Ishan Chatterjee Grade 8 – Dorseyville Middle School

Tissue Engineering Hottest job for 21 st Century

Replacing diseased or injured tissues with tissue constructs designed and fabricated for the specific needs of each individual patient. What is Tissue Engineering? Cells Growth factors Scaffold Culture Implant If needed, harvest cells from patient.

Introduction: Circulatory System Circulatory (Cardiovascular) system – Heart (“pump”) – Blood Vessels (“pipeline”) – Blood (carrier of O 2 & nutrients) Main function – delivery of oxygen-rich blood and nutrients to cells – Removal of CO 2 and waste products from cells Cardiovascular disease leading cause of death in US as of 2007 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 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 solution 3.B: 0.1 mL scaffold degradation product solution 4.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 10 7 cells per mL) Deionized sterile water 150 mL sterile sidearm flask 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 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

Procedure Scaffold Degradation Preparation (10% concentration) 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 Culturing 1. Saccharomyces cerevisiae yeast grown overnight in sterile YEPD media (nutrients) 2.Sample of overnight culture added to fresh media in sterile sidearm flask 3.Culture placed in incubator (30° C) until cell density of approximately 10 7 cells/mL reached 4.Culture diluted in sterile dilution fluid to concentration of approximately 10 5 cells/mL.

Procedure (contd.) Concentration Preparation 1.8 test tubes arranged in rack and labeled A1, A2, B1, B2, C1, C2, SC1, SC2 2.Yeast solution vortexed before adding to test tube 3.Materials pipetted into each test tube: Group AGroup BGroup C (control) Group SC (surface contact) Scaffold Solution1.0 mL0.1 mL0.0 mL1.0 mL (scaffold will be added directly on plate) Yeast Suspension0.1 mL Sterile Dilution Fluid8.9 mL9.8 mL9.9 mL Total Volume10.0 mL Concentration0.1%0.01%0.0% % (once scaffold has been added on plate)

Procedure (contd.) Scaffold Stock Group A (1.0 mL) A2 A1 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.) Plating 1.Test tubes vortexed to evenly suspend cells 2.4 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 concentration 4.Spreader bar sterilized using ethanol and flame 5.Solution spread around plate, using turntable, to coat the agar evenly 6.For group SC, 1.0 mL of scaffold solution added directly on plate before spreading Counting Colonies 1.Plates left at 20°C for 96 hours 2.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.) p < 0.01 Concentrations Colonies

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 dissolve 2.Use mammalian cells in place of yeast 3.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: National Heart, Lung, and Blood Institute: Article “What is Coronary Artery Disease?” URL: Pittsburgh Tissue Engineering Initiative: Article “Regenerative Medicine: Scaffold Guided,” URL: Wikipedia: Article “Coronary Heart Disease,” URL: Wikipedia: Article “Heart Disease,” URL:

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