Optimized Bombyx mori silk sericin biomaterial for retinal pigment epithelium regeneration.

Abstract

Purpose: A transplantable cell carrier is sought to restore the retinal pigment epithelium (RPE) in age-related macular degeneration. Preparations of Bombyx mori silk sericin (BMSS) have shown in vivo and in vitro protective, wound healing, and antioxidative properties; however, current isolation techniques lead to inconsistent structure and bioactivity. We present an optimized method for BMSS isolation and manufacture into a biomaterial capable ofsupporting RPE.Methods: Gravimetric yield analysis, SDS-PAGE, solubility, transparency, and the ability to support viable human adult RPE cells (ARPE-19) were determined for the BMSS films created from four increasing durations of high temperature high pressure (HTHP) extraction, without dialysis. The new preparations were compared to both the original HTHPmethod (30-min HTHP with 4-h dialysis) and to uncoated tissue culture plastic (TCP), as a positive control. The effect of water annealing the films was also investigated. The optimized method was tested as a biomaterial for humanembryonic stem cell derived-RPE (hESC-RPE) cells against Geltrex coated and uncoated transwell inserts (Fig. 1).Results: The yield was not adversely affected by increased duration of HTHP (n=5) and surpassed previous methods. The polypeptide component of BMSS degraded in a time-dependent manner, increasing solubility of the lyophilised product. Films were suitable for biomaterial applications if cast from freshly prepared solutions but not if reconstituted from lyophilised BMSS. Films prepared from both the new and original 30-min HTHP methods (and not annealed) performed comparably to TCP in terms of ARPE19 cell viability at 4 h and 7 days (2-way ANOVA, n=3, p>0.05). As dialysis proved unnecessary, the new 30 min method was selected for improved speed, yield, and theoretical retention of potentially bioactive components. Long term hESC-RPE established for 8 weeks upon optimized BMSS filmsdisplayed a monolayer culture (1-2 cells deep) with correct cobblestone morphology (Fig. 2) and pigmentation.Conclusions: Films prepared by the new optimized method of 30-min HTHP without dialysis or water annealing supported ARPE19 cells with viability equivalent to TCP controls, and outperformed Geltrex and uncoated transwells in developing correct hESC-RPE morphology and pigmentation