1. Engineering a TMJ Disc Danielle Lewis Louisiana Tech University REU, University of Louisiana at Lafayette Dr. David Mills Louisiana Tech University

2. What is a TMJ Disc?  Temporomandibul ar Joint Disc  Located between the base of the skull and the lower jaw  Allows for smooth opening and closing of the jaw

3. TMDs: Temporomandibular Disorders  Improper disc placement hampers proper jaw movement, called disc displacements  7-28% of adult population affected, mainly females between 18 and 25  No known treatment for disorder  In severe cases discs are completely removed and patients can no longer move jaw

4. http://www.jawjointpainrelief.com/what_is_TMJ.asp

5. Current Obstacles in Engineering a TMJ disc  Recreating the intricate cellular structure  3 regions: anterior band, intermediate zone, and posterior band  Anterior and posterior bands: interlaced collagen fiber bundles  Intermediate zone: aligned collagen fibers along with tiny fibrochondrocytes and small elastin fibers

6. Approach of the Mills Lab: Electrospun Scaffolds  Electrospinning: A high voltage is passed through a polymer solution inducing an electrostatic repulsion force A high voltage is passed through a polymer solution inducing an electrostatic repulsion force The polymer is pumped through an insulin syringe, the repulsion force results in the formation of a thin jet The polymer is pumped through an insulin syringe, the repulsion force results in the formation of a thin jet This jet is directed toward a grounded collection plate, the solvent evaporates before hitting the collection plate and results in the formation of a polymer scaffold This jet is directed toward a grounded collection plate, the solvent evaporates before hitting the collection plate and results in the formation of a polymer scaffold

7. My Research Plan  Culture bovine fibrochondrocytes (FBCs) in 3 different gel environments – agarose, collagen, alginate  Treat cells with growth factors to observe their effect on proliferation, cell survival and protein expression bFGF bFGF TGF alpha and beta TGF alpha and beta CTGF CTGF  Characterize the extracellular matrix being produced by the FBCs

8. Cell Isolations  Bovine FBCs were isolated from TMJ discs taken from cow skulls  Cells used in experiments were passage 6, slightly old but still exhibited the characteristic FBC shape

9. Gels: Collagen and Agarose  FBCs were suspended in both collagen and agarose gels and allowed to grow for several days, 18 day group and 8 day group

10. Fixing, Dehydration, and Paraffin Imbedding  Gels fixed in 2% paraformaldehyde  Gels were dehydrated by exposing them to a variety of ethanol solutions then infiltrated with pariffin to preserve them indefinitely  Gels were then imbedded in a paraffin block in preparation for creating slides  Next… Immunohistochemistry

11. Results  Simple examination under phase contrast showed that FBCs thrived better in collagen gels, cells attached and displayed characteristic shape

12. Is there any explanation for this observation?  Expected result  Collagen type I abundant in TMJ disc

13. Conclusions  Bovine fibrochondrocytes appeared to prefer the collagen gel environment over the agarose gel environment

14. Next Steps….  Begin staining gels for extra-cellular matrix proteins Hypothesis: FBCs grown in collagen gels will exhibit an increase in extra-cellular matrix proteins over those grown in agarose gels Hypothesis: FBCs grown in collagen gels will exhibit an increase in extra-cellular matrix proteins over those grown in agarose gels  Use this experiment as a control and continue by adding growth factors to FBCs in gel culture  Compare the amounts and types of extra- cellular matrix proteins found in gels, both with growth factors and without, to that found in actual disc and FBCs grown on electrospun scaffolds

15. Thank you…  Dr. Mills, Kanthi, Skylar, Deepak, Stephanie, Paul  Dr. Jones  Louisiana Tech for lab facilities in Carson Taylor and the BME building  National Science Foundation