The Effects of Exogenous TGF on the Development of the Temporomandibular Joint TMJ Lisa White1, Jaso

1
The Effects of Exogenous TGF-ß on the Development
of the Temporomandibular Joint (TMJ)Lisa White1,
Jason Roberson2, Michael Cimino3, and Dr. David
Mills41Graduate Student, School of Biological
Sciences, 2Graduate Student, School of Biological
Sciences, 3Undergraduate Student, School of
Biological Sciences, 4Professor, School of
Biological Sciences
Apoptosis
Introduction The temporomandibular joint
(TMJ) is a bilateral synovial joint formed by the
articulation of articular eminence and the
glenoid fossa of the temporal bone and the
condylar head of the mandible (Mills et al 1988)
(Christiansen Thompson 1990). These articular
surfaces undergo significant remodeling in
response to cranial growth and changing patterns
of dentition (Milam et al 1991). The articular
disc is morphologically uniform in shape at birth
with a high degree of cellularity and lack of
internal organization. Postnatally it is modeled
into a biconcave shape with defined regions
anterior and posterior band, intermediate zone,
and posterior attachment. The anterior band and
the posterior band are thicker regions of the
disc with the posterior band being the thicker of
the two (Scapino 1997). The fiber bundle system
and proteoglycan composition are also acquired
postnatally as the product of a temporally and
spatially coordinated sequence of matrix
synthesis and assembly. Structure and
function of the TMJ is interrelated in that the
structure often dictates the function of the
tissue or organ. Mechanical forces of muscular
origin act upon the TMJ and are significant
factors that interact with the cells comprising
the TMJ tissues to influence their growth and
adaptive responses (Hinton Carlson 1997).
Alterations in such activities as mastication and
incision, as well as other orofacial behaviors
can result in significant changes in the
mechanical loading of the joint (Hinton Carlson
1997). When these changes exceed a tolerable
range dictated by the structure of the joint,
function diminishes resulting in some type of
disorder, temporomandibular joint disorder
(TMD). The frequency of clinical signs of
TMD are estimated as high as 86 in the Western
industrialized countries with women making up 60
to 85 of all patients (Christiansen Thompson
1990) Symptoms of TMD may also include radiating
pain in the face, neck or shoulders painful
clicking, popping or grating sounds a change in
the way the upper and lower teeth fit together
and headaches, earaches, dizziness and hearing
problems (Institute of Dental and Craniofacial
Research 2004). The onset of most signs and
symptoms at ages after the peak craniofacial and
mandibular growth suggests that the maturation
processes may contribute to changes in the
ability of the cell populations to respond to the
remodeling demands of the TMJ force system (Yen
Carvalho 1997). The gross morphology of the
articular disc has been well characterized.
However, factors relating to or affecting growth
and development of the disc are not fully
understood. There are numerous factors involved
in tissue engineering and characterizing each of
them is beyond the scope of any one study. This
study has focused on two factors as they relate
to the growth and development of the disc. One
factor that is ubiquitous in that it is a
fundamental component of normal development and
homeostasis in all multicellular organisms is
apoptosis (Milligan Schwartz 1996) and the
other is transforming growth factor-beta (TGF-ß)
which is a critical regulator of many cellular
processes (Rosier et al 1998). There is
generally no consensus on the development or
pathology of the TMJ which has prompted
researchers to identify the factors that
facilitate the disease process. The frequency of
apoptosis as it relates to the development and
even the pathology of the TMJ, particularly the
articular disc, is not well characterized. Nor is
it fully understood as to what extent TGF-ß
stimulates or inhibits such processes. The
purpose of this study was to characterize the
effects that TGF-ß had on the frequency of
apoptosis during stages of maturation with the
long-term goal being to understand the mechanisms
behind growth and development of soft tissues
associated with complex joints. This study will
also provide a benchmark in which experiments
involving mechanical stresses and growth factors
can be compared in order to determine what
effects these factors play in disc modeling or
displacement.
Cell volume decreases, Cell density increases,
Blebs, spikes and blisters may appear on surface
DNA fragmentation occurs Chromatin condenses Cell
breaks up into apoptotic bodies
Cell count by treatment yielded that there was a
significant difference in the number of cells
based on treatment in the 4 day (t-test -2.049,
plt 0.0493) and 2 week (t-test 4.267 plt 0.0002)
disc. Analysis of the 1, 2, and 3 month disc
showed that there was not a significant
difference in total cell count within each of
these age groups based on treatment. By age there
was a significant difference among the different
age groups (plt0.0001). The 4 day age group
compared to 2 week, 1, 2, and 3 month (plt0.0001).
The 2 week age group compared to 1, 2, and 3
month age groups (plt0.0001). No significant
difference was observed between the age groups 1
and 2 months
3 mo PB Toluidine Blue
The nuclei in both the experimental and control
groups and through out each region of the four
day disc are small, oval or spindle shaped which
is characteristic of fibroblast. This morphology
was also observed in the two week control group.
However, the nuclei in the experimental group are
more characteristic of chondrocytes having a
rounded nuclei and a vacuolated cytoplasm. These
cells also appear in isogenous groups which is
also indicative of chondrocytes. The one month
experimental group exhibited larger nuclei as
well as an apparent increase in the formation of
isogenous groups. Characteristic chondrocyte-like
cells were observed in the control and
experimental groups of the two and three month
disc with no observable difference in the overall
morphology of the nuclei.
There is a sharp increase in the length of the
disc between the ages of 2 weeks and 1 month
followed by a plateau between ages 1 and 3
months. The width of the disc has a steady
increase in the rate of growth between 2 weeks
and 3 months.
1 mo TGF-ß

• Methods and Materials
• Experimental Animal
• Twenty New Zealand White rabbits (CKC Farms,
  Pitkin, Louisiana) ages 4 days, 2 weeks, 1 month,
  2 months, and 3 months were studied. This animal
  model is best suited for studying the TMJ because
  domestic rabbits have several structural and
  functional characteristics that are closely
  comparable to humans and their disc are similar
  in overall form and in internal structure (Mills
  et al 1994). Four animals per age group were
  examined and each group consisted of littermates.
  Animals were sacrificed in CO2 chamber according
  to Louisiana Tech University's Animal Care
  policy. Disc were extracted using aseptic
  techniques and placed in 35 mm Petri dishes
  containing Dulbecco's modified eagle's media
  (DMEM) and antibiotic/antimycotic.
• TGF-ß
• After initial collection of tissues, the
  entire conditioned medium was replaced with 3 ml
  DMEM containing 10 fetal bovine serum and
  antibiotic/antimycotic (complete media), and
  incubated for 48 hours in 95 air and 5 CO2 at
  37C to replenish nutrients to the cells.
• Day three of the organ culture, media was
  transferred from each dish into 5 ml transport
  tubes and 3 ml of complete media was added to the
  control group in the absence of TGF-ß and media
  containing exogenous TGF-ß was added to the
  experimental group. The concentration being
  5ng/ml of TGF-ß which has provided optimal
  affects for other studies (Morales and Roberts
  1988). The tissues were then incubated for 24
  hours.
• Day four of the organ culture, media was
  transferred from each dish into 5 ml transport
  tubes. The discs were immediately placed in 4
  formalin for approximately 48 hours, dehydrated
  in a series of graded ethanols, embedded in
  paraffin, and sectioned on a transverse plane at
  6 to 7 microns with at a 10 degree angle. All
  media collected after each of the incubations was
  stored at -80o C for future studies.
• For each of the samples, picro-sirius red,
  nuclear fast red, toluidine blue and labeling of
  DNA fragments was performed on serial sections.
• Histochemistry
• Toluidine blue was used for the investigation of
  proteoglycan content and distribution.
• Picro-Sirius red was used for collagen content
  and distribution.
• Nuclear fast red was used for identification of
  nuclei in samples.
• In Situ Detection of Apoptosis
• In situ detection of apoptosis was preformed
  using the Oncogene BrdUTP-FragEL Kit (Sigma, St.
  Louis, MO) according to the manufacturer's
  instructions. This protocol is designed to label
  the 3 OH ends of DNA fragments produced during
  apoptosis.

4 day IZ control
4 day IZ experimental
Active MMP 1
2 week IZ control
2 week IZ experimental
Discussion Based on results so far, TGF-ß has
been shown to stimulate cell proliferation during
the early stages in development and to advance
chondrogenic differentiation of cells
particularly during the two week and one month
stages of development. This differentiation also
seems to correspond to the same period of
development when the disc has a substantial
increase in overall length. Interestingly, this
two-week stage of development coincides with the
transition from suckling to hard diet
mastication. Although the results are still
pending for apoptosis, it is expected that the
number of cells positive for apoptosis will
increase especially in the developmental groups
which have shown to be most affected by
TGF-ß. Preliminary results suggest that the
coordinated sequence of MMP expression is also
altered during early stages of discal
development. The authors wish to acknowledge Ray
Jarvis and Dr. Jim Spaulding for their technical
assistance with this project.
4 day disc
2 week disc
1 month IZ control
1 month IZ experimental
2 month IZ control
2 month IZ experimental
1 month disc
2 month disc
3 month disc
Picro-Sirius Red Picrosirius red staining is used
to demonstrate collagen fiber orientation under
polarizing light microscopy. In the four day
disc there is very little collagen fiber
orientation. By week two the collagen fibers
begin to assemble into a parallel orientation
within the intermediate zone (IZ) and branch out
in the posterior band (PB) and the anterior band
(AB). The fiber bundle system increases in size
as the disc matures. At age 2 months, the regions
of the disc are very distinct with major
branching in the PB. By age three months this
disc is fully matured. The IZ fiber bundle
system is arranged in a parallel fashion which
branches into both band regions. However, the
fiber bundle system in the PB resembles more of a
basket-like weave.
3 month IZ control
3 month IZ experimental