Abstract
Patients with severe limbal damage and limbal stem cell deficiency are a therapeutic challenge. We evaluated four decellularization protocols applied to the full-thickness and half-thickness porcine limbus, and we used two cell types to recellularize the decellularized limbi. The results demonstrated that all protocols achieved efficient decellularization. However, the method that best preserved the transparency and composition of the limbus extracellular matrix was the use of 0.1% SDS applied to the half-thickness limbus. Recellularization with the limbal epithelial cell line SIRC and human adipose-derived mesenchymal stem cells (hADSCs) was able to generate a stratified epithelium able to express the limbal markers p63, pancytokeratin, and crystallin Z from day 7 in the case of SIRC and after 14–21 days of induction when hADSCs were used. Laminin and collagen IV expression was detected at the basal lamina of both cell types at days 14 and 21 of follow-up. Compared with control native limbi, tissues recellularized with SIRC showed adequate picrosirius red and alcian blue staining intensity, whereas limbi containing hADSCs showed normal collagen staining intensity. These preliminary results suggested that the limbal substitutes generated in this work share important similarities with the native limbus and could be potentially useful in the future.
Highlights
Numerous diseases, including trauma, infections, congenital malformations, degeneration, and other conditions, may affect the transparency of the human cornea and cause blindness [1]
Analysis of the different decellularization protocols studied in this work revealed that the four protocols were able to efficiently decellularize the porcine corneal limbus
The present report is a preliminary work, our results suggest that these bioartificial limbi display several similarities with the native limbus and, could be potentially useful for the treatment of limbal stem cell deficiency (LSCD)
Summary
Numerous diseases, including trauma, infections, congenital malformations, degeneration, and other conditions, may affect the transparency of the human cornea and cause blindness [1]. Cultured limbal stem cells can be grafted as isolated cells or by using different types of carriers and biomaterials such as the human amniotic membrane, fibrin, collagen, or synthetic biopolymers [9,10,11] Alternative approaches such as the use of cultured oral mucosa keratinocytes have been proposed for LSCD treatment [12]. The fine structure of the human cornea and corneal limbus is very complex, and alternative approaches including the development of transparent bio-inks and complex design protocols are in need to generate efficient limbal substitutes using bioprinting [14,15]. The use of decellularization methods applied to the native limbi provides the specific morphology, structure, and protein composition of the corneal limbus [30], and offers the opportunity of obtaining adequate limbal scaffolds for use in tissue engineering.
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