Decellularization of human stromal refractive lenticules for corneal tissue engineering.

Gary Hin-Fai Yam,Yu-Chi Liu,Nur Zahirah Binte M Yusoff,Tze-Wei Goh,Jodhbir S Mehta,Melina Setiawan,Xiao-Wen Lee

doi:10.1038/srep26339

Abstract

Small incision lenticule extraction (SMILE) becomes a procedure to correct myopia. The extracted lenticule can be used for other clinical scenarios. To prepare for allogeneic implantation, lenticule decellularization with preserved optical property, stromal architecture and chemistry would be necessary. We evaluated different methods to decellularize thin human corneal stromal lenticules created by femtosecond laser. Treatment with 0.1% sodium dodecylsulfate (SDS) followed by extensive washes was the most efficient protocol to remove cellular and nuclear materials. Empty cell space was found inside the stroma, which displayed aligned collagen fibril architecture similar to native stroma. The SDS-based method was superior to other treatments with hyperosmotic 1.5 M sodium chloride, 0.1% Triton X-100 and nucleases (from 2 to 10 U/ml DNase and RNase) in preserving extracellular matrix content (collagens, glycoproteins and glycosaminoglycans). The stromal transparency and light transmittance was indifferent to untreated lenticules. In vitro recellularization showed that the SDS-treated lenticules supported corneal stromal fibroblast growth. In vivo re-implantation into a rabbit stromal pocket further revealed the safety and biocompatibility of SDS-decellularized lenticules without short- and long-term rejection risk. Our results concluded that femtosecond laser-derived human stromal lenticules decellularized by 0.1% SDS could generate a transplantable bioscaffold with native-like stromal architecture and chemistry.

Highlights

Damage-associated molecular pattern (DAMP) molecules, which could be removed by proper decellularization[15,16]
Sodium dodecylsulfate (SDS, ionic detergent) efficiently solubilizes cellular components, conflicting data have shown its consequences on extracellular matrix (ECM) disruption[19,20,21,22]
After treatment using various decellularization protocols and deturgescence with glycerol, the lenticules were first analyzed for the spectral transmittance (380 to 780 nm wavelength)

Summary

Introduction

Damage-associated molecular pattern (DAMP) molecules, which could be removed by proper decellularization[15,16]. As most decellularization procedures are shown to have negative effects on extracellular matrix (ECM), this may compromise the biomechanical and optical properties of stromal lenticules and hamper their potential corneal applications. Sodium dodecylsulfate (SDS, ionic detergent) efficiently solubilizes cellular components, conflicting data have shown its consequences on ECM disruption[19,20,21,22]. Like freezing and thawing, pressurization and voltage, have direct impact on ECM structure and are expensive to perform. In view of these variable results, we performed a comprehensive comparison of various protocols and optimized a feasible method to decellularize thin human stromal lenticules. Our work was aimed to generate acellular thin lenticules with preserved transparency and stromal architecture as a bioscaffold suitable for recellularization and stromal implantation

Methods

Results

Conclusion