Characterization of the Stiffness and Cytotoxicity of Poly(ethylene glycol) Diacrylate Hydrogels for Retinal Tissue Engineering

Tyler DiStefano1, Corina White2, Dr. Ronke Olabisi2 1Department of Mechanical Engineering, The Cooper Union, New York, NY 2Department of Biomedical Engineering, Rutgers University, Piscataway, NJ

INTRODUCTION AND BACKGROUND

PEG-DA Hydrogels Prepared: Ø  1X and 2X concentration 3.4kDa Ø  1X and 2X concentration 5kDa Ø  1X and 2X concentration 10kDa Ø  1X and 2X concentration 20kDa Ø  1X and 2X concentration 4-ARM

KEY FINDINGS

EXPERIMENTAL DESIGN

RESULTS AND DISCUSSION

•  Dry Age-related Macular Degeneration (AMD) is a condition that compromises visual function due to its debilitating effects on mechanical and transport properties within the blood-retinal barrier.[1]

•  Dry AMD occurs from the build up of carrier lipoproteins that transport essential vitamins and glycosaminoglycans (GAGs) to photoreceptors across Bruch’s membrane.[1]

•  No treatment exists for dry AMD, and is the leading cause of blindness in developed countries.[2]

•  Clinical trials entail a localized injection in the eye without a scaffold, which leads to cell migration and low viability[3]

•  Poly(ethylene glycol) diacrylate (PEG-DA) is a seemingly attractive biocompatible polymer, which is widely used in hydrogel research applications.[4]

•  Hydrogel composition corresponds to characteristic material properties: o  Molecular weight and concentration of

PEG-DA affects Young’s Modulus

•  BROADER GOAL OF RESEARCH: Ø  Understand if Young’s modulus of PEG-DA cultured hydrogels enhances co-

cultured stem cells differentiate into Retinal Pigment Epithelium (RPE) cells.

•  SPECIFIC GOAL OF RESEARCH: Ø  Characterize Young’s modulus of different PEG-DA hydrogels and determine RPE

cell viability on cultured substrates.

PHASE II:

PHASE III:

CELL VIABILITY RESULTS

20kDa, 1X Concentration 10kDa, 2X Concentration

3.4kDa, 1X Concentration 5kDa, 2X Concentration

FIGURE 1 – Dry AMD within the blood-retinal barrier.

Source: https://www.scienceofamd.org/wp-content/uploads/2012/01/slide_5_resize.jpg

FIGURE 4 –Young’s modulus for double-network PEG-DA hydrogels

FIGURE 7 – Day 1 cell viability in various environments

FIGURE 5 –Young’s modulus for surface patterned PEG-DA hydrogels

FUTURE WORK

REFERENCES

ACKNOWLEDGEMENTS •  A very special thank you to Dr. Ronke Olabisi, Corina White, and the rest of the lab members for a warm

welcome into the Biomedical Engineering laboratory. •  Thank you to Dr. Evelyn Erenrich and Dr. David Shreiber of Rutgers University for their support and

acceptance into the RiSE/REU program. •  Thank you to the NSF for the organization and funding of the REU program in Cellular Bioengineering: From

Biomaterials to Stem Cells: NSF EEC 1262924.

•  Increase the concentration of RGD within the PEG-DA hydrogels, and examine if the higher concentration of RGD supports cell life on the hydrogels on and after Day 7.

•  Analyze stem cell differentiation into Retinal Pigment Epithelium (RPE) cells on cultured substrates: o  Expose stem cells to a direct co-culture with RPE cells o  Expose stem cells to an indirect co-culture with PEG-DA

microencapsulated RPE cells

PHASE I:

•  Chosen Hydrogel Scaffolds to seed ARPE-19 on: Ø  20kDa PEG-DA, 1X concentration, EY = 60kPa Ø  10kDa PEG-DA, 2X concentration, ~5EY = 280kPa Ø  3.4kDa PEG-DA, 1X concentration, ~10EY = 500kPa Ø  20kDa PEG-DA, 2X concentration, ~20EY = 1100kPa Ø  Tissue Culture Plastic (Control)

•  Use 3mM RGD tripeptide (arginine-glycine-aspartic acid) to attach ARPE-19 cells onto the hydrogel

•  Live/Dead Stain at Days 1, 7, and 14 to determine cell viability

MECHANICAL TESTING RESULTS

FIGURE 3 – Young’s modulus for single-network PEG-DA hydrogels

FIGURE 8 – Correlation between Young’s modulus and ARPE-19 viability

Ø  Correlation between Young’s modulus and cell viability reveals that an increased material stiffness is more likely to support RPE cell life

Ø  Day 7 indicated no cell viability, which was thought to be from too low of an RGD concentration within the hydrogel

•  Single network PEG-DA hydrogels are on average three times more stiff when the chemical concentration doubles.

•  Double network PEG-DA hydrogels have a Young’s modulus that is approximately the sum of the composing moduli.

•  Topographical patterns do not have a statistically significant effect on the mechanical properties of PEG-DA hydrogels.

•  ARPE-19 cells are more viable on hydrogels with a stiffness similar to that of a healthy Bruch’s membrane.

FIGURE 6 – Live/Dead stain of ARPE-19 cells

[1] Curcio, Christine. Johnson, Mark. “Structure, Function, and Pathology of Bruch’s Membrane” Anatomy and Physiology: Basic Science and translation to Therapy (2013) 465-481 [2] National Institute of Health, National Eye Institute. Fact Sheet: Leading Causes of Blindness in the U.S. Web. 20 June 2014. [3] Macular Degeneration Partnership. Dry AMD Clinical Trials. Web. 3 July 2014. [4] Mazzoccoli, Jason. Feke, Donald. Baskaran, Harihara. Pintauro, Peter. “Mechanical and Cell Viability Properties of Crosslinked Low and High Molecular Weight Poly(ethylene glycol) Diacrylate Blends.”Journal of Biomedical Materials Research. (2010) 558-566