Engineering a TMJ DiscEngineering a TMJ Disc

Danielle LewisDanielle LewisLouisiana Tech University REU, University of Louisiana at LafayetteLouisiana Tech University REU, University of Louisiana at Lafayette

Dr. David MillsDr. David MillsLouisiana Tech UniversityLouisiana Tech University

What is a TMJ Disc?What is a TMJ Disc? TemporomandibulTemporomandibul

ar Joint Discar Joint Disc Located between Located between

the base of the the base of the skull and the lower skull and the lower jawjaw

Allows for smooth Allows for smooth opening and opening and closing of the jawclosing of the jaw

TMDs: TMDs: Temporomandibular DisordersTemporomandibular Disorders

Improper disc placement hampers proper Improper disc placement hampers proper jaw movement, called disc displacementsjaw movement, called disc displacements

7-28% of adult population affected, mainly 7-28% of adult population affected, mainly females between 18 and 25females between 18 and 25

No known treatment for disorderNo known treatment for disorder In severe cases discs are completely In severe cases discs are completely

removed and patients can no longer move removed and patients can no longer move jawjaw

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

Current Obstacles in Engineering a Current Obstacles in Engineering a TMJ discTMJ disc

Recreating the intricate cellular structureRecreating the intricate cellular structure 3 regions: anterior band, intermediate zone, and 3 regions: anterior band, intermediate zone, and

posterior bandposterior band Anterior and posterior bands: interlaced collagen fiber Anterior and posterior bands: interlaced collagen fiber

bundlesbundles Intermediate zone: aligned collagen fibers along with Intermediate zone: aligned collagen fibers along with

tiny fibrochondrocytes and small elastin fiberstiny fibrochondrocytes and small elastin fibers

Approach of the Mills Lab:Approach of the Mills Lab:Electrospun ScaffoldsElectrospun Scaffolds

Electrospinning:Electrospinning: A high voltage is passed A high voltage is passed

through a polymer solution through a polymer solution inducing an electrostatic inducing an electrostatic repulsion forcerepulsion force

The polymer is pumped The polymer is pumped through an insulin syringe, through an insulin syringe, the repulsion force results in the repulsion force results in the formation of a thin jetthe formation of a thin jet

This jet is directed toward a This jet is directed toward a grounded collection plate, grounded collection plate, the solvent evaporates the solvent evaporates before hitting the collection before hitting the collection plate and results in the plate and results in the formation of a polymer formation of a polymer scaffoldscaffold

My Research PlanMy Research Plan Culture bovine fibrochondrocytes (FBCs) in 3 Culture bovine fibrochondrocytes (FBCs) in 3

different gel environments – agarose, collagen, different gel environments – agarose, collagen, alginatealginate

Treat cells with growth factors to observe their Treat cells with growth factors to observe their effect on proliferation, cell survival and protein effect on proliferation, cell survival and protein expressionexpression bFGFbFGF TGF alpha and betaTGF alpha and beta CTGFCTGF

Characterize the extracellular matrix being Characterize the extracellular matrix being produced by the FBCs produced by the FBCs

Cell IsolationsCell Isolations

Bovine FBCs were isolated from TMJ discs Bovine FBCs were isolated from TMJ discs taken from cow skullstaken from cow skulls

Cells used in experiments were passage 6, Cells used in experiments were passage 6, slightly old but still exhibited the slightly old but still exhibited the characteristic FBC shapecharacteristic FBC shape

Gels:Gels:Collagen and AgaroseCollagen and Agarose

FBCs were suspended in both collagen FBCs were suspended in both collagen and agarose gels and allowed to grow for and agarose gels and allowed to grow for several days, 18 day group and 8 day several days, 18 day group and 8 day groupgroup

Fixing, Dehydration, and Paraffin Fixing, Dehydration, and Paraffin ImbeddingImbedding

Gels fixed in 2% paraformaldehydeGels fixed in 2% paraformaldehyde Gels were dehydrated by exposing them Gels were dehydrated by exposing them

to a variety of ethanol solutions then to a variety of ethanol solutions then infiltrated with pariffin to preserve them infiltrated with pariffin to preserve them indefinitelyindefinitely

Gels were then imbedded in a paraffin Gels were then imbedded in a paraffin block in preparation for creating slidesblock in preparation for creating slides

Next… ImmunohistochemistryNext… Immunohistochemistry

ResultsResults

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

Is there any explanation for this Is there any explanation for this observation?observation?

Expected resultExpected result Collagen type I abundant in TMJ discCollagen type I abundant in TMJ disc

ConclusionsConclusions

Bovine fibrochondrocytes appeared to Bovine fibrochondrocytes appeared to prefer the collagen gel environment over prefer the collagen gel environment over the agarose gel environmentthe agarose gel environment

Next Steps….Next Steps…. Begin staining gels for extra-cellular matrix Begin staining gels for extra-cellular matrix

proteins proteins Hypothesis: FBCs grown in collagen gels will exhibit Hypothesis: FBCs grown in collagen gels will exhibit

an increase in extra-cellular matrix proteins over an increase in extra-cellular matrix proteins over those grown in agarose gelsthose grown in agarose gels

Use this experiment as a control and continue by Use this experiment as a control and continue by adding growth factors to FBCs in gel cultureadding growth factors to FBCs in gel culture

Compare the amounts and types of extra-cellular Compare the amounts and types of extra-cellular matrix proteins found in gels, both with growth matrix proteins found in gels, both with growth factors and without, to that found in actual disc factors and without, to that found in actual disc and FBCs grown on electrospun scaffoldsand FBCs grown on electrospun scaffolds

Thank you…Thank you…

Dr. Mills, Kanthi, Skylar, Deepak, Dr. Mills, Kanthi, Skylar, Deepak, Stephanie, PaulStephanie, Paul

Dr. JonesDr. Jones Louisiana Tech for lab facilities in Carson Louisiana Tech for lab facilities in Carson

Taylor and the BME buildingTaylor and the BME building National Science FoundationNational Science Foundation