Evaluation of Small Intestine Grafts Decellularization Methods for Corneal Tissue Engineering

Abstract

Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.

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

“…Tissue sections 5-lm thick were obtained with a microtome. To determine the effectiveness of cell removal (decellularization efficiency), the number of remaining nuclei in control and decellularized corneas was quantified by using 4,6-diamidino-2-phenylindole (DAPI) staining (Sigma-Aldrich) on deparaffinized tissue sections, 13 and the percentage of cell removal was determined for DPC samples. The number of remaining nuclear debris was determined per field.…”

“…For quantitative analysis of the ECM, histochemistry was carried out to detect the main fibrillar and nonfibrillar ECM components on tissue sections as previously described by Oliveira et al 13 (all reagents were purchased to Sigma-Aldrich):…”

“…On the other hand, we recently demonstrated that the use of decellularized extracellular matrix (ECM) obtained from extraocular tissues, such as the small intestine could permit generating bioengineered cornea substitutes with potential clinical usefulness and translational perspectives. 13 Among all available decellularization methods, those based on the use of detergents are likely the most simple and inexpensive, although none of these methods have been tried for decellularization of whole corneas including the sclerocorneal limbus (SCL), which is essential for cornea cell physiology and regeneration. This could be of interest if limbal stem cells are used in the future to repopulate the SCL of decellularized corneas.…”

Ex vivo construction of a novel model of bioengineered bladder mucosa: A preliminary study

“…For this purpose, small tissue fragments were obtained, washed twice with phosphate‐buffered saline and stained with the LIVE/DEAD assay reagents (calcein/AM and ethidium homodimer‐1; Invitrogen, Carlsbad, CA, USA) following the protocols provided by Invitrogen. The number of alive and dead cells was determined by using a fluorescence microscope, and automatically quantified using ImageJ software as previous reported . Average and standard deviation values were obtained for 7‐ and 14‐days bioengineered samples.…”

Progress in developing decellularized bioscaffolds for enhancing skin construction

“…82 The use of intestinal scaffolds has been extensively evaluated owing to cytokines and growth factors, such as FGF and TGF-β, 83 that are preserved after decellularization. 2(C,D)], and many studies have reported reconstruction of distinct organs, such as the cornea, 38 urethra, 84 vagina, 85 lung, 86 and heart, 87 using decellularized intestinal scaffolds. 2(C,D)], and many studies have reported reconstruction of distinct organs, such as the cornea, 38 urethra, 84 vagina, 85 lung, 86 and heart, 87 using decellularized intestinal scaffolds.…”