Detection of Limbal Stem Cells Adhered to Melt Electrospun Silk Fibroin and Gelatin-Modified Polylactic Acid Scaffolds

Abstract

Limbal stem cells (LSCs) are of paramount importance in corneal epithelial tissue repair. The cornea becomes opaque in case of limbal stem cell deficiency (LSCD), which may cause serious damage to the ocular visual function. There are many techniques to restore damaged epithelium, one of which is the transplantation of healthy cultured LSCs, usually onto a human amniotic membrane or onto bio-based engineered scaffolds in recent years. In this study, melt electrospun polylactic acid (PLA) was modified by silk fibroin or gelatin and further cultured with LSCs originating from three different donors. In terms of physicochemical properties, both modifications slightly increased PLA scaffold porosity (with a significantly larger pore area for the PLA/gelatin) and improved the scaffolds’ swelling percentage, as well as their biodegradation rate. In terms of the scaffold application function, the aim was to detect/visualize whether LSCs adhered to the scaffolds and to further determine cell viability (total number), as well as to observe p63 and CK3 expressions in the LSCs. LSCs were attached to the surface of microfibers, showing flattened conformations or 3D spheres in the formation of colonies or agglomerations, respectively. All scaffolds showed the ability to bind the cells onto the surface of individual microfibers (PLA and PLA/gelatin), or in between the microfibers (PLA/silk fibroin), with the latter showing the most intense red fluorescence of the stained cells. All scaffolds proved to be biocompatible, while the PLA/silk fibroin scaffolds showed the highest 98% viability of 2.9 × 106 LSCs, with more than 98% of p63 and less than 20% of CK3 expressions in the LSCs, thus confirming the support of their growth, proliferation and corneal epithelial differentiation. The results show the potential of these bio-engineered scaffolds to be used as an alternative clinical approach.

Materials Design Innovations in Optimizing Cellular Behavior on Melt Electrowritten (MEW) Scaffolds

“…Several studies have explored the optimization of water-based electrowriting parameters such as voltage, flow rate, and concentration of the ink solution. Researchers have investigated various natural polymeric materials, including gelatin, [73,74] silk fibroin (SF) [75][76][77][78] alginate, and collagen, and for their suitability in water-based electrowriting. Synthetic polymers like polyvinyl alcohol (PVA), [79,80] have also been investigated, and these studies often focus on achieving specific mechanical and biological properties for the resulting fibers or structures.…”

Fabrication of Silk Fibroin‐Derived Fibrous Scaffold for Biomedical Frontiers

“…The addition of SF resulted in the enhancement of fiber diameter (100.33 ± 14.05 μm), porosity (51.68 ± 19.39%), swelling percentage (>30%), and biodegradation rate (36.31 ± 11.29% weight loss in 7 days). Further improved performance was observed in terms of LSCs cell viability (2.9 × 10 6 at 98%) and gene expression (>98% for p63 and <20% for CK3) [39] Promnil et al developed a PLA-based electrospun scaffold where SF was added to improve bio adhesion and degradability. The dissolved SF underwent electrospinning with a 20 kV voltage, 2.0 mL h −1 flow rate, and 15 cm collector distance.…”

Patterned collagen films loaded with miR-133b@MBG-NH2 for potential applications in corneal stromal injury repair

“…These will help and support postoperative endogenous host tissue reconstruction, and contribute to clinical compliance for easy application and use. In view of this, various biopolymers such as collagen [9][10][11], gelatin [12], chitosan [8], and silk fibroin [13][14][15][16] have been tested as implant biomaterials for corneal bioengineering. Collagen, as the main component of corneal stroma, has physiological and immunological characteristics similar to natural cornea [17].…”