1. Scaffold: Toxic Effects on Microflora
Luke J. Barrante, Grade 10
PJAS
Central Catholic High School

2. Tissue Engineering (TE)
An emerging field of medical practice and research, launched in Pittsburgh
Regenerates damaged human tissue by combining human stem cells, cell growth factors, and porous scaffolds
No scarring

3. Applications Tissue replacement for Battlefield wounds Burn grafting
Disease
Trauma
Congenital problems
Battlefield wounds
Burn grafting
Transplantation
Improved organ
performance
Lee Spievack, 2005
Week Week Week Week 4

4. The Scaffold
Our body possesses a natural scaffold, or extra cellular matrix (ECM)
Tissue engineering often uses growth factors and cells infused onto synthetic extra cellular matrix
These synthetic scaffolds include polyglycolic acid (PGA), tri-calcium phosphate (TCP), and Poly(vinyl alcohol) (PVA) hydrogels

5. Requirements of the Scaffold
Biocompatibility
Porosity
Mechanics (strength, elasticity, rigidity, etc.)
Biodegradability- degradation products must be nontoxic, leave the body without rejection, and be replaced by natural ECM

6. PVA Hydrogel Scaffold
Biomaterial suitable for tissue mimicking, vascular cell culturing, and vascular implanting
Suitable as soft tissue substitutes
Similar micro-structures as porcine liver tissues
5000x

7. Microbial flora
The aggregate of microorganisms- fungi, bacteria, and archaea- that thrive on human tissues
Present mainly in the digestive tract and aid in human digestion, defend against harmful organisms
Microbial behavior changes, reflecting the state of the body

8. Escherichia coli (E. coli)
Gram-negative, facultatively anaerobic, rod-shaped bacterium
Strains are usually harmless; present in lower intestine of warm- blooded organisms
The most widely studied prokaryotic model organism and an important species in the fields of biotechnology and microbiology

9. Purpose
To determine whether PVA hydrogel scaffold degradation products have a toxic effect on the human microflora, represented by E. coli

10. Hypothesis
Experimental hypothesis: degraded PVA hydrogel scaffold will have a significant toxic effect on E. coli survivorship. Null hypothesis: degraded PVA hydrogel scaffold will not have a significant toxic effect on E. coli survivorship.

11. Materials Deionized water Ethanol PVA Hydrogel Scaffold Vortex
Escherichia coli DH5-alpha (10^5 cells/mL, obtained from Doonan Lab, CMU)
0.22 micron syringe filters and 10mL syringe
Scalpel
Macro and micro pipettes + tips
15 mL Sterile conical tubes with Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl)
Spreader bars
Sterile test tubes
Incubator
LB-agar plates

12. Procedure
1 g. PVA scaffold was divided into pellets with a scalpel and added to 9 mL deionized water to create a 10% stock solution.
PVA scaffold solution was degraded by incubator and vortex over a 1 month period.
Scaffold was sterile filtered.
8.9 mL deionized water was pipetted into 4 sterile test tubes.
1 mL, 0.1 mL, 0.01 mL, and 0 mL PVA hydrogel scaffold stock was pipetted into each of the four test tubes.
0.1 mL E. coli was added to each tube, making a dilution of 10^3 cells/ mL.
Each tube received an appropriate amount of deionized water to be filled to a total volume of 10 mL.
0.1 mL of solution from each of the four groups was spread onto six agar plates with sterile spreader bars.
Plates were incubated for 24 hours, and resulting colonies counted.

13. Procedure Control 0.01% 0.1% 1% PVA Hydrogel Scaffold 0 mL 0.001 mL
E. Coli
Deionized Water
9.9 mL
9.899 mL
9.89 mL
9.8 mL

14. Results P-value: 0.93202 E. coli survivorship Number of colonies 450
400
350
300
250
200
150
100 50
Number of colonies
% Concentration of PVA Scaffold

15. Conclusion
Since the p-value was above the alpha of 0.05, the variation for all groups is insignificant.
The experimental hypothesis is rejected; the null that PVA scaffold degradation products do not significantly affect E. coli survivorship is accepted.
These findings suggest that PVA hydrogel scaffold degradation products may be nontoxic to microflora in human tissue regeneration therapy.

16. Limitations Extensions
Only one microbe (E. coli) was tested
Only one scaffold type could be obtained
Narrow range of concentrations
The experiment only determines biocompatibility after degradation.
Test does not simulate the environment of the human body
Only one time of exposure and exposure type
Different microflora will react uniquely to the scaffold
Test multiple microbes (staph, fungi, protozoa)
Test the biocompatibility of multiple scaffolds
Wider range of concentrations
Test multiple scaffold structures, porosity, and degradation rate
Simulate realistic conditions: 37ᵒ C, variable infusion
Test multiple models of the microflora; follow-up study: test scaffold toxicity on human cell survivorship

17. References
 "What Is Tissue Engineering." What Is Tissue Engineering. N.p., n.d. Web. 27 Jan < Environ/Projects00/tissue/What%20is%20Tissue%20Engineering.htm>.
 "Tissue Engineering Scaffold." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 25 Jan Web. 27 Jan <
"Clinical Infectious Diseases." Resistant Escherichia Coli- We Are What We Eat. N.p., n.d. Web. 27 Jan <
Chan, B. P., and K. W. Leong. "Scaffolding in Tissue Engineering: General Approaches and Tissue- specific Considerations." European Spine Journal. Springer-Verlag, n.d. Web. 27 Jan <
"Search Results: 276,039 Results Found." 276,039 Search Results. N.p., n.d. Web. 27 Jan < &_sort=r&_st=13&view=c&md5=afbb2c6e7199b7c f9ab8b516b&searchtype=a>.
"Coursera - Free Online Courses From Top Universities." Coursera. N.p., n.d. Web. 27 Jan <
"Stem Cells." Medical News Today. MediLexicon International, n.d. Web. 27 Jan <

18. Results E. coli Survivorship P-value: 0.93202
500
450
400
350
300
250
200
150
100 50
Number of E. coli colonies
Control % PVA % PVA % PVA
Trial Trial Trial Trial Trial Trial 6

19. Groups
Count
Sum
Average
Variance
Control 6
2420
0.01% PVA
2401
0.1% PVA
2453
1% PVA
2382
397
633.2
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
Within Groups
21151 20
Total 23