Decellularization of porcine corneas and repopulation with human corneal cells for tissue‐engineered xenografts

Abstract

. Purpose: To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering. Methods: Corneal cells were isolated from human corneoscleral rims. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra‐acetic acid (EDTA, 0.1%), aprotinin (10 KIU/ml) and 0.3% sodium dodecyl sulphate. Haematoxylin–eosin (HE) and 4,6‐diamidino‐2‐phenylindole (DAPI) staining were performed to confirm removal of the corneal cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using DNA Purification Kit (Fermentas, St. Leon‐Rot, Germany). Alcian blue staining was carried out to analyse the structure of the extracellular matrix (ECM). Corneal stromal cells were injected into the DPCs; limbal corneal epithelial cells and corneal endothelial cells were seeded onto the anterior and posterior surfaces of the DPCs, respectively. Evaluation was undertaken at days 14 and 30. The phenotypical properties of the cultivated corneal cells were investigated using Immunolocalization of type I collagen, keratocan, lumican, cytokeratin 3 (AE5) and type VIII collagen. Results: Haematoxylin–eosin and DAPI staining showed efficient elimination of porcine corneal cells, whereas alcian blue confirmed gross preservation of the ECM. The quantitative analysis of the DNA content showed a significant reduction (mean before decellularization: 75.45 ± 13.71 ng/mg; mean after decellularization: 9.87 ± 2.04 ng/mg, p < 0.001). All three types of corneal cells were efficiently cultured and expanded on the DPCs. Conclusions: Decellularized porcine corneas might serve as a potential scaffold for tissue engineering of the cornea, possibly providing xenogenic substrate for corneal transplantation.

Enhanced Regeneration of Corneal Tissue via a Bioengineered Collagen Construct Implanted by a Nondisruptive Surgical Technique

“…Alternatively, efforts by a number of investigators have focused on the use of de-cellularized, chemically-treated pig corneas as an alternative to human donor corneal tissue [10][11][12][13]. While this approach has shown promising results in animal models, the standardized procurement, decellularization methods, storage procedures, and material certification for human use are lacking and can lead to varying outcomes, for example with respect to transparency of the final constructs [10,13].…”

“…While this approach has shown promising results in animal models, the standardized procurement, decellularization methods, storage procedures, and material certification for human use are lacking and can lead to varying outcomes, for example with respect to transparency of the final constructs [10,13]. Moreover, traces of cytotoxic chemicals used to remove all traces of DNA from the corneas may remain in the constructs [10], the consequences of which are unclear.…”

“…The surgery minimizes disturbance of the epithelium and maintains corneal shape and curvature without suturing, thereby avoiding stimulation of an aggressive wound healing response. Additionally, FLISK can be considered a therapeutic procedure for replacement of diseased or damaged native stroma, in contrast to micropocket insertion models [10,12,13] where native tissue is not excised thus leading to an unnatural refraction and stromal architecture, inflammation, and the need for surgical suturing and use of postoperative steroids. In this study, it was found that the BPC implants maintained the shape and structure of the host cornea while supporting rapid healing and a limited in-growth of host stromal cells, which began to degrade the implanted collagen scaffold and produce new host collagen in less than 8 weeks.…”

Biocompatibility Evaluation Criteria for Novel Xenograft Materials: Distribution and Quantification of Remnant Nucleic Acid and Alpha-Gal Epitope

“…These functional properties, combined with low antigenicity [37], make TC a potential biomaterial for the production of heart valve substitute as well as tissue patches. However the presence of the alphaGal gene in ocular surface tissue could be decisive for the rejection of the implanted xenograft [38]. Up to now, a qualitative evaluation of such xenoantigen in corneal tissue has been limited to three fish species [39][40][41]: our results implement this knowledge.…”

“…Scaffolds derived from mammalian tissues have been used to repair or replace a variety of damaged or diseased tissues including cardiac [1,2], esophageal [3,4], dermal [5], musculotendinous [6,7] and ocular [8], among others. Results of preclinical and clinical studies have varied from success [9][10][11] to complete failure [12][13][14][15][16][17].…”