Abstract
Purpose :
Dry age-related macular degeneration (AMD) is the leading cause of blindness in people over 60 years, and to-date has no effective treatment. Researchers have used electrospun membranes to support retinal pigment epithelium cells (RPEs) to treat dry AMD. It remains unclear if mimicking the microenvironment of the top 2 layers of native Bruch’s membrane (BM), namely laminin and the inner collagenous layer (ICL) can support functional RPEs. Our aim was first, to fabricate electrospun nanofibrous membranes (ENMs) with physical properties similar to the ICL. Second, evaluate invitro laminin adsorption on the ENMs and their subsequent influence on RPE proliferation and functionality.
Methods :
ENMs were fabricated via electrospinning, and coated with 20μg/mL of laminin. ENM fibre diameter, thickness and porosity were determined by SEM. Laminin adsorption was determined by a micro-BCA™ assay. ENM surface roughness, surface stiffness and modulus were measured using AFM. In invitro studies, RPEs were seeded (10,000/cm2) on ENMs and assessed at various time points via transepithelial resistance (TEER), q-PCR and immunohistochemistry.
Results :
ENMs with average fiber diameters ≤ 70nm, thicknesses < 1.4μm, and porosities > 45% were fabricated via electrospinning, thereby mimicking the ICL (Fig1A). The 70nm fiber diameter helped create ENMs with high surface roughnesses (Fig1B). Due to a thermodynamically favorable state, PLLA based ENMs adsorbed high amounts of laminin (Fig1C), which in short-term culture, significantly increased RPE attachment and proliferation (Fig1D). qPCR (Fig2C) and immunohistochemical (Fig2D) assessments showed all the ENMs to support expression of signature RPE markers. In long-term culture, PLLA based ENMs supported functional RPE monolayers, which exhibited polygonal morphology and apical microvilli (Fig2A), high TEER (Fig2B) and phagocytic activity (Fig2D).
Conclusions :
We successfully fabricated ICL-like scaffolds, coated with laminin, mimicking the top 2 layers of native BM. The PLLA based ENMs not only accelerated RPE cell proliferation, but also promoted RPE functionality. Biomimetic, laminin coated PLLA based ENMs are therefore a potential candidate to be used as scaffolds for the transplantation of RPEs for treating dry AMD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.