Scaffold Degradation Product Toxicity

Effects on a Eukaryotic Cell Model

By: Olumuyiwa Idowu & Darcy Diago

Bones that lose minerals after age 30

Fallible spinal disks

Muscles that losemass and tone

Leg veins prone to varicosity

Joints that wear

Shorter limbsand stature

Forward-tilting upper torso

Curved neck with enlarged vertebrae

Thicker disks

Extra muscles and fat

Leg veins with more check valves

Larger hamstringsand tendons

Knee able to bendbackward

Thicker bones

Larger ears

If Humans Were Built to Last

Adapted from Olshansky, Carnes, Butler, Sci Am 2001 Mar

Current Design Alternative Design

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

RightCellsRightCells

RightHormones

RightHormones

RightECM

(Scaffolds)

RightECM

(Scaffolds)

The Basic Three Rs of Tissue EngineeringThe Basic Three Rs

of Tissue Engineering

Signaling Molecules

Matrices Cells

Healing

Normal Wound RepairRequires a Balance of Processes

CellsHormones

Matrix(Scaffolds)

Culture Implant

If needed, harvest cells from patient.

Applying the Three R’sfor Tissue Engineering

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,

A useful tissue engineering matrix ...

must be bio-compatible.

Printed Matrix Fibrin

Matrix with microspheres

Matrix with hormone-laden microspheres

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.

Procedure1. 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 107 cells/mL.

4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 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 103 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.

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

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.