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Thesis Defense
1. Mechanostimulated Wharton’s Jelly Stem Cells Seeded into Human Umbilical Veins for Tendon Tissue Engineering Applications Warren Yates Oral Defense of Master’s Thesis Department of Bioengineering
3. Tendon injuries affect thousands each year >230,000 Achilles tendon injuries in US in 2002 Non-surgical treatments Rest: removes cause Cryotherapy: reduces inflammation, slows metabolism Eccentric strengthening: promotes normal tissue structure? NSAIDs: reduces inflammation through inhibition of prostaglandin synthesis Local corticosteroids: reduces inflammation, slows protein synthesis Orthotics: removes cause Therapeutic ultrasonography: thermal effects, blood flow Extracorporeal shock therapy: reduces pain, helps blood flow Surgery often required, especially for gap defects HUV+ WJSCs might serve as tendon implant Introduction
4. Why the HUV? Longitudinal stiffness comparable to tendons/ligaments (>10 MPa) Tensile strength 10-fold higher than seeded collagen gels (1.56±1.04 MPa) Often discarded Immunologically favorable Introduction
5. Rat Mesenchymal Stem Cells (MSCs) in HUV Seeding density: 1 million cells/mL Stimulation: 150g/scaffold (~2% strain) Frequency of stretch: 0.01667 Hz (1 per min) Previous Work
10. Human Wharton’s jelly stem cells (WJSCs) in HUV Seeding density: 1 million cells/mL Stimulation: 150 g/scaffold (~2% strain) Frequency of stretch: 0.00833 Hz (0.5 stretch/min) 0.01667 Hz (1 stretch/min) 0.03333 Hz (2 stretch/min) Duration of stretch: 0.5 hr/day 1 hr/day 2 hr/day Current Research - Methods
11. Preparation of HUV scaffolds 8.5 cm sections isolated from placenta Mounted, frozen on steel mandrels Lathed to wall thickness of 750µm Inverted, trimmed in length Decellularized Multistage washes SDS, H2O, EtOH, Peracetic Acid, PBS Current Research - Methods
12. Preparation of WJCs Explants of Wharton’s jelly of human umbilical cord Cultured on T75 flasks using α-MEM Used at passage 3 Current Research - Methods P0 WJSCs migrating out of explant, 20x
26. For short-term culture, 1 hour of low- or medium-frequency stimulation each day is favorable Tenomodulin expression increases with stimulation in a frequency dose-independent manner Collagen I expression increases most dramatically following less rigorous stimulation Conclusions
27. Lower seeding densities (WJSCs very aggressive) Presence of growth factors (e.g. FGF-4) Scaffold material (e.g. PLA tubes) Longer experiments 7 days 14 days More genes MMP-1 (acts on fibrillar collagen) TGF-β VEGF COX2 Fibronectin Elastin Future Directions
28. Drs. Sikavitsas, Nollert, McFetridge Dr. Rita Abousleiman Alan Miles My lab associates, who often help one way or another: Vi, Sam, Bonnie, Brandon, Erica, Mike, Joe, Jeff, Marc, Selda, Zehra Anyone who has helped me Acknowledgements
29. Pennati G. Biomechanical properties of the human umbilical cord. Biorheology 2001;38:355–66. Cronkite A. Tensile strength of human tendons. AnatRec1936;64:173–86. Noyes FR, Grood ES. The strength of the anterior cruciate ligament in humans and rhesus monkeys. J Bone Joint Surg1976;58:1074–82. Abousleiman R, Reyes Y, McFetridge P, Sikavitsas V. Tendon Tissue Engineering Using Cell-Seeded Umbilical Veins Cultured in a Mechanical Stimulator. Tissue Engineering: Part A 2009;15:787-795 Abousleiman R, Reyes Y, McFetridge P, Sikavitsas V. The Human Umbilical Vein: A Novel Scaffold for Musculoskeletal Soft Tissue Regeneration. Artif Organs 2008; 32:735-742. Dalkowski A, Schuppan D, Orfanos CE & Zouboulis CC. Increased expression of tenascin C by keloids in vivo and in vitro. Br J Dermatol 1999; 141: 50–56. Kaarteenaho-Wiik R, Lakari E, Soini Y, Pollanen R, Kinnula VL & Paakko P Tenascin expression and distribution in pleural inflammatory and fibrotic diseases. J HistochemCytochem 2000; 48: 1257–1268. Mackie EJ, Halfter W & Liverani D Induction of tenascin in healing wounds. J Cell Biol 1988; 107: 2757–2767. Riley G. Tendinopathy—from basic science to treatment. Nature ClinPrac Rheum 2008; 4(2): 82-89 Liu X, Wu H, Byrne M, Krane S, Jaenisch R. Type III collagen is crucial for collagen I fibrillogenesis and for normal cardiovascular development. PNAS USA 1997; 94: 1852-1856 Edom-Vovard F, Schuler B, Bonnin M-A, Teillet M-A, Duprez D. Fgf4 positively regulates scleraxis and tenascin expression in chick limb tendons. Dev Biol 2002; 247: 351-366 Many, Many More References
31. ΔΔCT Method CTis the cycle at which fluorescence crosses the threshold value (exponential growth) ΔCT is the difference between reference (GAPDH) CT and CT of interest ΔΔCT is the difference between ΔCT of the gene of interest (control group) and ΔCT of the same gene (different experimental group) Relative expression: 2- ΔΔCT
32. ΔΔCT Method During exponential growth phase, gene is approximately doubled each cycle If gene has lower expression in control group, ΔΔCT will be positive and vice versa Hence 2-ΔΔCT represents the approximate factor by which expression differs
37. Flow Cytometry: CD 34: hematopoietic SC marker CD 90: found in neuronal cells, some CD 34+ cells, some fetal liver cells and fetal thymocytes, fibroblasts, activated endothelial cells, and some leukemia cells HLA-ABC: member of MHC class I molecules Current Research
38. Flow Cytometry: CD 44: glycoprotein expressed on mesenchymal cells, interacts with collagens and MMPs CD 105: found on endothelial cells HLA-DR: member of the MHC II molecules Current Research
Editor's Notes
Unstretched, seeded HUVs vs. stretched, seeded HUVs in bioreactor
