Generation of a Biomimetic Substitute of the Corneal Limbus Using Decellularized Scaffolds

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.

Recent Approaches to the Modification of Collagen Biomatrix as a Corneal Biomatrix and Its Cellular Interaction

“…In this review, the search successfully finalized the studies related to the modification of collagen biomatrix and its effect on CECs or LESCs. To summarize the selected articles from 2012 to 2022, eight studies aimed to develop a new formulation for collagen biomaterials [ 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 ], whereas twelve studies aimed in improving or characterizing current collagen biomaterials, including the improvement of fabrication methods to produce better collagen biomatrix [ 27 , 35 , 51 , 58 , 59 , 60 , 61 , 76 , 77 , 78 , 79 , 80 ].…”

Optical Behavior of Human Skin Substitutes: Absorbance in the 200–400 nm UV Range

“…In all cases, positive and negative control tissues were used, and samples were counterstained using Harry’s hematoxylin and coverslipped. To histologically evaluate the dermis development, relevant extracellular matrix components (ECM) were assessed by identifying collagen fibers and proteoglycans using alcian blue (AB) and picrosirius red (PS) histochemistry, respectively, as previously reported [ 28 ] (reagents from Panreac AppliChem, Barcelona, Spain). Normal human native skin samples (CTR) were used as controls for all these analyses.…”

Recent Approaches of Collagen Biomatrix Modification as a Corneal Biomatrix and its Cellular Interaction

“…In this review, the search was successfully finalized the studies related to the modification of collagen biomatrix and its effect on CECs or LESCs. To summarize the selected articles from 2012 to 2022, eight studies aimed to develop a new formulation for collagen biomaterials [68][69][70][71][72][73][74][75], whereas 12 studies aimed in improving or characterizing current collagen biomaterials, including the improvement of fabrication methods to produce better collagen biomatrix [27,35,51,[58][59][60][61][76][77][78][79][80].…”

“…A study by Park and colleagues also decellularized corneal stromal tissue from bovine eyeballs and produced three dimensional (3D) bioprinting decellularized collagen sheet (3D-BDCS), which could re-epithelialize the damaged epithelial layer within a few days [71]. Moreover, the decellularization of the porcine limbus and re-cellularized Statens Seruminstitut Rabbit Cornea (SIRC) limbal epithelial cell line and human adiposederived mesenchymal stem cells (hADSCs) could produce a biomatrix with high content of collagen IV, and are able to regenerate stratified epithelium [75]. This is due to collagen IV affecting CECs transcriptional factor, which plays a significant role in CECs adhesion and migration properties [92,93].…”