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Scaffold Degradation Product Toxicity Effects on a Eukaryotic Cell Model

Explore toxicity effects of scaffold degradation products on a eukaryotic cell model using Saccharomyces cerevisiae. Understand LD50 levels and implications for human tissue engineering.

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Scaffold Degradation Product Toxicity Effects on a Eukaryotic Cell Model

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

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

  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

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

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

  6. 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,

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

  8. 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.

  9. Procedure • Saccharomyces cerevisiae was grown overnight in sterile YEPD and media. • Samples of the overnight culture was added to fresh media in a sterile sidearm flask. • 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 107 cells/mL. • The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL. • 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%. • 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 103 cells/mL. • The solutions were mixed by vortexing and allowed to sit at room temperature for 15 minutes. • After vortexing to evenly suspend cells, 100 µL aliquots were removed from the tubes and spread on YEPD plates. • The plates were incubated at 30 degrees for 48 hours. • The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

  10. 250 200 150 100 50 0 164 Surviving Colonies 155 56 0 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% Concentration % of Scaffold Degradation Product Scaffold Degradation Product Effects on Yeast Survivorship 82 LD-50

  11. 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.

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