1. Scaffold Degradation Product Toxicity Effects on a Eukaryotic Cell Model By: Olumuyiwa Idowu & Darcy Diago

2. Bones that lose minerals after age 30 Fallible spinal disks Muscles that lose mass and tone Leg veins prone to varicosity Joints that wear Shorter limbs and stature Forward-tilting upper torso Curved neck with enlarged vertebrae Thicker disks Extra muscles and fat Leg veins with more check valves Larger hamstrings and tendons Knee able to bend backward Thicker bones Larger ears If Humans Were Built to Last Adapted from Olshansky, Carnes, Butler, Sci Am 2001 Mar Current Design Alternative Design

3. Still thousands die while waiting for a transplant, and thousands more aren’t even on the list. 400 bill: ½ of national health care bill goes to patients with organ failure, or tissue loss

5. Right Cells Right Cells Right Hormones Right Hormones Right ECM (Scaffolds) Right ECM (Scaffolds) The Basic Three Rs of Tissue Engineering The Basic Three Rs of Tissue Engineering Signaling Molecules Matrices Cells Healing Normal Wound Repair Requires a Balance of Processes

6. Cells Hormones Matrix (Scaffolds) Culture Implant If needed, harvest cells from patient. Applying the Three R’s for Tissue Engineering

7. Scaffolds Allow cell attachment and migration Deliver and retain cells and biochemical factors Enable diffusion of vital cell nutrients and expressed products Exert certain mechanical and biological influences to modify the behavior of the cell phase Need a certain porosity, biodegradability,

8. A useful tissue engineering matrix... must be bio-compatible. Printed Matrix Fibrin Matrix with microspheres Matrix with hormone-laden microspheres

9. Purpose To simulate a biocompatibility assay that assesses the toxicity of a scaffold’s degradation products Specifically, various concentrations of degradation products were exposed to SC (yeast) Why Use Yeast? The yeast species Saccharomyces cerevisiae has been used in baking and fermenting alcoholic beverages for thousands of years. It is also important as a model organism in modern cell biology research, as it is the most thoroughly researched eukaryotic microorganism. Scientists have used Saccharomyces cerevisiae for information on the biology of the eukaryotic cell.

10. Procedure 1.Saccharomyces cerevisiae was grown overnight in sterile YEPD and media. 2.Samples of the overnight culture was added to fresh media in a sterile sidearm flask. 3.The culture was placed in an incubator (30°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/mL. 4.The culture was diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/mL. 5.Scaffold Degradation product ‘A’ (along with extra SDF) was pipetted into sterile test tubes containing sterile dilution fluid (SDF), creating the final concentrations of 0%, 0.1%, 1%, and 10%. 6.100 µL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 10 3 cells/mL. 7.The solutions were mixed by vortexing and allowed to sit at room temperature for 15 minutes. 8.After vortexing to evenly suspend cells, 100 µL aliquots were removed from the tubes and spread on YEPD plates. 9.The plates were incubated at 30 degrees for 48 hours. 10.The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

11. 0%.5% 1% 1.5% 2% 2.5% 3% 3.5% 4% 4.5% 5% 5.5% 6% 6.5% 7% 7.5% 8% 8.5% 9% 9.5% 10% 10.5% 250 200 150 100 50 0

12. Conclusion After testing 0%, 0.1%, 1% and 10% concentrations of SDP “A” cell survival decreased as concentration increased, at a fairly constant rate. The Lethal Dose 50% (LD50) was found to be scaffold degradation concentration of 0.65%. Thus, it appears that the scaffold degradation product “A” has a direct toxic affect on eukaryotic cells, indicating that this type of scaffold might not be suitable for human tissue engineering. To further understand the effects of scaffold degradation products, the scaffolds should be tested on mammalian and human cell lines.