detergent-Based Protocols on Decellularization of Corneas With Sclerocorneal Limbus. Evaluation of Regional Differences

Miguel González-Andrades et al. Transl Vis Sci Technol. .

Abstract

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 = 0.0174; r = 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.

Keywords: cornea regions; decellularization protocols; extracellular matrix; sclerocorneal limbus; tissue engineering.

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Figures

Figure 1.
Figure 1.
Decellularization efficiency based on histological analysis using DAPI staining of NPC and DPC subjected to the different decellularization protocols. The percentage of remaining cells or cell debris is shown for each group of samples and for the anterior (blue), medium (red), and posterior (green) areas of the cornea (n = 3) using NPC as control. TX100: Triton X-100.
Figure 2.
Figure 2.
ECM components preservation of NPC and DPC subjected to the different decellularization protocols. Illustrative images correspond to samples decellularized for 48 hours using 0.1% concentrations of each agent. For each protocol, four images are shown corresponding to the immunohistochemical staining using alcian blue (AB), PAS, picrosirius red staining (PSR), and polarized picrosirius red staining (pPSR). Scale bar: 300 μm.
Figure 3.
Figure 3.
Collagen preservation as determined by PSR histochemical staining of NPC and DPC subjected to the different decellularization protocols. The percentage of remaining collagen fibers is shown for each group of samples and for the anterior (blue), medium (red), and posterior (green) areas of the cornea (n = 3) using NPC as control.
Figure 4.
Figure 4.
Analysis of collagen fibers orientation based on the evaluation of mean resultant vector (MRV) using the SurfCharJ ImageJ plug-in in NPC and DPC subjected to the different decellularization protocols. For each sample, the MRV is shown for the anterior (blue), medium (red), and posterior (green) areas of the cornea (n = 3).
Figure 5.
Figure 5.
Proteoglycans preservation as determined by AB histochemical staining of NPC and DPC subjected to the different decellularization protocols. The percentage of remaining proteoglycans is shown for each group of samples and for the anterior (blue), medium (red), and posterior (green) areas of the cornea (n = 3) using NPC as control.
Figure 6.
Figure 6.
Glycoproteins preservation as determined by PAS histochemical staining of NPC and DPC subjected to the different decellularization protocols. The percentage of remaining glycoproteins is shown for each group of samples and for the anterior (blue), medium (red), and posterior (green) areas of the cornea (n = 3) using NPC as control.
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