2014 AAO Annual Session Milo Hellman Research Award, Harry Sicher Research Award, Thomas
M. Graber Awards of Special Merit
The Milo Hellman Research Award, Harry Sicher Research Award and Thomas M. Graber Awards of
Special Merit lectures will be held on Monday, April 28 in the Ernest N. Morial Convention Center Room
353 from 2:15pm-4:00pm. Continuing education credit is available for attending these lectures.
Milo Hellman Research Award
2:15pm-2:35pm
Integrating Biology and Imaging of Temporomandibular Joint Health and Disease
Lucia HS Cevidanes, DDS, MS, PhD
University of Michigan
Objective: To investigate 3D morphological variations and local and systemic biomarker profiles in
subjects with diagnosis of temporomandibular joint osteoarthritis (TMJ OA).
Methods: Twenty-eight patients with long-term history of TMJ OA (39.9 ± 16 years), 12 patients at initial
consult diagnosis of OA (47.4 ± 16.1 years) and 12 healthy controls (41.8 ± 12.2years), recruited from the
university clinic and through advertisement, underwent a clinical diagnosis exam by an orofacial pain
specialist using the RDC guidelines. All participants were female and had Cone beam CT scans taken,
and 12 OA and 12 age and gender matched controls also had TMJ arthrocentesis and venipuncture.
Levels of 50 synovial fluid and plasma biomarkers of arthritic inflammation were measured with custom
Quantibody protein microarrays. Shape Analysis MANCOVA tested statistical correlations between
biomarker levels and variations in condylar surface morphology.
Results: The OA average condylar surface model was significantly smaller in all dimensions except its
anterior surface, with the areas indicative on bone resorption being localized along the articular surface,
particularly in the lateral pole. Synovial fluid levels of ANG, GDF15, TIMP-1, CXCL16, MMP-3 and MMP-7
cytokines gave interactions with the bone apposition of the anterior surface of the OA condyles. Plasma
levels of ENA-78, MMP-3, PAI-1, VE-Cadherin, VEGF, GM-CSF, TGFb1, IFNg, TNFa, IL-1a, and IL-6
cytokines resulted in interactions with bone resorption and flattening reshaping of the lateral pole of the
OA condyles. Expression levels of ANG were correlated with the articular surface morphology in healthy
controls.
Conclusions: Condylar morphology of the TMJ OA and control samples were statistically significant
different at the superior articular surface, particularly at the lateral pole. Synovial fluid levels of ANG,
GDF15, TIMP-1, CXCL16, MMP-3 and MMP-7 interacted with bone apposition and plasma levels of ENA78,MMP-3, PAI-1, VE-Cadherin, VEGF, GM-CSF, TGFb1, IFNg, TNFa, IL-1a, and IL-6 cytokines resulted
in interactions with bone resorption.
Harry Sicher Research Award
2:35pm-2:55pm
PTH/PTHrP Receptor Signaling in Osterix-Expressing Progenitors Is Essential for Root Formation
Wanida Ono, DDS, PhD
Harvard Medical School
Dental root formation is a dynamic process involving definite steps of epithelial-mesenchymal
interactions. Thus far, how dental mesenchymal cells originate, choose various fates and participate in
biomineralization during this process has been incompletely understood. Parathyroid hormone-related
protein (PTHrP) and its PTH/PTHrP receptor (PPR) play crucial roles in organogenesis, and PPR
haploinsufficiency results in primary failure of tooth eruption in humans. In this study, using transgenic
mouse models, we sought to identify how osterix-expressing (Osx+) progenitors participate in root
formation by a lineage-tracing system, and understand how PTHrP/PPR signaling regulates these
progenitors by conditionally deleting the receptor in these cells. The descendants of postnatal Osx + cells
robustly contributed to root formation by differentiating into all associated cell types. Conditional deletion
of PPR in Osx-lineage cells led to significant truncation of the root together with agenesis of the
periodontal ligament and ankylosis to the alveolar bone. PPR-deficient progenitors exhibited defective
cementoblast differentiation that formed thick cellular cementum on the coronal root surface. Therefore,
these data suggest that PPR signaling in Osx+ dental follicle progenitors is essential for orchestrated
cementoblast differentiation and root formation, underscoring the importance of PTHrP/PPR system in
maintaining the periodontal homeostasis in various situations such as orthodontic tooth movement.
Thomas M. Graber Awards of Special Merit
2:55pm-3:15pm
Osteoclast-Derived Exosomes: Novel Regulators of Bone Remodeling and Markers of Resorption
Nancy Huynh, DDS
University of Florida
Introduction: Histologic studies reveal that 90% of orthodontically treated teeth have root resorption.
Radiographic techniques are significantly less sensitive and require additional radiation exposure to the
patient. Therefore, non-invasive methods of screening for root resorption will prove highly useful in the
field of orthodontics. Recently, our group found that approximately 54% of proteins detected in gingival
crevicular fluid (GCF) are exosome proteins. Exosomes are 30-100 nm vesicles that have been shown
to be secreted by many mammalian cells and are involved in intercellular communication. To date, there
have been no published findings of osteoclast-derived exosomes. We hypothesized that osteoclasts
secrete exosomes, and the composition of these exosomes change based on the activation state of the
osteoclasts. We further hypothesized that osteoclast-derived exosomes are involved in bone remodeling.
Methods: Osteoclasts were grown from mouse marrow or RAW 264.7 cells by treatment with
recombinant RANKL and CSF-1. Bone slices were labeled with biotin using NHS-LC-biotin. Exosomes
were isolated using published protocols. We used transmission electron microscopy and antibodies
against the exosome markers CD63 and EpCAM to detect exosomes. Antibodies against subunits of
vacuolar proton-ATPase (V-ATPase) were used to examine differences in exosomes secreted by active
vs. inactive osteoclasts. We tested the biologic activity of osteoclast-derived exosomes by counting
TRAP-positive giant cells with and without the addition of exosomes.
Results: Immunoblots confirmed the presence of CD63 and EpCAM. Transmission electron microscopy
revealed numerous vesicles of 30-100nm. The E- and a3-subunits of V-ATPase appeared in exosome
fractions from active osteoclasts and not in exosomes from inactive osteoclasts. Exosomes from
osteoclasts resorbing biotinylated bone slices contained biotinylated peptides. Exosome-treated cultures
had significantly increased counts of TRAP+ giant cells (73% more) compared with untreated cultures.
Conclusions: Osteoclasts produce exosomes that change in composition when osteoclasts become
activated. These exosomes carry the ability to stimulate osteoclast differentiation. We hope that future
studies will prove the value of osteoclast-derived exosomes as novel diagnostic tools for bone and/or root
resorption, and perhaps as molecular nanodevices for regulating orthodontic tooth movement.
3:20pm-3:40pm
Local Delivery of Recombinant RANKL Protein Enhances Root Resorption and Orthodontic Tooth
Movement in Sprague-Dawley Rats
Sarah M. Smith, DMD, MS
University of Michigan
Receptor activator of nuclear factor kB (RANKL) activates osteoclastogenesis and enhances bone
resorption. Local delivery of RANKL-Fc in a rat model of orthodontic tooth movement resulted in
increased root resorption with and without orthodontic springs, and significantly increased mesial
orthodontic molar movement.
Introduction: Bone resorption is the rate-limiting step in orthodontic tooth movement. Enhanced tooth
movement rates may be achieved using bioactive mediators such as receptor activator of nuclear factor
kB (RANKL), which stimulates osteoclastogenesis and subsequent bone resorption. However, the same
processes that activate bone resorption may also contribute to external root resorption. We examined the
effect of RANKL injections on bone resorption, the rate of tooth movement and root resorption in a rodent
model of orthodontic tooth movement.
Materials and Method: Maxillary molars of adult male Sprague-Dawley rats were moved mesially using a
calibrated nickel-titanium orthodontic spring (OS) attached to the maxillary incisors. Vehicle (drug control),
low dose (0.1 mg/kg) and high dose (0.5 mg/kg) recombinant RANKL-Fc were injected mesial to the first
molars in control (Non-OS) and OS rats immediately prior to and during tooth movement. Injections were
given every three days for 24 days total. Maxillary impressions were taken every three days for
orthodontic tooth movement measurements. Rats were sacrificed prior to and at the end of the
experiment, the maxilla retrieved and analyzed for bone and root changes by histology,
immunohistochemistry and quantitative micro-computed tomography.
Results: High dose RANKL OS rats showed higher rates and magnitudes of molar movement than
Vehicle OS and low dose RANKL OS rats, which had similar amounts of orthodontic tooth movement
through the experimental period. Overall high dose RANKL OS rats had 43% greater molar movement
than Vehicle and low dose RANKL OS animals at day 24 (p<0.01). The molar-to-incisor movement ratio
was also significantly greater in high dose RANKL OS rats compared to Vehicle and low dose RANKL
groups. Histologic evidence of diminished bone quality and increased amount and severity of root
resorption was noted in molars subjected to OS or RANKL or both, which corresponded with increased
staining of osteoclasts. Quantitative measures of bone quality with -CT demonstrated effects of both
OS and high dose RANKL particularly on connectivity density, trabecular numbers and trabecular
thickness.
Conclusions: Local delivery of RANKL enhanced osteoclastogenesis as well as the rate and magnitude
of molar tooth movement, while also increasing local root resorptive processes in a dose-dependent
manner. Our data suggest that biological modulation of tooth movement rates is feasible and more
importantly, provides a novel model for studying orthodontic root resorption to better understand its
mechanisms and identifying diagnostic biomarkers for clinical use.
3:40pm-4:00pm
Mesio-Distal Tip and Facio-Lingual Torque Outcomes in Computer-Assisted Orthodontic Treatment
Tharon L. Smith, DDS, MS
University of Illinois - Chicago
Introduction: To investigate the accuracy of the three dimensional clinical outcome of the root inclination
(facio-lingual torque) and angulation (mesio-distal tip) of SureSmileTM (SS) treated cases.
Methods: Initial SS CBCT (therapeutic or initial CBCT), SS therapeutic model (initial model), SS target
model (plan or simulation) and post-treatment CBCT (outcome or final CBCT) were collected for 40
consecutively finished SS cases. DolphinTM 3D root analysis software was used to measure the tip and
torque values for SS target model and post-treatment CBCT of 30 randomly selected cases.
Discrepancies between these variables were compared against the mean discrepancies of the initial
CBCT and the initial model for ten randomly selected cases, which represented a baseline for expected
mean discrepancy of like samples. One sample t-tests were conducted to assess if there was a
difference between the planned tooth tip and torque and their treatment outcomes. T-tests were further
used to assess these discrepancies by the individual tooth type (i.e. maxillary central incisor, maxillary
lateral incisor, etc.).
Results: The results indicate statistically significant differences between the target and clinically
achieved outcomes for both the tooth tip and torque overall across majority of tooth types.
Conclusion: Although statistically different, the overall mean discrepancy of the SS target models to the
outcome CBCT were within the clinically acceptable range (±2.5˚). Though the mean outcome
discrepancies were found to be statistically significant across several tooth types, their clinical
significance (beyond ±2.5˚) was limited to the maxillary and mandibular second molars for tip, and the
maxillary second molar and mandibular central and lateral incisors for torque. Overall, the tip outcomes
were closer to the plan than the torque outcomes for most of the tooth types.