Scaffold: Toxic Effects on Microflora Luke J. Barrante, Grade 10 PJAS 2015-2016 Central Catholic High School

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

Applications Tissue replacement for Battlefield wounds Burn grafting Disease Trauma Congenital problems Battlefield wounds Burn grafting Transplantation Improved organ performance Lee Spievack, 2005 Week 1 Week 2 Week 3 Week 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

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

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

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

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

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

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.

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

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.

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

Results P-value: 0.93202 E. coli survivorship Number of colonies 450 400 350 300 250 200 150 100 50 403 400 409 397 Number of colonies 0 0.01 0.1 1 % Concentration of PVA Scaffold

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.

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

References  "What Is Tissue Engineering." What Is Tissue Engineering. N.p., n.d. Web. 27 Jan. 2016. <http://www.rpi.edu/dept/chem-eng/Biotech- 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. 2016. Web. 27 Jan. 2016. <http://www.cdc.gov/>. "Clinical Infectious Diseases." Resistant Escherichia Coli- We Are What We Eat. N.p., n.d. Web. 27 Jan. 2016. <http://cid.oxfordjournals.org/content/49/2/202.full>. 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. 2016. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2587658/>. "Search Results: 276,039 Results Found." 276,039 Search Results. N.p., n.d. Web. 27 Jan. 2016. <http://www.sciencedirect.com/science?_ob=ArticleListURL&_method=list&_ArticleListID=- 929394716&_sort=r&_st=13&view=c&md5=afbb2c6e7199b7c4737089f9ab8b516b&searchtype=a>. "Coursera - Free Online Courses From Top Universities." Coursera. N.p., n.d. Web. 27 Jan. 2016. <https://www.coursera.org/course/tissue101>. "Stem Cells." Medical News Today. MediLexicon International, n.d. Web. 27 Jan. 2016. <http://www.medicalnewstoday.com/info/stem_cell/>.

Results E. coli Survivorship P-value: 0.93202 500 450 400 350 300 250 200 150 100 50 Number of E. coli colonies Control 0.01% PVA 0.1% PVA 1% PVA Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6

Groups Count Sum Average Variance Control 6 2420 403.3333333 1490.266667 0.01% PVA 2401 400.1666667 643.7666667 0.1% PVA 2453 408.8333333 1462.966667 1% PVA 2382 397 633.2 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 458.3333333 3 152.7777778 0.144463881 0.932021278 3.098391212 Within Groups 21151 20 1057.55 Total 21609.33333 23