Scaffold and Cell Engineering of Artificial Bruch s Membrane

Abstract

Purpose : Our main goals are to design and create and improve functional and artificial Bruch s membranes through established bioengineering techniques.

Methods : We fabricated novel ultrathin 3-dimensional(3D) nanofibrous membranes from Polycaprolactone, and Polycaprolactone/5% gelatine and Polycaprolactone/30% gelatin by an advanced clinical-grade needle-free-electrospinning process. The nanofibrillar 3D networks highly mimicked the fibrillar architecture of the native inner collagenous layer of human BM. ARPE-19 cells (n 15,000) were plated onto the surface and the RPE reattachment, apoptosis, and proliferation ratios were determined on the modified surfaces. Cells were cultured up to 17 days to determine the surface coverage. Ultrastructure of the modified Bruchs membrane and RPE morphology were studied with transmission and scanning electron microscopy. Triplicate wells were used to calculate the average reattachment, apoptosis, and proliferation ratios and the final fate of RPE cells seeded
onto each substrate. MTT assays were performed in order to evaluate ARPE cell viability and citotoxicity of the membranes. SEM studies were performed in duplicate only.

Results : Data from all experiments were pooled and expressed as the mean SD. The reattachment, apoptosis, and proliferation ratios and surface coverage on different substrates and RPE cell populations were analyzed in pairs by the Dunn’s multiple comparison test 28. A confidence level of P 0.05 was considered to be statistically significant. ARPE19 cells grown on our nanofibrous membranes in a similar mannes to native human RPE. They exhibited a correctly orientated monolayer with polygonal cell shape and abundant sheet-like microvilli on their apical surfaces after several days of culture. ARPE 19 cells built tight junctions and expressed RPE65 protein. MTT assays demostrate that our artificial membrane is not toxic for ARPE19 cells.

Conclusions : Age-related changes that impair RPE repopulation of Bruch s membrane can be significantly reversed by combined cleaning and ECM protein coating of the ICL. Our artificial membranes may imitate the natural BM to such extent that they allow for the engineering of in vivo-like human RPE monolayer and maintaining its biofunctional characteristics. Such artificial membranes may be a promising vehicle for a functional RPE cell monolayer implantation in the subretinal space in patients with AMD or SD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.