1. Scaffold Degradation Part II Effect of Ionic Strength on Scaffold Biodegradability Jay Sehgal North Allegheny Intermediate High School

2. Scaffold Degradation Background Information Scaffold - temporary framework used to support a structure In tissue engineering, used to temporarily support the growth of tissue

3. Replacing diseased or injured tissues with tissue constructs designed and fabricated for the specific needs of each individual patient. What is Tissue Engineering/ Regenerative Medicine?

4. Spinal Cord Upper and Lower Jaw Upper and Lower Jaw Limb An Ultimate Vision for Regenerative Medicine: Complete Tissue Regeneration Tail Heart

5. Signaling Molecules Matrices Cells Healing Normal Wound Repair

6. Right Cells Right Cells Right Hormones Right Hormones Right ECM Right ECM The Basic Three R’s of Tissue Engineering The Basic Three R’s of Tissue Engineering

7. Cells Hormones Matrix Culture Implant If needed, harvest cells from patient. Guided Tissue Repair

8. Scaffold Degradation Introduction Scaffold degradation rate is important to tissue engineering If the rate is not studied and regulated, scaffold may interfere with the successful replacement of biological functions

9. Scaffold Degradation Introduction The rate of degradation of scaffolding material can be affected by factors such as: –Temperature –pH –Size –Weight –Degree of porosity –Physical stresses

10. Scaffold Degradation Synopsis of Part I The purpose of Scaffold Degradation Part I was to determine whether or not the scaffold’s biodegradation would be affected if it’s environment was acidic or basic. Hypothesis: If the pH of the scaffold’s environment is acidic or basic, then the biodegradation process will be affected. Polycaprolactone (PCL) was incubated for 14 days in acidic, neutral, and basic environments. The degradation was monitored. The data concluded that the more acidic the environment was, the faster the PCL biodegraded. The data supported the hypothesis.

11. Scaffold Degradation Scaffolding Materials Polylactic acid (PLA): –Biodegradable polyester –Sutures –Drug delivery device –Stents –Medium degradation rate Polyglycolic acid (PGA): –Biodegradable polyester –Sutures –Drug delivery device –Faster degradation rate

12. Scaffold Degradation PCL Polycaprolactone (PCL) – biodegradable polyester Created by polymerization of caprolactone using a catalyst and heat Sutures Drug delivery device Slower degradation rate

13. Scaffold Degradation Ionic Strength (NaCl) Ionic strength is a function of all ions present in a solution NaCl’s ionic strength is equal to it’s concentration Molar Mass (NaCl)≈58.5g/mol 1M solution can be diluted to obtained levels of high or low ionic strength 1M solution of NaCl=58.5g NaCl + 1.0L H 2 0

14. Scaffold Degradation Purpose Ionic strength of NaCl in the body’s natural cellular matrix doesn’t vary much from zero Knowing the affects of various ionic strength’s on a PCL’s degradation is beneficial in designing scaffolds in vitro Using this knowledge, the rate of degradation can be regulated

15. Scaffold Degradation Hypothesis Null Hypothesis If biodegradable PCL scaffolds will be submerged in water with varying ionic strengths, then the biodegradation will not be affected.

16. Scaffold Degradation Materials Polycaprolactone (PCL): 3.2g NaCl Tetrahydrofuran (THF): 32mL Teflon-coated muffin tins Stirrer plates and stirrer bars 15mL test tubes Large flasks and beakers Distilled water Incubator Fume hood Drying oven Electronic metric scale Volumetric flasks

17. Scaffold Degradation Procedure Create Scaffolds Day 1 –32mL THF added to 3.2g PCL in large flask –Flask stopped and placed under fume hood on stirrer overnight Day 2 –8mL solution into Teflon-coated muffin tin mold –7.2g NaCl stirred in until slurry formed. –Repeated for 3 more molds –Molds placed under fume hood to allow THF to evaporate overnight

18. PCL being dissolved by THF on stirrer

19. Day 3 –Scaffolds removed from mold and flipped –Allowed THF to evaporate overnight Day 4 –Scaffolds placed in large beaker of H 2 0 to leech out NaCl –Leeched for 2 days –Water changed 2x daily Scaffold Degradation Procedure Create Scaffolds

20. Scaffold before degradation

21. Scaffold Degradation Procedure Create Scaffolds Day 6 –Scaffolds removed from water and towel dried –Each scaffold cut into 8 pieces –Pieces were placed in drying oven overnight

22. Scaffolds in drying oven

23. Scaffold Degradation Procedure Degradation 3 sets of tubes: –Set A—100mM (10 tubes) –Set B—10mM (10 tubes) –Set C—DW Control (10 tubes) 1 Molar stock solution –100mL DW –5.85g NaCl 100mM solution –90mL DW –10mL 1M stock 10mM solution –90mL DW –10mL 100mM solution

24. Dried scaffolds weighed and data recorded Test tubes filled with appropriate solution Scaffolds placed in all 30 tubes Tubes capped and incubated at 37° C (body temp) for 3 days Scaffolds removed, washed, dried, and weighed Scaffold Degradation Procedure Degradation

25. Test tubes ready for incubation

26. Scaffold Degradation Data Analysis Average Initial Mass (g) Average Final Mass (g) Average % Change 100mM.0924.0919.62803% 10mM.0904.0898.68679% 0 (control).0917.0914.34112%

27. Scaffold Degradation Data Analysis

28. Scaffold Degradation Conclusion Results from 10mM PCL and 100mM PCL show significantly more biodegradation than distilled water control Data does not support hypothesis PCL biodegrades faster in environments with NaCl ionic strength’s of 10mM and 100mM

29. Scaffold Degradation Possible Sources of Error Degradation time not long enough to determine full effects of NaCl ionic strength on PCL NaCl not fully leeched out, remained part of mass Concentration of molar stock solutions not perfectly accurate

30. Scaffold Degradation Future Research Test degradation rate at different ionic strength levels Test degradation rate and bio- compatibility of scaffolds using live cells Test strength of scaffolds with varying factors Investigate degradation with varying degrees of porosity