A Toxicity Analysis of Cardiovascular Scaffold

DegradationIshan Chatterjee

Grade 8 – Dorseyville Middle School

Tissue Engineering

Hottest job for 21st Century

Replacing diseased or injured tissues with tissue constructs designed and fabricated for the specific needs of each individual patient.

What is Tissue Engineering?

CellsGrowth factors

Scaffold Culture Implant

If needed, harvest cells from patient.

Introduction: Circulatory System• Circulatory (Cardiovascular) system

– Heart (“pump”)– Blood Vessels (“pipeline”)– Blood (carrier of O2 & nutrients)

• Main function– delivery of oxygen-rich blood

and nutrients to cells– Removal of CO2 and waste

products from cells

Cardiovascular disease leading cause of death in US as of 2007

StentBalloon Angioplasty

Introduction: Coronary Artery Disease• Plaque builds up inside arteries causing

occlusion• Oxygen-rich blood cannot reach the heart

resulting in heart attack• #1 killer in US - One death every 72 seconds• Current treatment

– Balloon angioplasty– Stent– Bypass

• Drawbacks– Restenosis (re-narrowing of

the artery)– Clots on stents– Others

Introduction: Scaffolds

• Type varies with purpose, type of cell being produced, and where to be implanted

• Polyurethane scaffold used to replace sections of blood vessels

• Properties– Strength and elasticity– Biocompatibility– Biodegradability– Non-toxic breakdown materials

Objective • To test whether breakdown materials from

scaffold degradation show any toxic effect on yeast cells (used in place of human cells)– Yeast cells have similar properties to human cells

such as its DNA replication, recombination, cell division, and metabolism

– Effect measured by counting number of surviving yeast colonies after exposure to different concentrations of scaffold degradation products

Hypothesis

• Null hypothesis: Scaffold degradation products will not affect yeast survivorship

• Alternate hypothesis: Scaffold degradation products will affect yeast survivorship

• Single factor Analysis of Variance (ANOVA) + Dunnett’s test for comparing multiple treatment groups against control– Null hypothesis rejected if p-value < 0.05

Experimental Design• Four groups, one control plus three treatment:

1. C: Control: no scaffold degradation product solution

2. A: 1.0 mL scaffold degradation product solution3. B: 0.1 mL scaffold degradation product solution4. SC: Surface contact (1.0 mL scaffold solution

spread onto agar prior to cell plating: allows for longer exposure time)

• 8 replicates per group

Materials and Apparatus• 0.5 g Polyurethane scaffold• Saccharomyces cerevisiae yeast

(grown to 107 cells per mL)• Deionized sterile water• 150 mL sterile sidearm flask• 10 -10 mL sterile capped test tubes

with Sterile Dilution Fluid (SDF) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl)

• Test tube rack• 32 YEPD agar plates(1% yeast

extract, 2% peptone, 2% glucose (dextrose), 1.5% agar)

• Spreader bar• Plating turntable• Vortex • Incubator

• 100 - 1000 µL pipette• 0.1 – 1 mL pipette• 1 – 10 mL pipette• 95% Ethanol• Microburner• Felt-tip marker• Klett Spectrophotometer• 15 mL sterile conical tubes• YEPD media (1% yeast extract, 2%

peptone, 2% glucose (dextrose)• Sterile pipette tips• 0.22 micron syringe filters + 10 mL

syringe

Saccharomyces cerevisiae yeast

ProcedureScaffold Degradation Preparation (10% concentration)1. 0.5 g polyurethane scaffold matrix sterilized by immersion

in 95% ethanol 2. Sterile scaffold sectioned using a razor blade and scissors 3. Scaffold sections transferred to 10 mL of sterile deionized

water in a 15 mL sterile polystyrene conical tube and left to dissolve for three months

Yeast Culturing4. Saccharomyces cerevisiae yeast grown overnight in sterile

YEPD media (nutrients)5. Sample of overnight culture added to fresh media in sterile

sidearm flask6. Culture placed in incubator (30° C) until cell density of

approximately 107 cells/mL reached7. Culture diluted in sterile dilution fluid to concentration of

approximately 105 cells/mL.

Procedure (contd.)Concentration Preparation1. 8 test tubes arranged in rack and labeled A1, A2, B1, B2, C1,

C2, SC1, SC22. Yeast solution vortexed before adding to test tube3. Materials pipetted into each test tube:

Group A Group B Group C (control)

Group SC(surface contact)

Scaffold Solution 1.0 mL 0.1 mL 0.0 mL 1.0 mL (scaffold will be added directly on plate)

Yeast Suspension 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Sterile Dilution Fluid 8.9 mL 9.8 mL 9.9 mL 9.9 mL

Total Volume 10.0 mL 10.0 mL 10.0 mL 10.0mL

Concentration 0.1% 0.01% 0.0% 0.0 - 0.1% (once scaffold has been added on plate)

Procedure (contd.)Scaffold Stock

Group A (1.0 mL)

A2A1

Plate 1

Plate 2

Plate 3

Plate 4

Plate 1

Plate 2

Plate 3

Plate 4

Group B (0.1 mL)

B1 B2

Plate 1

Plate 2

Plate 3

Plate 4

Plate 1

Plate 2

Plate 3

Plate 4

Group C (control)

C1 C2

Plate 1

Plate 2

Plate 3

Plate 4

Plate 1

Plate 2

Plate 3

Plate 4

Group SC (1.0 mL)

SC1 SC2

Plate 1

Plate 2

Plate 3

Plate 4

Plate 1

Plate 2

Plate 3

Plate 4

Procedure (contd.)Plating1. Test tubes vortexed to evenly suspend cells2. 4 YEPD agar plates assigned to solution in each test tube (A1, A2,

etc.)3. From each test tube, 0.1 mL pipetted onto each of the four plates

– thus, 8 plates for each concentration4. Spreader bar sterilized using ethanol and flame5. Solution spread around plate, using turntable, to coat the agar

evenly6. For group SC, 1.0 mL of scaffold solution added directly on plate

before spreadingCounting Colonies7. Plates left at 20°C for 96 hours8. Resulting colonies counted and recorded using felt-tip marker to

mark colonies already counted– Each colony assumed to have arisen from one cell

Results(Average of 8 plates)

p < 0.01

p < 0.05

Results (contd.)

Control (0 mL) Surface Contact (1.0 mL)0

20

40

60

80

100

120

140

160

180

81.625

161.25

Number of Colonies After 96 Hours (Average of 8 Plates)

p < 0.01

Concentrations

Co

lon

ies

Conclusion

• Null hypothesis of no effect on yeast survivorship was rejected for all treatment conditions including surface contact (all p-values < 0.05)

• Significant growth effect observed for chosen concentrations of scaffold degradation materials

• Growth effect appears to be reversed when concentration increases above certain value– # colonies in A (0.1%) significantly lower than #

colonies in B (0.01%) [p < 0.05]

Sources of Error

1. Scaffold did not fully dissolve after being crushed, vortexed, and left for 3 months

2. Yeast may not have provided accurate representation of human cells

3. Possible minor measurement errors

Experiment Extensions

1. Let scaffold fully dissolve2. Use mammalian cells in place of yeast3. Wider range of concentrations of scaffold

solution to better understand relationship between concentration and toxicity/growth

References• An Outreach Education Manual in Tissue Engineering, Pittsburgh Tissue

Engineering Initiative • MayoClinic.com: Article “Coronary Angioplasty and Stenting: Opening

Clogged Heart Arteries,” URL: http://www.mayoclinic.com/health/angioplasty/HQ00485

• National Heart, Lung, and Blood Institute: Article “What is Coronary Artery Disease?” URL: http://www.nhlbi.nih.gov/health/dci/Diseases/Cad/CAD_WhatIs.html

• Pittsburgh Tissue Engineering Initiative: Article “Regenerative Medicine: Scaffold Guided,” URL: http://www.ptei.org/interior.php?pageID=84

• Wikipedia: Article “Coronary Heart Disease,” URL: http://en.wikipedia.org/wiki/Coronary_artery_disease

• Wikipedia: Article “Heart Disease,” URL: http://en.wikipedia.org/wiki/Heart_disease

Acknowledgements

• Pittsburgh Tissue Engineering Initiative• Mr. Mark Krotec• Mr. Peter Shiner• Ms. Priya Ramaswami• My mother and father

Thank You!