ORIGINAL ARTICLE

Cystatins and cathepsin B during orthodontic

tooth movement
Seung-Hoon Rhee,a Junghee Kang,b and Dong-Seok Nahmc
Suwon and Seoul, Korea

Introduction: The lysosomal cysteine protease cathepsin B is known to play an important role in the
resolution of organic matrix, a final step in bone resorption. Cystatins function as an inhibitor of cathepsin B.
Determining the correlation between cathepsin B and cystatin levels in gingival crevicular fluid at various
times might provide a better understanding of both the dynamics and the metabolic stages of orthodontic
tooth movement. Methods: Human gingival crevicular fluid was collected at the distal sulcus from the
canines of persons not in orthodontic treatment, in retention, and in retraction at various times (initial, 1 day,
1 week, and 1 month postretraction). Cathepsin B and its inhibitor, cystatin, were found with fluorometry.
Results: The level of cathepsin B was varied in the retraction group; this was different from the retention and
the nonorthodontic groups. Significant initial decreases after force application and subsequent increases by
1 month posttreatment were observed in the retraction group. The variations and differences among groups
were negatively correlated with cystatin. Conclusions: The balance between enzyme and inhibitor might
reflect the clinical status of orthodontic tooth movement and provide valuable information for the assessment
of recall intervals and retention procedures. (Am J Orthod Dentofacial Orthop 2009;135:99-105)

G
ingival crevicular fluid (GCF) comprises en-
zymes, metabolic by-products, serum proteins,
and other substances related to alveolar bone
metabolism.1 Changes in the composition of GCF have
previously been studied as a noninvasive means of
checking the biologic activity of bone in humans.
Because GCF levels can represent the biologic condi-
tion of bone, the characterization of GCF content might
provide a better understanding of both the dynamics
and the metabolic status of orthodontic tooth move-
ment.1-7 One such component of GCF, cathepsin B, is
an intracellular lysosomal cysteine proteinase that can
degrade extracellular components including collagen
and protein turnover in the lysosomal system.8 It is also
known to play an important role in the initiation and
perpetuation of inflammatory processes. Moreover, this
enzyme is a biologic marker reflecting the clinical state
of inflammatory disease. For instance, in periodontitis,
cathepsin B activity was found in GCF, and the
expression levels of this enzyme were suggested to be
a
Private practive, Suwon, Korea.
b
Former graduate student, Department of Pharmacy, Ajou University School of
Medicine, Suwan, Korea.
c
Professor, Department of Orthodontics, School of Dentistry, Seoul National
University, Seoul, Korea.
Reprint requests to: Dong-Seok Nahm, Department of Orthodontics, Seoul
National University School of Dentistry, 28 Yonkun-dong, Chongro-gu, Seoul
110-749, Korea (R.O.K.); e-mail, dsnahm@snu.ac.kr.
Submitted, November 2005; revised and accepted, October 2006.
0889-5406/$36.00
Copyright © 2009 by the American Association of Orthodontists.
doi:10.1016/j.ajodo.2006.10.029
a good predictor of the clinical category of the dis-
ease.9-13
The accumulation of cathepsin B in GCF has been
shown to increase with orthodontic tooth movement.6
When orthodontic force was applied, histologic and
histochemical findings showed that cathepsin was in-
creased around osteoclast, and cathepsin would decom-
pose the exposed collagen fiber and collagen degrada-
tion by-product.7,14 Although cathepsin B could be
detected during orthodontic tooth movement, there
were no signs of severe inflammation or bone destruc-
tion such as periodontitis in most cases.
We investigated whether GCF-localized cathepsin
B (and its potential clinical value) might be altered by
endogenous inhibitors. Peptide proteinase inhibitors or
cystatins are cysteine proteinase inhibitors belonging to
the MEROPS inhibitor family I25, clan IH (1,2,3).15
They mainly inhibit enzymes belonging to peptidase
families C1 (papain) and C13 (legumain). Cystatin is an
effective inhibitor of cathepsin B.16,17 The cystatin
family is classified as follows: (1) type 1 cystatin is an
intracellular cystatin in the cytosol of many cell types
but also localized in body fluids at significant concen-
trations; (2) type 2 cystatin is mainly extracellular
secreted polypeptides synthesized with a 19-28 residue
signal peptide, and it is broadly distributed and found in
most body fluids; (3) type 3 cystatin is a multidomain
protein, and the mammalian representatives of this
group are the kininogens; (4) type 4 cystatins are
unclassified cystatins, cystatin-like proteins found in
99
100 Rhee, Kang, and Nahm
many organisms, including plant phytocystatins, fetuin
in mammals, insect cystatins, and a puff adder venom
cystatin. This final cystatin is known to inhibit metal-
loproteases of the MEROPS peptidase family M12
(astacin/dadmalysin).
Cystatin activity and interaction with cathepsin
have been reported in the GCF of patients with peri-
odontal disease, but little information is available
concerning correlations between cathepsin B and cys-
tatin during orthodontic tooth movement at various
times.10,18 The comparisons between cathepsin B and
cystatins before, during, and after tooth movement can
be made clinically. Therefore, this study was conducted
to measure GCF and determine alterations in cathepsin
B and cystatins levels during orthodontic force appli-
cation in nonorthodontic treatment, treatment, and re-
tention groups of patients. Furthermore, we sought to
evaluate the correlation (if any) between the release of
cathepsin and its inhibitor cystatin while orthodontic
force is applied.
MATERIAL AND METHODS
Three groups were designated as nonorthodontic,
treatment (canine retraction), and retention. Seventeen
patients who required canine retraction (14 women, 3
men; mean age, 24 years 1 month) were selected as the
canine retraction group. The retention group included
38 retainer patients (25 women, 13 men; mean age, 20
years 2 months; average time wearing the retainer, 4.5
months) who had received orthodontic treatment with
extraction of the 4 premolars. All patients had clear
medical histories, including lack of antibiotic therapy
within the past 3 months and no use of anti-inflamma-
tory drugs in the month before the study. They were
periodontally healthy, with generalized probing depths
ⱕ3 mm and no radiographic evidence of periodontal
bone loss. They completed an informed consent form
and needed first premolar extractions and canine distal
tooth movement as part of their orthodontic treatment
plan (canine retraction group). Students of Ajou Uni-
versity School of Medicine (Suwon, Korea) who had
never received orthodontic treatment and had normal
occlusion were selected as the nonorthodontic treat-
ment group (25 men, 9 women; mean age, 22 years 10
months).
Maxillary canines moving distally were identified
in each subject of the canine retraction group. After
initial leveling, the canine was retracted with Energy
Chain Elastics (Rocky Mountain Orthodontics, Denver,
Colo). We used 0.020-in stainless steel Tru-arch orth-
odontic wire (G&H Wire, Greenwood, Ind) and a .022 ⫻
.028-in slot bracket (Roth prescription, American Orth-
odontics, Sheboygan, Wis). Approximately 100 to 150 g
American Journal of Orthodontics and Dentofacial Orthopedics
January 2009
of force was applied.19 The patients were restricted from
taking any drugs during the experiment. To control the
development of bacterial plaque, the patients received
oral hygiene instructions.
At the distal sulcus of the retracted canine, GCF
was collected, and the Periotron (model 8000, Ora-
Flow, Smithtown, NY) scores were recorded immedi-
ately before force application (initial) and after the
following time: 1 day, 1 week, and 1 month postretrac-
tion. In the retention and nonorthodontic treatment
groups, GCF at the distal sulcus of the maxillary canine
was collected, and the Periotron scores were recorded
immediately.
The sites under investigation were isolated with
cotton rolls and gently dried with an air syringe. Paper
strips (Periopaper, OraFlow) were placed in the gingi-
val crevice until light resistance was felt and allowed to
remain in place for 30 seconds. To measure GCF
volume, the Periotron was used, after calibration with
human serum. The strips were then placed individually
in 300 ␮L of 20 mmol/L phosphate buffer, pH 6.0,
containing 0.15 mol/L sodium chloride, 1.0 mmol/L
EDTA, 1 mmol/L dithiothreitol (DTT), and 0.1%
Tween 20. Samples were centrifuged 3 times over a
30-minute period, the strips were removed, and the
eluate was centrifuged for 5 minutes at 3000 g. The
supernatants were separated and frozen at ⫺20°C until
analysis.
The activity of cathepsin B in the GCF eluate was
determined by fluorometric assay according to estab-
lished protocols.9,10 Thirty microliters of eluate was
added to 450 ␮L of 0.1 M 2-(N-morpholino)-ethane-
sulphonic acid buffer, pH 5.5, containing 0.1% Triton-
X100, 2.0 mmol/L DTT, and 0.2 mg per milliliter of
soybean trypsin inhibitor (Sigma-Aldrich Korea, Seoul,
Korea). After 10 minutes of preincubation at 40°C, 20
␮L of 1.0 mmol/L Z-Val-Lys-Lys-Arg-AFC (7-amino-
4-trifluoromethyl caumarine) Substrate (Enzyme Sys-
tems Products, Livermore, Calif) was added. The reaction
was allowed to proceed at 40°C for 40 minutes and then
stopped with 1 mL of 1 mmol/L of iodoacetic acid. The
concentration of liberated AFC was read by using a
microplate fluorescence reader (FL 600, Bio-Tek Instru-
ments, Winooski, Vermont) with an excitation wave-
length of 360 nm and an emission wavelength of 508 nm.
Enzyme activity was initially calculated in micro units
(picomoles of substrate hydrolyzed per minute) and com-
pared with the measured activity of purified human liver
cathepsin B (Calbiochemicals, Nottingham, United King-
dom) under the same conditions.
Total cystatin activity was determined as the extent
of papain inhibition by GCF.10,18 The eluates were
diluted individually and alkalinized by treatment with
American Journal of Orthodontics and Dentofacial Orthopedics Rhee, Kang, and Nahm 101
Volume 135, Number 1

Table I. Descriptive statistics of GCF volume (␮L), cathepsin B activity (␮U/␮L), and cystatins (ng/␮L)
Left Right

Group Variable n Mean SD n Mean SD

No treatment GCF volume 34 0.43 0.22 34 0.40 0.22

Cathepsin B 34 55.22 30.32 34 67.47 24.42
Cystatin 34 372.23 136.35 34 316.79 154.28
Initial GCF volume 17 0.46 0.30 17 0.53 0.30
Cathepsin B 17 79.24 48.56 17 90.57 48.93
Cystatin 17 195.84 185.77 17 156.52 165.27
Day GCF volume 17 0.48 0.24 17 0.39 0.22
Cathepsin B 17 44.98 32.95 17 54.46 36.69
Cystatin 17 283.78 205.10 17 255.42 203.97
Week GCF volume 17 0.43 0.27 17 0.35 0.21
Cathepsin B 17 72.25 39.33 17 82.49 41.64
Cystatin 17 270.37 194.60 17 205.59 202.87
Month GCF volume 17 0.42 0.28 17 0.57 0.25
Cathepsin B 17 79.32 37.41 17 100.06 48.77
Cystatin 17 242.76 214.83 17 203.42 198.83
Retention GCF volume 38 0.44 0.28 38 0.41 0.25
Cathepsin B 38 70.81 44.56 38 77.62 46.63
Cystatin 38 266.04 206.12 38 244.37 187.53

120.00 300.00 inhibition curve was prepared by using chicken egg

white cystatin in concentrations of 0 to 500 ng. Enzy-
100.00
matic activity was calculated by comparison with the
250.00 standard curve and expressed as nanogram equivalents
80.00
CaB(L)
CaB(R)
of egg white cystatin.
Cys(L)
Cy s(R)
200.00 STATISTICAL ANALYSIS
60.00

The means and standard deviations of the measure-

40.00 150.00
Initial Day W eek Month
Fig 1. Mean value diagram of cathepsin B activity
(␮U/␮L) and cystatin levels (ng/␮L) of the left and right
sides.
200 mmol/L sodium hydroxide (NaOH) for 5 minutes
at 4°C followed by incubation for 10 minutes at 40°C
(ratio of NaOH: sample ⫽ 2:5). The eluate was then
neutralized by the addition of 200 mmol/L hydrogen
chloride (HCl) (ratio of HCl: NaOH ⫽ 1:1). Fifty
microliters of each pretreated sample was added to 2 ng
of twice-crystallized papain (Sigma-Aldrich Korea) in
0.1 mol phosphate buffer, pH 6.0, containing 0.1%
Triton-X 100 and 10 mmol/L DTT. After 10 minutes of
preincubation at 40°C, the reaction was started with 20
␮L of 1.0 mmol/L Z-Phe-Arg-7-amido-4-methylcou-
marine (Z-Phe-Arg-AMC, Calbiochemicals) to give a
total volume of 0.5 mL. The reaction was stopped after
30 minutes by the addition of 1 mL of iodoacetic acid
(1 mmol/L). Liberated AMC was measured using a
fluorescence reader (360 nm/460 nm). A standard
ments of GCF volume, cathepsin B, and cystatins of the
left and right sides were calculated for the nonorth-
odontic, retention, and retraction groups at the initial, 1
day, 1 week, and 1 month times. The left and right
cystatin and cathepsin B values were plotted separately
to determine whether there were variations. Repeated
measures ANOVA was used to test the significance of
differences between time intervals in the retraction
group. The relationships between enzyme and inhibi-
tors were analyzed by correlation tests with Excel
statistics (Microsoft, Redmond, Wash) and SPSS for
Windows software (SPSS, Chicago, Ill).
RESULTS
GCF volumes were recorded as follows: no treat-
ment (left, 0.43 ␮L; right, 0.40 ␮L), initial (left, 0.46
␮L; right, 0.53 ␮L), 1 day (left, 0.48 ␮L; right, 0.39
␮L), 1 week (left, 0.43 ␮L; right, 0.35 ␮L), 1 month
(left, 0.42 ␮L; right, 0.57 ␮L), and retention group (left,
0.44 ␮L; right, 0.41 ␮L). Cathepsin B activity of the
nonorthodontic treatment group was left, 55.22 ␮U/␮L,
and right, 67.47␮U/␮L. In the retention group, it was
left, 70.81 ␮U/␮L, and right, 77.62 ␮U/␮L. In the
102 Rhee, Kang, and Nahm American Journal of Orthodontics and Dentofacial Orthopedics
January 2009

Table II. Repeated measured ANOVA test for cathepsin B and cystatin variations at different times in the retraction
group
Effect Value F Hypothesis df Error df Significance

Cathepsin
Pillai’s trace 0.548 12.121 3 30 0.000
Wilks lambda 0.452 12.121 3 30 0.000
Hotelling’s trace 1.212 12.121 3 30 0.000
Roy’s largest root 1.212 12.121 3 30 0.000
Cystatin
Pillai’s trace 0.249 3.310 3 30 0.033
Wilks lambda 0.751 3.310 3 30 0.033
Hotelling’s trace 0.331 3.310 3 30 0.033
Roy’s largest root 0.331 3.310 3 30 0.033

Cathepsin B Correlation between Cathepsin B & Cystatin (Left side) 316.79 ng/␮L; and left, 266.04 ng/␮L, and right,
200
244.37 ng/␮L, respectively. Interestingly, cystatins val-
ues in the retraction group showed opposite variations
150 to cathepsin B activity. Smaller initial cystatin values
(left, 195.84 ng/␮L; right, 156.52 ng/␮L) were elevated
Initial to left, 283.73 ng/␮L, and right, 255.42 ng/␮L, by 1 day
100
Day
week
and then reduced slowly over time (left, 270.37 ng/␮L,
Month and right, 205.59 ng/␮L at 1 week; and left, 242.76
50 ng/␮L, and right, 203.42 ng/␮L at 1 month). These
results are shown in Table I and Figure 1.
0
To test whether there were any significant differ-
0 200 400 600 ences between cystatin and cathepsin B at the various
Cystatin
time intervals in the retraction group, repeated mea-
Cathepsin B Correlation between Cathepsin B & Cystatin (Right side) sured ANOVA was applied. The Mauchly test of
200 sphericity showed significances of 0.234 for cathepsin
B and 0.511 for cystatin. We then applied the multi-
150
variate test of the SPSS for Windows software to test
within-subject effects (Table II). The results showed
Initial
Day
significant differences of cathepsin B (significance ⫽
100 Week .000) and cystatin (significance ⫽ .033) between the
Month
time intervals in the retraction group (P ⬍0.05). But
50 there was no difference between the left and right sides
of cathepsin B (significance ⫽ 0.249) and cystatin
(significance ⫽ 0.414) levels. The scattergram shows
0
0 200 400 600 the negative correlation between cystatin and cathepsin
Cystatin
B at the different time intervals on both sides (Fig 2).
The negative correlation was significant at the 0.01
Fig 2. Scattergram (upper, left side; lower, right side)
between cystatins and cathepsin B in the retraction
level (2-tailed) with the Pearson correlation test of the
group to visualize correlations. SPSS software (Table III).

retraction group, the activity at the initial time was left,
79.24 ␮U/␮L, and right, 90.57 ␮U/␮L. After 1 day, it
decreased to left, 44.98 ␮U/␮L, and right, 54.46 ␮U/
␮L, but then recovered to left, 72.25 ␮U/␮L, and right,
82.49 ␮U/␮L, by 1 week and, finally, to left, 79.32
␮U/␮L, and right, 100.06 ␮U/␮L by 1 month. The
cystatins levels in the nonorthodontic treatment and
retention groups were left, 372.23 ng/␮L, and right,
DISCUSSION
Fluctuations of GCF volumes were seen at the
different treatment times and locations, but a relation-
ship was not established. None of these volumes was
classified as the periodontitis state (according to the
guidelines of the Periotron’s manufacturer). For all
subjects, gingival health was good, and plaque accu-
mulation was minimal. Increased GCF volume during
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 135, Number 1
Table III. Pearson correlation coefficient and significance between cathepsin B and cystatins
Group Test
No treatment (n ⫽ 34) Pearson correlation
Significance (2-tailed)
Initial (n ⫽ 17) Pearson correlation
Significance (2-tailed)
Day (n ⫽ 17)
Pearson correlation
Significance (2-tailed)
Week (n ⫽ 17) Pearson correlation
Significance (2-tailed)
Month (n ⫽ 17) Pearson correlation
Significance (2-tailed)
Retention (n ⫽ 38) Pearson correlation
Significance (2-tailed)
*Correlation significant at the 0.01 level (2-tailed).
orthodontic tooth movement was reported by Samuels
et al20 and Pender et al,21 but Sugiyama et al6 reported
a different result, that GCF volume around the orth-
odontically moved tooth was similar to that in healthy
subjects.
We performed our experiments slightly differently
from the study of Sugiyama et al6 with respect to
retracted canines after initial leveling. Canine retraction
usually begins after initial leveling during the normal
treatment process; therefore, we collected GCF after
initial leveling and used round wire for retraction to
help reduce friction, and subsequently decreased the
applied force to a light force. A recent study reported
that cathepsin K was initially detected in osteoclasts of
alveolar bone on the pressure side during experimental
tooth movement, but not on the tension side.7 Based on
this observation, GCF collection from the distal pres-
sure side might effectively show cathepsin B activity.
The amount and activity of cathepsin B in the
treatment group were similar to those of periodontitis
groups.10,22-24 Mineral removal occurred to the same
extent in orthodontic bone turnover as in bone destruc-
tion from periodontitis.25 By comparing our results to
previous reports, it can be stated that organic matrix
removal in orthodontic bone turnover also takes place
similarly to periodontal breakdown in periodontitis.
The retention group showed slightly elevated ca-
thepsin B activities in GCF (Table I); this indicates that
the periodontal structure during retention might still be
unstable. The average retention period in this investi-
gation was 4.5 months. Miyajima et al,25 studying lactic
acid secretion from GCF, suggested that the retention
period should be longer than 12.6 months to decrease
secretion. We surmise that cathepsin B activity might
also decrease as the retention period is extended.
Rhee, Kang, and Nahm 103
Cystatin
Left Right
⫺.888(*) ⫺.886(*)
0 0
⫺.900(*) ⫺.890(*)
0 0
⫺.955(*) ⫺.961(*)
Cathepsin B
0 0
⫺.911(*) ⫺.882(*)
0 0
⫺.957(*) ⫺.972(*)
0 0
⫺.937(*) ⫺.914(*)
0 0
The variation in cathepsin B levels in the retraction
group was an interesting finding (Fig 1). The average
cathepsin B activity was initially high but decreased by
41.5% a day after force application. Levels returned to
91.2% after 1 week, coinciding with the time when the
power of the elastic module was diminished. Thereaf-
ter, the activity recovered slowly to 105.6% at 1 month
postretraction. In the study of Sugiyama et al,6 GCF
levels of cathepsin B for treated teeth were significantly
higher than the contralateral and antagonist control
teeth at 24 hours. This difference could be because of
the different sites of GCF collection. After applying
retracted force, the periodontal membrane of the distal
pressure side is compressed and the blood supply
decreased. This causes ischemia of the periodontal
ligament and subsequently might reduce the GCF
substances. This event will be followed by indirect
bone resorption so that the bone resorption pathways
and the formation and activation of osteoclasts will
continue on the pressure side of the alveolar bone.7
Cathepsin B activity recovered within 1 month, a result
that might be clinically related to the tendency of many
orthodontists to prefer a 1-month recall term.
The detected amount of cystatin was higher than in
previous periodontitis studies10,18; this suggests that
orthodontic bone remodeling is different from the
periodontal disease process. The increase of cystatin
might help to limit excessive bone resorption during
orthodontic bone remodeling and, furthermore, be help-
ful for controlled bone turnover.
The rapid increase of cystatin levels was followed
by a slow decline until 1 month postretraction but never
attained levels as low as before force application (Fig
1). The rapid increase of the inhibitor might explain the
reduction of cathepsin B activity after 1 day. This
104 Rhee, Kang, and Nahm
variation was negatively correlated with cathepsin B
(Fig 2, Table III). The difference of cystatin between
the nonorthodontic treatment and the retention groups
could imply that a longer retention process should be
required, as previously mentioned.
Ideally, a comprehensive study of cathepsin B
enzyme and inhibitors in biologic material should
assess all possible bound and free forms, but in practice
this is not possible because of the small volumes of
GCF available. We therefore compared active cathep-
sin B assayed with a peptide substrate as in a previous
study.10 The procedure for cystatin measurement was
also based on enzyme activity (inhibition of papain)
after sample pretreatment to inactivated cysteine pro-
teinases and dissociated enzyme-inhibitor complexes,
thus ensuring that both free and bound forms were
measured. Although these procedures could not guar-
antee the exact correlation of data from the various
assays, they enabled broad comparisons of the levels of
GCF components and assessed trends in their relation-
ships. Further work might require more definitive
clarification of the types of enzymes and inhibitors and
their relationships.
CONCLUSIONS
The activity of cathepsin B in GCF was determined
by fluorometric assay, and total cystatins were deter-
mined from the extent of papain inhibition in the GCF
samples.
1. Both activities showed significant variations at
different times in the treatment groups.
2. There was a negative correlation between cystatin
and cathepsin B activity at the 0.01 significance
level (2-tailed).
From this investigation, it can be inferred that the
balance between enzyme and inhibitors reflects the clini-
cal status of orthodontic tooth movements, the assessment
of recall intervals, and retention procedures.
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