The Effects of Scaffold Rigidity on Retinal Pigment Epithelial Cells - PowerPoint PPT Presentation

The Effects of Scaffold Rigidity on Retinal Pigment Epithelial Cells Corina White Symposium on Biomaterials Science 24 October 2016

Background Physiology The retina is the light-responsive tissue layer at the back of the eye where the transduction of light signals to vision begins. Photo- Optic receptors Nerve Retinal Fiber Pigment Bipolar Epithelium Neuron Choroid Sclera Retina Cornea Pupil Lens Optic Iris Nerve Cillary Body Rod Ganglion Choroid Cone Cell Cell Cell Bruch’s Membrane 2 Modified from: http://webvision.med.utah.edu/book/part-i-foundations/simple-anatomy-of-the-retina/

Background Physiology The Bruch’s Membrane (BM) and Retinal Pigment Epithelium (RPE) are crucial in maintaining a viable and functional neural retina. KEY FUNCTIONS OF BM & RPE • • Provide Physical Support Phagocytosis of Retinal Waste • • Regulate Transport Secrete Proteins • Absorb Excess Light 3 http://www.sens.org/sites/srf.org/files/Anuj_Fig_2jpg.jpg

Aged Retina Several changes to the retina occur naturally with aging and are characteristically present during age-related macular degeneration. NUMBER OF CASES EXPECTED TO DOUBLE BY 2050 AGED HEALTHY Interrupted Junctions Drusen Changes of BM Aged/Diseased Phenotype • Thickness of membrane increases • Decreased phagocytic • Higher level of collagen cross- activity linking • Altered regulation of • Increased presence of lipids transport • 4 Appearance of drusen • Altered protein expression

Current Approaches & Challenges There are several hurdles that must be overcome in order for current approaches to be translational. APPROACH CHALLENGES • Monolayer does not form • Long term efficacy still under investigation • Does not address altered transport Bolus Free and mechanical properties Cell Injection • In vitro cell studies on aged BM indicate poor attachment, morphology, and viability [1,2] • Cell-Scaffold Inflammatory response in vivo • “De - differentiation” of transplanted Implants RPE cells [3,4] [1] Lu, B., et al. Stem Cells, 2009. 27 (9): p. 2126-35. [2] Sun, K., et al. Mol Vis, 2007. 13 : p. 2310-9. PMID: 18199972 5 [3] Diniz, B., et al. Invest Ophthalmol Vis Sci, 2013. 54 (7): p. 5087-96. PMC3726243 [4] Christiansen, A.T., et al. Stem Cells Int, 2012. 2012 : p. 454295. PMC3328168

Aim 1: Modulus - Approach Synthetic polymer scaffolds with varying moduli were fabricated. Fabricate and characterize Culture RPE cells on scaffolds of various scaffolds to investigate elastic moduli effects of modulus Pre-polymer solution Glass Mold UV Polymerization UV LIGHT Glass Slides Spacer Pre-polymer solution • Poly(ethylene glycol) diacrylate (PEGDA) • Argenine-Glycine-Aspartic Acid-PEG-Acrylate • Photoinitiator • Buffer 6

Results Scaffold modulus is tunable through polymer molecular weight and concentration. 1400 Young's Modulus (kPa) 1x 2x 1200 Modulus of Native BM [5] 1000 800 600 400 200 0 20 10 5 3.4 PEGDA Molecular Weight (kDa) 7 [5] Candiello, Joseph. FEBS Journal 274.11 (2007): 2897-908.

Results RPE cells qualitatively show different adhesion patterns on different moduli scaffolds. DEAD Day 7 (Ethidium Homodimer-1) A CONTROL Tissue Culture Polystyrene DAPI (TCPS) B – High Modulus C – Low Modulus B C SCAFFOLDS 1000 kPa 60 kPa 8 8 DECREASING STIFFNESS

Results The metabolic activity of RPE cells significantly decreases on a low modulus scaffold during 14 day culture. HIGH MODULUS LOW MODULUS TCPS # 1.2 * METABOLIC ACTIVITY (Normalized to Day 1) 1 0.8 0.6 0.4 0.2 0 Day 7 Day 14 Day of Culture 9

Results Cells cultured on substrates with different moduli exhibited significant differences in gene expression compared to control. 4 HIGH * MODULUS 3.5 LOW FOLD CHANGE MODULUS 3 * 2.5 * 2 1.5 * 1 0.5 0 IL-6 MCP-1 IL-8 SMAD3 CRALBP 10

Summary & Conclusions Scaffold modulus affects cell adhesion, metabolic activity, and expression and can be tuned to optimize post-transplantation survival and function. Growing cells on a viscoelastic surface compared to TCPS affects the cells. Scaffolds with a modulus that mimics native BM stiffness demonstrate: • More homogenous cell attachment • Higher RPE metabolic activity CONCLUSIONS • Scaffold modulus can be tuned to control cell behavior and, with a deeper understanding of the effects, it can be optimized to increase post- transplantation survival and function 11

The Effects of Scaffold Rigidity on Retinal Pigment Epithelial Cells Corina White Symposium on Biomaterials Science 24 October 2016