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

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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

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The Effects of Scaffold Rigidity on Retinal Pigment Epithelial Cells

Corina White Symposium on Biomaterials Science 24 October 2016

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Background Physiology

Modified from: http://webvision.med.utah.edu/book/part-i-foundations/simple-anatomy-of-the-retina/

The retina is the light-responsive tissue layer at the back of the eye where the transduction of light signals to vision begins.

Optic Nerve Cillary Body Iris Lens Pupil Cornea Sclera Choroid Retina

Bruch’s Membrane Ganglion Cell Optic Nerve Fiber Bipolar Neuron Choroid Cone Cell Rod Cell Retinal Pigment Epithelium Photo- receptors

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The Bruch’s Membrane (BM) and Retinal Pigment Epithelium (RPE) are crucial in maintaining a viable and functional neural retina.

Background Physiology

Provide Physical Support
Regulate Transport
Phagocytosis of Retinal Waste
Secrete Proteins
Absorb Excess Light

KEY FUNCTIONS OF BM & RPE

http://www.sens.org/sites/srf.org/files/Anuj_Fig_2jpg.jpg

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Several changes to the retina occur naturally with aging and are characteristically present during age-related macular degeneration.

Aged Retina NUMBER OF CASES EXPECTED TO DOUBLE BY 2050

Drusen Interrupted Junctions

HEALTHY AGED

Changes of BM

Thickness of membrane increases
Higher level of collagen cross-

linking

Increased presence of lipids
Appearance of drusen

Aged/Diseased Phenotype

Decreased phagocytic

activity

Altered regulation of

transport

Altered protein expression

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Current Approaches & Challenges Bolus Free Cell Injection Cell-Scaffold Implants

APPROACH CHALLENGES

Monolayer does not form
Long term efficacy still

under investigation

Does not address altered transport

and mechanical properties

In vitro cell studies on aged BM

indicate poor attachment, morphology, and viability [1,2]

Inflammatory response in vivo
“De-differentiation” of transplanted

RPE cells [3,4]

There are several hurdles that must be overcome in order for current approaches to be translational.

[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 [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

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Synthetic polymer scaffolds with varying moduli were fabricated.

Aim 1: Modulus - Approach Fabricate and characterize scaffolds of various elastic moduli Culture RPE cells on scaffolds to investigate effects of modulus

Poly(ethylene glycol)

diacrylate (PEGDA)

Argenine-Glycine-Aspartic

Acid-PEG-Acrylate

Photoinitiator
Buffer

Glass Slides Spacer

UV LIGHT

Pre-polymer solution Glass Mold UV Polymerization

Pre-polymer solution

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Scaffold modulus is tunable through polymer molecular weight and concentration.

Results

200 400 600 800 1000 1200 1400 20 10 5 3.4

Young's Modulus (kPa) PEGDA Molecular Weight (kDa)

1x 2x Modulus of Native BM [5] [5] Candiello, Joseph. FEBS Journal 274.11 (2007): 2897-908.

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RPE cells qualitatively show different adhesion patterns on different moduli scaffolds.

DECREASING STIFFNESS

60 kPa 1000 kPa Tissue Culture Polystyrene (TCPS)

Day 7

CONTROL SCAFFOLDS

DEAD (Ethidium Homodimer-1) DAPI

C B A

B – High Modulus C – Low Modulus Results

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0.2 0.4 0.6 0.8 1 1.2 Day 7 Day 14

METABOLIC ACTIVITY (Normalized to Day 1) Day of Culture

HIGH MODULUS LOW MODULUS TCPS

*

The metabolic activity of RPE cells significantly decreases on a low modulus scaffold during 14 day culture.

Results

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Cells cultured on substrates with different moduli exhibited significant differences in gene expression compared to control.

Results

0.5 1 1.5 2 2.5 3 3.5 4

IL-6 MCP-1 IL-8 SMAD3 CRALBP FOLD CHANGE

HIGH MODULUS LOW MODULUS

* * * *

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Scaffold modulus affects cell adhesion, metabolic activity, and expression and can be tuned to optimize post-transplantation survival and function.

Summary & Conclusions 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 Growing cells on a viscoelastic surface compared to TCPS affects the cells.

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