Characterization of Gingival Crevicular Fluid During Orthodontic Tooth Movement
Conditions: Healthy, Maxillary First Premolar Tooth Extraction, Canine Retraction
Sponsor: University of Illinois at Chicago

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Gingival crevicular fluid characterization during orthodontic treatment 
 
 
 
Phimon Atsawasuwan  
Associate Professor 
Department of Orthodontics, College of Dentistry,  
University of Illinois at Chicago 
V3; 6/22/2017-Recent IRB approval 10/3/2018-10/2/2021 
 

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Gingival crevicular fluid and orthodontic treatment 
During Orthodontic tooth movement, teeth are moved through alveolar bone under 
applied forces. The applied mechanical loading force must be transferred to the alveolar 
bone via periodontal ligament (PDL) [1]. This process of mechanotransduction stimulates 
bone remodeling during which osteoblasts produce bone on the tension side and 
osteoclasts resorb bone on the compression side of the PDL [2-6]. Complex interactions 
between osteoblasts and osteoclasts involve numerous biologic molecules including 
cytokines and growth factors [7]. During the tooth movement, the expression of cytokines 
such as interleukin (IL)-1β, IL-6, IL-8, prostalglandin E2, RANKL and MMP1 in PDL will 
be up-regulated [8-10]. The sequence of events from the mechanotransduction 
commanding the tightly controlled accomplishment of osteogenesis attention sides and 
osteoclastogenesis at compressive sides is not completely understood [11, 12].  
 
The gingival crevicular fluid (GCF) is a transudate of interstitial tissues which is 
produced by an osmotic gradient [13] and it is released into the crevicular crevices at a 
flow rate of about 3 ul/h [14]. Orthodontic treatment is triggered by an inflammatory 
process and it has been hypothesized that the quantification of specific biomarker within 
the GCF can be determined using Periotron [15]. However contrasting results have been 
reported in the literature, which studies showing both increased or unchanged GCF 
volumes incident to orthodontic treatment.  
Given that the orthodontic treatment is triggered by a set of inflammatory cytokines 
which are released into the crevicular fluid during the mechanical loading, and its 
homeostasis is dependent on mechanical stimulation. An understanding of the  biological 
response of crevicular fluid to mechanical loading could further advance the knowledge 
of orthodontic treatment.  
 
In this study, we will investigate the biological response of gingival crevicular fluid 
before and after the initial wire placement of orthodontic treatment to determine the 
differentially expressed genes and proteins related to mechanotransduction.  
 
Materials and Methods 
 
Eligibility criteria for the collection of gingival crevicular fluid 
 
One Hundred Healthy, non-smoking patients aged 11-35 years old who require 
orthodontic treatment with first premolar extraction at the orthodontic clinic at College of 
Dentistry, University of Illinois at Chicago (UIC) will be included. The informed consent 
will be presented to patients and parent(s) [legal guardian(s)] and signed before the 
procedure. The patient will be screened for the inclusion criteria at time of consent. The 
written assent will also be obtained from minor subjects. Before the treatment, periapical 
radiographs will be taken on both maxillary canines to verify the root length and 
morphology so the pre-fabricated power arms on the canine brackets will be closed to 
center of resistance of the canines. Fixed orthodontic appliances (0.018-in; MBT 
prescription) will be placed in the subject with an auxiliary vertical slot in the maxillary 
canine brackets (Dentsply International, York, NY) to accommodate the prefabricated 
power arms. The subjects will be referred for extraction of the maxillary first premolars. A 
temporary anchorage device will be placed between maxillary second premolar and 
maxillary first molar, 5 mm from alveolar crest for anchorage and reference purposes. 
The teeth in maxillary arch will be leveled and aligned before canine retraction. Once 

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0.016x0.016 stainless steel archwires (Dentsply International) are placed, the canine 
retraction will be achieved using calibrated 70-g [127] nickel-titanium closing-coil springs 
(Dentsply International) connected from a temporary anchorage device to the power arm 
on the canine brackets that allowed application of the force closer to the center of 
resistance of the tooth.  
 
Collection of gingival crevicular fluid 
 
Patients will be seated in the dental chair in an upright position. The selected site 
will be air-dried and isolated with cotton rolls. Without touching the marginal gingiva, 
supragingival plaque will be removed to avoid contamination of the paper strips. The 
absorbent paper strips will be placed gently in the sulcus and the absorbed paper strips 
will be pooled and placed in a sterile eppendorf tube containing 200 ul of phosphate buffer 
saline. The collected sample will be coded with unique ID numbers and kept at -80C until 
analyzed. The key for the IDs will be kept in a locked cabinet in Rm#237B , College of 
Dentistry. The patient will be informed not to take NSAIDs but paracetamol after the 
orthodontic treatment. The collection of GCF will be performed before bracket placement, 
before canine retraction, 2 weeks after canine retraction, 5 weeks after canine retraction 
and 7 weeks after canine retraction.  
 
Compensation for the study 
 
The subjects will be given the money compensation for travel expense for $100 
after the completion of the sample collection.  
 
 
Intraoral scanning 
 
The patients’ occlusion and jaws will be scanned using an intraoral digital scanner. 
The intraoral scanning will be performed at the highest resolution at each studied 
timepoint. The scanning data will be exported as stereolithography binary file format (.stl) 
and imported to a sophisticated processing software package (Geomagic® Control 14, 
Geomagic®, Research Triangle Park, NC, USA). The miniscrews and palatal rugae 
structure will be used as reference points 
 
Wire placement and orthodontic treatment 
 
The patients will be bonded with brackets and 0.016x0.016” stainless steel wire 
will be placed and ligated onto the brackets with rubber rings. The patient will be 
scheduled to come back for the orthodontic treatment according to the sample collection 
time and once a month after the study. 
 
ELISA 
 
The GCF samples will first be homogenized for 30 s and centrifuged for 5 min at 
1500 g to elute. The elute will then used as sample for ELISA estimation from GCF 
samples. The GCF sample will be detected IL1, IL6, IL8, OPG and RANKL. The ration of 
OPG and RANKL will be calculated and determine the potential of PDL to activate 
osteoclasts. 
 
microRNA analysis 

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Total RNA will be extracted from the stored GCF samples using miRNeasy 
serum/plasma kit (Qiagen, Valencia, CA). Quantity of the total RNA will be determined by 
NanoDrop spectrophotometer (Thermoscientific). The total RNA will be subjected to 
quantitative realtime RT-PCR using Taqman® microRNA RT-PCR assays specific to hsa-
miR-21, -29a, b and c. hsa-let-7d, g and i will be used as an internal control [112, 129]. 
The samples from each subject will be analyzed using a 7900HT realtime PCR system 
(Applied Biosystems). 
 
Statistical Analysis 
All the analyses will be performed in triplicate for three independent experiments 
to confirm reproducibility of the results.  Quantitative data will be presented as 
means ± SD from three independent experiments. Wilcoxon Sign-ranked analysis will be 
used to determine the mean difference before and after treatment at P = 0.05.  
 
References 
 
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