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Received: 14 December 2018    Accepted: 14 December 2018

DOI: 10.1111/ocr.12285

SUPPLEMENT ARTICLE

Clastic cells in orthodontic treatment: Translational challenges

and recent advances

Wellington J. Rody Jr1  | Estela L. Truzman2 | Desmond T. Foster2 | 

Leigh N. Smith2 | Fernanda G. Rocha3 | Heather L. Sorenson4 | Shannon M. Wallet4 | 
L. Shannon Holliday2

1
Department of Orthodontics and Pediatric
Dentistry, Stony Brook University, Stony
Brook, New York
2
Department of Orthodontics, University of
Florida, Gainesville, Florida
3 osteoclast-­like cells on dentin (“odontoclasts”) is a pathological side effect of ortho-
Department of Oral Biology, University of
Florida, Gainesville, Florida
4
School of Dental Medicine, East Carolina
University, Greenville, North Carolina
Correspondence
Wellington J. Rody Jr, School of Dental
Medicine, Stony Brook University, Stony
Brook, NY.
Email: wellington.rody@
stonybrookmedicine.edu
Structured Abstract
Objectives: Orthodontic treatment consists of numerous appliance activations that
rely on stimulation of osteoclasts at alveolar bone sites. However, the action of
dontic treatment. The aim of this article is twofold: (a) To report preliminary results
from ongoing cell culture experiments to identify unique markers of dentin resorp-
tion, and (b) To discuss our work using nanoparticle tracking analysis (NTA) and ex-
osomes for developing biological fluid-­based biopsies to monitor clastic cell activity.
Setting and sample population: Twelve healthy volunteers in permanent dentition.
Material and methods: For the in vitro experiments, murine clastic cell precursors
were cultured on dentin or bone slices for 7 days and phage-­display biopanning was
used to identify molecular surface differences between osteoclasts and odonto-
clasts. In the human study, gingival crevicular fluid (GCF) samples were collected
using different tools and analysed for protein and exosome recovery.
Results: Biopanning generated antibody fragments that were uniquely reactive to
odontoclasts. Numerous nanoparticles in the size range of exosomes were detected
in all of the human GCF samples.
Conclusions: Our results support that there are molecular differences between os-
teoclasts and odontoclasts. Emerging technologies may allow the use of exosomes in
GCF as a clinical tool to detect markers of root resorption.

KEYWORDS
exosomes, gingival crevicular fluid, odontoclasts, osteoclasts, phage display

1 | I NTRO D U C TI O N
Orthodontic treatment consists of numerous appliance activations
that are sometimes planned without full appreciation for biological
issues. The remarkable plasticity of the tooth's supporting alveolar
bone permits orthodontic tooth movement; however, it is important
to remember that the pattern of bone resorption differs significantly
induced tooth movement.1-3 In
between physiologic and force-­
orthodontic tooth movement, the alveolar bone is initially removed
by osteoclasts located in the marrow spaces, a process called ‘under-
mining resorption’.1 This happens because during force application
the periodontal ligament (PDL) undergoes necrosis and needs a few
days to be restored histologically. 2,3
Since bone resorption is accomplished by multinuclear, giant cells,
called osteoclasts, it can be concluded that the appearance of these
cells at specific sites is essential to orthodontic tooth movement.1,2

180  |  wileyonlinelibrary.com/journal/ocr

© 2019 John Wiley & Sons A/S. Orthod Craniofac Res. 2019;22(Suppl. 1):180–185.
Published by John Wiley & Sons Ltd
RODY Jr et al.
Unlike osteoblasts that have mesenchymal origin, osteoclasts are of
hematopoietic origin. When bone is stimulated to resorb, a complex
multistep process is initiated in order to recruit and maintain osteo-
4
clasts in the periodontium during tooth movement. The literature on
osteoclast recruitment in rodents reports remarkably similar periods
of 3-­
4 days between orthodontic force application and peak ele-
2,5
vations in osteoclast numbers at alveolar bone sites. The lifespan
of osteoclasts in rodents is short as they usually die by apoptosis in
about 4 days.3 Interestingly, the decline in osteoclast numbers during
force-­induced tooth movement is followed by an immediate increase
in clastic cell activity on the root surface as shown by many histological
studies.2,3,6 The molecular pathway that orchestrates this shift from
osteoclast activity to odontoclast activity is still obscure and so are
the phenotypic differences between these two types of clastic cells.
While several biological features are shared by clastic cells that resorb
bone (osteoclasts) and clastic cells that resorb dentin (odontoclasts),
differences have been identified as well, that likely result from cell's
7
interactions with distinct mineralized matrices.
Harnessing our understanding of clastic cell mechanisms and
phenotypes not only will improve the clinical management of or-
thodontic tooth movement, but may also allow the development
of novel approaches to minimize side effects such as root resorp-
tion. Studies have emerged in recent years showing that exosomes
secreted by clastic cells may be involved in the regulation of bone
remodelling 8,9 and dentin resorption.10,11 Exosomes are minute ves-
icles (30-­150 nm in diameter) that can be found in saliva and gingival
crevicular fluid (GCF).12 Thus, the assessment of mineralized tissue
resorption using biofluids is an important research topic since it may
allow for the development of non-­invasive methods to monitor bone
and dentin resorption over time. In this article, we will review our ef-
forts to identify differences between osteoclasts and odontoclasts
and report the results of some in vitro investigations designed to
examine the translational feasibility of identifying novel markers
of clastic cell activity for screening and therapeutics. In addition,
we will show evidence that exosomes in GCF may be detected by
nanoparticle tracking analysis (NTA) and describe the technical chal-
lenges associated with this approach.
2 | PH E N OT Y PE S O F C L A S TI C C E LL S
For the past six years, our research group has developed a working
hypothesis that cells resorbing dentin and cells resorbing bone have
different phenotypes. Note that while the consensus on what to call
these clastic cell types is still evolving,7,11 we will refer to them as ‘os-
teoclasts’ and ‘odontoclasts’ in this publication. Indeed, much contro-
versy exists about the role of different mineralized matrix types in the
terminal differentiation of multinuclear clastic cells. Although bone
and dentin share a similar inventory of proteins and minerals, they are
13
distinct mineralized matrices. Morphological differences between
‘clastic’ cells have long been noted including decreased size and nu-
clei number in odontoclasts relative to osteoclasts.7,14 In addition, the
regulation of dentin resorption is influenced by different hormones
|
      181
and drugs when compared to bone resorption.15,16 For instance, in-
domethacin seems to enhance dentin resorption but doesn't interfere
as much with bone resorption during tooth movement.16 Towards this
end, a cell culture workflow was implemented in which primary mu-
rine bone marrow cells were cultured on bone and dentin slices and
allowed to mature and begin resorption as described previously.11 We
then employed two independent yet complementary screening ap-
proaches that enabled not only the interrogation of osteoclasts and
odontoclasts but also the identification of secreted factors in condi-
tioned media that may be unique to one cell type or the other (Fig. 1).
Phage-­display biopanning was used in an attempt to identify mo-
lecular surface differences between osteoclasts and odontoclasts.
Readers are referred to a current review for a comprehensive descrip-
tion of this molecular biology technique.17 Traditionally, researchers
use phage-­display biopanning to screen for engineered antibody frag-
ments (named single-­chain variable fragment-­scFv) that bind to a par-
ticular molecule of interest. We have modified the traditional protocol
to screen for antibodies that bind uniquely to odontoclasts, but not
osteoclasts, in an attempt to identify surface molecules that are very
specific for odontoclast activity. Adhering to all relevant Institutional
Animal Care and Use Committee (IACUC) regulations at the University
of Florida, clastic cell precursors were isolated from the bone marrow
of C57BL/6 mice and cultured on dentin or bone slices for 7 days. In
order to identify scFvs that reacted with molecules associated with
odontoclasts but not osteoclasts, we utilized the Tomlinson J (TJ) li-
brary, which is a phage display library that encodes for over 100 million
different scFv fragments encoded in an ampicillin resistant phagemid
vector.18 Initially, the osteoclast-­reactive scFvs were absorbed out
while odontoclast-­
reactive scFvs were enriched. Following three
rounds of panning, the odontoclast-­reactive antibodies were eluted
(Figure 1A). In preliminary experiments, thirty colonies containing
antibody fragments reactive to odontoclasts were identified and we
confirmed the reactivity of three of these scFvs to protein lysates from
odontoclasts. Interestingly, two of these antibodies were also non-­
reactive to osteoclasts (Figure 1B). These findings are promising and
may contribute to the development of tools for in vivo characteriza-
tion of odontoclasts, which in turn can drive the development of tar-
geted therapies for dental root resorption in the future.
Exosome fractions from conditioned media of osteoclasts and
odontoclasts were analysed for differences in protein expres-
sion by one-­
dimensional (1D) and two-­
dimensional (2D) liquid
chromatography-­mass spectrometry (LC-­MS/MS). The results of
the 1D analysis were previously reported by our group.11 Briefly, a
proprietary polymer (ExoQuick TC, System Biosciences, Palo Alto,
CA) was combined with cell conditioned media to pellet the exo-
somes overnight, which were then analysed by LC-­MS/MS. The
study showed different proteome profiles between exosomes from
osteoclasts and odontoclasts, thus suggesting that matrix type
may play a role in the final differentiation of clastic cells. A total of
40 exosomal proteins were uniquely present in osteoclast media,
while 6 proteins were unique to odontoclast media. More recently,
our group has completed the 2D LC-­MS/MS analysis (manuscript
in preparation). The data from the 2D analysis are consistent with
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182       RODY Jr et al.

F I G U R E   1   A, Cell culture workflow to search for molecules that can differentiate between osteoclasts and odontoclasts. Bone marrow-­
derived cells are grown on bone (‘osteoclasts’) or on dentin (‘odontoclasts’). Screening of cell surface markers was carried out by phage-­
display biopanning. Briefly, ‘osteoclast’ reactive antibodies were absorbed out (1) while ‘odontoclast’ reactive antibodies were enriched
(2). Following three rounds of absorption and panning (3), odontoclast reactive antibodies were eluted (4). Liquid chromatography–mass
spectrometry (LC-­MS/MS) analysis of exosomes was used to identify markers in conditioned media that were differentially expressed during
dentin resorption. B, Preliminary results of the modified phage-­display biopanning protocol. The figure on the left confirms that biopanning
generated approximately 30 colonies containing antibody fragments (scFvs) reactive to odontoclasts. The reactivity of 3 antibody fragments
was tested by modified western dot blot using lysates from odontoclasts and osteoclasts (figure on the right). Note that the antibodies
scFv01 and scFv02 are uniquely reactive to odontoclasts

the initial 1D study but provide a much larger database of pro-
teomic differences between osteoclasts and odontoclasts. From
a biological standpoint, these proteomic studies contribute to a
better understanding of clastic cell phenotypes. From a clinical
perspective, the proteomic findings may be significant in providing
fundamental new knowledge for the development of a biomarker
panel for early diagnosis of root resorption before the damage can
be visualized with diagnostic imaging.
3 |  LI Q U I D B I O P S Y FO R C L A S TI C C E LL
AC TI V IT Y
Dysregulated bone or dentin resorption is associated with a
host of oral diseases including attachment loss and external root
resorption.7 Currently, standard diagnosis of these conditions
relies on x-­r ays or computed tomography scans. However, these
methods usually detect the problem at an advanced stage where
significant tissue damage had occurred. Rather, the expression of
biomarkers in GCF has been recognized as a promising non-­invasive
tool for clastic cell activity surveillance during orthodontic treat-
ment.12,19,20 Gingival crevicular fluid is defined as a transudate of
interstitial fluid and/or inflammatory exudate. Gingival crevicular
fluid initially appears to be a more efficient means of fluid analysis
compared to saliva because its proteomic composition is far less
complex and provides a clearer picture of localized biological pro-
cesses. Nevertheless, significant limitations also exist. 21 A major
problem is that a limited amount of GCF (1-­5 μL) can be obtained
from a single site. Given that liquid biopsies are usually performed
using millilitres of biofluids (serum, urine, saliva), this represents a
RODY Jr et al. |
      183

formidable challenge. Therefore, studies have been inconclusive
due to several challenges in obtaining adequate amounts of non-­
contaminated GCF and analysing the molecules expressed at low
levels within it. 20,21
Multiple techniques have been developed to collect GCF and
22
each has its advantages and disadvantages. Amongst the most
popular collection methods are micropipette, or capillary tubing, and
21
absorbent filter paper strips. The micropipette collection method
is carried out by holding a micropipette at the entrance of the gingi-
val crevice for an extended period of time (5-­30 minutes) and allow-
ing GCF to migrate into the tube. 23 In theory, this technique allows
collection of uncontaminated native sample whose volume can be
accurately determined. The most glaring drawback of the micropi-
pette collection method is the time-­consuming process of which the
collection tube must be held at the entrance of the gingival crev-
ice. An additional drawback is the difficulty of removing the sample
from the micropipette without introducing contaminants. Collection
of GCF via filter paper is conducted by inserting the strip 1-­2 mm
into the gingival sulcus for 30-­60 seconds. 21,22 In comparison to
collection with capillary tubing, collection with paper strips is much
faster but complete elution of GCF components from the strip can
be difficult. In addition, the difficulty of eluting the GCF components
from the strip varies depending on the type of strip used by the in-
vestigator. 24 In a recent cross-­sectional clinical study done at the
University of Florida, 25 our group tested three collection methods
of GCF and observed that the use of filter membrane (Durapore,
Millipore Corp., Bedford, MA) and filter paper (Periopaper, Oraflow
Inc., Plainview, NY) were significantly better at capturing proteins
than the micropipette (Figure 2). The improved performances by
Periopaper and Duropore were probably in part attributable to the
skill and patience (both on the part of the clinician and the subjects)
required to obtain samples by micropipette.
Research in the biomarker field suggests that the sample com-
plexity in liquid biopsy can be reduced if the exosome fraction is
targeted. 26 Exosomes are rich in biomarker molecules, including pro-
teins and RNAs. Although the cargo of exosomes varies according
to the cell of origin, they do express common proteins on their sur-
face (such as CD9, CD63 and CD81) that allow for the development
of techniques to capture them in biofluids. 27 It is well documented
that exosomes are found in saliva28; however, the presence of exo-
somes in GCF has just begun to be explored. Our group previously
observed that 54% of the proteins detected in GCF have been re-
ported to be found in exosomes, and 37 of them were significantly
upregulated at sites of dentin resorption. 20 Although the sample size
of 11 subjects was relatively small and exosomes were not directly
observed, this study pioneered the exploration of exosomes in GCF
research. More recently, a study published by Atsawasuwan et al12
provided direct evidence that exosome tracking may be a suitable
alternative to monitor osteoclast activity in GCF during tooth move-
ment. These advances open a new window of opportunity in the
discovery of novel biomarkers of mineralized tissue resorption.
Nanoparticle tracking analysis (NTA) has recently emerged as a
“gold standard” for the detection and enumeration of small parti-
cles, including exosomes. 29 In this technique, the particle size is de-
termined by following its Brownian motion, which is in turn related
to particle size in a solution with a specific viscosity at a specific

F I G U R E   2   Bar graph showing higher median concentrations of proteins in gingival crevicular fluid (GCF) samples collected with Durapore
and Periopaper. Gingival crevicular fluid was obtained from the labial side of the upper central incisors in twelve healthy volunteers using
three methods: micropipette, durapore and periopaper. Sampling was carried out in one single visit and the research protocol was approved
by the University of Florida Internal Review Board (UF-­IRB # 201600476). Protein concentration in each sample was quantified in duplicate
by ultraviolet spectroscopy using the NanoDrop 2000c Spectrophotometer (Thermo Scientific, Waltham, MA). The results indicated a
statistically significant advantage in the use of Periopaper and Durapore to recover proteins from GCF when compared to Micropipettes
(P < 0.05). There was no significant difference (NS) in protein recovery between Durapore and Periopaper. Wilcoxon signed rank was used
for pairwise comparisons
 |
184       RODY Jr et al.
temperature according to the Stoke-­Einstein equation.30 As the laser
beam passes through the liquid sample, light is scattered from the
particle which can then be visualized under a microscope in real time
based on its Brownian motion. Our experiments were carried out
using a Nanosight NS300 (Malvern Instruments, UK) equipped with
a 488 nm laser beam. Videos of the nanoparticles were recorded
by a built-­in high-­resolution camera, while the NTA 2.3 software
(Nanosight, Amesbury, UK) calculated particle size and concentra-
tion. Interested readers are referred to Gercel-­Taylor et al29 for a
more detailed theoretical description of this method. The GCF sam-
ples were eluted from the collection device by centrifugation using
100 μL of phosphate buffer saline (PBS) as previously described in
the literature. 20
Our initial step was to test different dilutions of the GCF stock
solution to ensure a constant flow of the sample through the laser
beam. Our experiments showed that 30 μL of the GCF stock solu-
tion further diluted in 500 μL of PBS showed the best performance
by providing a refreshed population of nanoparticles for analysis
with fewer air bubbles and less background scattering. The samples
were manually introduced into the equipment using a 1 mL syringe
connected to a push-­fit elbow Luer connector and three video re-
cordings were carried out for each sample. Noise and background
measurements were determined by running control samples of PBS
eluted from the GCF collection devices.
There are several important challenges with NTA in GCF re-
search. First, background particles are difficult to eliminate.
Despite using highly purified dilution media and nano filters, our
experience with nanoparticle tracking of exosomes in GCF has
been that there are always background particles. We are currently
testing GCF and control (dilution media) samples of the various
methods (Durapore, Periopaper and Micropippete) to determine
which has the lowest level of background particles and which is
most effective at isolating exosomes from GCF. Our preliminary
analysis indicates that a large number of nanoparticles in the size
range of exosomes can be observed in the GCF; however, collec-
tion methods also produce background particles (Figure 3). In gen-
eral, manufacturers of everything from plastic ware to filters have
not been challenged to create their products so that nanoparti-
cles are not present or are not shed. Until recently such nanopar-
ticles would have not been detected; however, for exosome
research, this level of background particles becomes a problem
until nanoparticle free reagents and disposables become available.
Another important caveat with this technique is that the theoret-
ical and absolute levels of detection of GCF exosomes are around
50 nm in diameter. In our own studies by electron microscopy,
we have found that roughly half of the exosomes detected have
a diameter less than 50 nm. Thus, the percentage of these very
small exosomes that are actually detected by NTA is still unknown.
Finally, NTA by itself is unable to determine the cellular origin of
the exosomes. Labelling the vesicles with odontoclast-­
specific
markers will be important to allow root resorption surveillance in
the clinical setting.
4 | CO N C LU S I O N S
A growing number of studies indicate that the mineralized matrix
type contributes to the final differentiation of osteoclast-­like cells;
F I G U R E   3   Characterization of
exosomes in gingival crevicular fluid (GCF)
and control samples using Nanoparticle
Tracking Analysis (NTA). The graphs
on the left show size distribution
and concentration of nanoparticles
recovered from a GCF sample collected
with Periopaper and a control sample
containing only dilution media. The
images on the right are representative
screenshots of the NTA videos used in
the evaluation of the respective samples.
Particle size between 30 and 150 nm
(box) is consistent with exosome size. The
concentration of nanoparticles in this size
range tends to be lower in the control
sample than in the GCF sample; however,
some background noise can be detected.
Note that the Y-­axes of the equipment
output graphs are not in the same scale
RODY Jr et al.
however, the ability of clastic cells to acquire specific phenotypic
characteristics is still very controversial. Our previous studies sup-
port that there are molecular differences between osteoclasts
and odontoclasts. Exosomes released by clastic cells appear to be
promising candidates to carry clinically useful biomarkers. Emerging
technologies, such as NTA, may allow assessment of differences in
exosomes present in GCF to be carried out in the clinical setting to
detect early biomarkers for root resorption.
AC K N OW L E D G E M E N T S
This work was supported by the American Association of
Orthodontists Foundation (AAOF) and the University of Florida
College of Dentistry (UFCD).
C O N FL I C T O F I N T E R E S T S
The authors have stated that there is no conflict of interests.
ORCID
Wellington J. Rody Jr  https://orcid.org/0000-0002-5496-7642
L. Shannon Holliday  https://orcid.org/0000-0002-0844-1965
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How to cite this article: Rody WJ Jr, Truzman EL, Foster DT,
et al. Clastic cells in orthodontic treatment: Translational
challenges and recent advances. Orthod Craniofac Res.
2019;22(Suppl. 1):180‐185. https://doi.org/10.1111/ocr.12285
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