Effect of nonsurgical periodontal therapy on crevicular fluidlevels of Cathepsin K in periodontitis

Garima Garg *, A.R. Pradeep, Manoj Kumar Thorat

Department of Periodontics, Government Dental College and Research Institute, Fort, Bangalore 560002, Karnataka, India

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 0 4 6 – 1 0 5 1

a r t i c l e i n f o

Article history:

Accepted 26 August 2009

Keywords:

Gingival crevicular fluid

Cathepsin K

Periodontal health

Gingivitis

Chronic periodontitis

Scaling and root planing

a b s t r a c t

Objectives: Cathepsin K (CTSK), predominantly expressed in osteoclasts, is a potent extra-

cellular matrix degrading enzyme that plays a critical role in osteoclast-mediated bone

resorption. Its increased gingival crevicular fluid (GCF) levels in periodontal disease have

been reported in a previous study. The present study has been carried out to assess the role

of CTSK in periodontal disease and to determine the effect of periodontal treatment on CTSK

concentration in GCF.

Design: 60 subjects were divided into three groups (n = 20) based on gingival index (GI),

probing pocket depth (PPD) and clinical attachment loss (CAL): healthy (group I), gingivitis

(group II) and chronic periodontitis (group III). A fourth group (group IV) consisted of 20

subjects from group III, 6–8 weeks after nonsurgical periodontal therapy (scaling and root

planing). GCF samples collected from each patient were quantified for CTSK using ELISA.

Results: The mean CTSK concentration in GCF was found to be the highest in group III, i.e.

55.55 pmol/l. The mean CTSK concentration in GCF in group I and group II was 5.95 pmol/l

and 6.90 pmol/l respectively. The mean CTSK concentration in GCF in group IV decreased to

11.15 pmol/l, slightly more than that in groups I and II.

Conclusions: GCF CTSK levels increased in periodontitis and correlated negatively with

clinical parameters like GI, PPD and CAL. CTSK levels decreased after nonsurgical treatment

of periodontitis. Thus, CTSK can be considered as a ‘marker of osteoclastic activity’ in

periodontal disease and also deserves further consideration as a therapeutic target.

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1. Introduction

Periodontal diseases are initiated by Gram-negative tooth-

associated microbial biofilms that elicit a host response, with

resultant osseous and soft tissue destruction. Mediators

produced as a part of host response that contribute to tissue

destruction include proteinases, cytokines and prostaglandins.1

Peptidases/ proteinases are enzymes that catalyze the

cleavage of peptide bonds, fundamental to almost every

aspect of life like digestion, blood coagulation, fibrinolysis,

processing of preproproteins such as collagen, immune

function, development and apoptosis.2

* Corresponding author. Tel.: +91 9740144987.E-mail address: garg_gg@yahoo.co.in (G. Garg).

0003–9969/$ – see front matter # 2009 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2009.08.007

Lysosomes contain a number of hydrolases including a

considerable number of proteases. Among the latter, the best

known are the cathepsins, which are involved in a number of

important biological processes.3 They can be divided into four

families: cysteine proteases, aspartic proteases, serine pro-

teases and tripeptidyl peptidase I.4 Most of the cathepsins are

cysteine proteases; however, cathepsins D and E, napsin A and

B are aspartic proteases and cathepsin G is a serine protease.3

Cysteine cathepsins are primarily involved in the intracel-

lular breakdown of proteins in lysosomes, where up to 50%

of proteins are degraded.5 Cathepsin K (CTSK), an acidic

cysteine endoproteinase which is predominantly expressed

d.

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 0 4 6 – 1 0 5 1 1047

inosteoclasts is a potent extracellular matrix degrading enzyme

and play a critical role in osteoclast-mediated bone resorption.6

Literature reports involvement of CTSK in various dis-

orders associated with bone resorption such as osteoporosis,

Paget’s disease,7 diffuse sclerosing osteomyelitis of mandible8

and a critical role of CTSK in the pathogenesis of Rheumatoid

arthritis (RA).9,10 Abnormally high CTSK production was

reported in ankylosing spondylitis11 and atherosclerosis.12

Gene expression of CTSK in osteoclasts result in increased

bone resorptive activity and decreased bone formation in

insulin dependent diabetes mellitus in rats.13 CTSK promotes

the growth and metastasis of tumour cells.14 CTSK is involved

in the modulation of amyloid deposits in amyloidosis15 and

may be involved in the pathogenesis of obesity by promoting

adipocyte differentiation.16 Pycnodysostosis, a human disease

caused by congenital deficiency of CTSK, indicates the crucial

role of this protease in the functional maturation of

osteoclasts.17

Increased CTSK mRNA was detected in mononuclear and

multinuclear osteoclasts on the pressure side of the alveolar

bone of rat after orthodontic force application.18 Strbac et al.

reported elevated levels of CTSK in the gingival crevicular fluid

(GCF) in patients with peri-implantitis.19 Mogi and Otogoto

reported elevated concentration of CTSK in the GCF of chronic

periodontitis affected patients as compared to healthy

individuals, suggesting the contribution of CTSK to osteoclas-

tic bone destruction in periodontal disease.20

Thus, in view of the aforementioned findings, this clinico-

biochemical study was designed to estimate the CTSK levels in

GCF from subjects with clinically healthy periodontium,

gingivitis, chronic periodontitis [before and after periodontal

treatment, scaling and root planing (SRP)] and to know the

effect of periodontal treatment, SRP, on CTSK concentration to

confirm the role of CTSK in periodontal disease progression.

2. Materials and methods

The study population consisted of 60 age and gender balanced

subjects (30 females and 30 males; age range: 25–42 years)

attending the outpatient section of the Department of

Periodontics, Government Dental College and Research

Institute, Bangalore, Karnataka, India. Written informed

consent was obtained from those who agreed to participate

voluntarily. Ethical clearance was obtained from the institu-

tion’s Ethical Committee. Subjects with aggressive period-

ontitis, diseases of bone such as arthritis (rheumatoid and

osteoarthritis), osteoporosis, osteolytic bone metastasis, his-

tory of menopause (in women) or any other systemic disease

which can alter the course of periodontal disease, history of

smoking, medication like cyclosporine A, bisphosphonates,

hormone replacement therapy, steroids, calcium or vitamin D,

antibiotics, anti-inflammatory drugs or history of periodontal

therapy in the preceding 6 months, were excluded from the

study.

Each subject underwent a full mouth periodontal probing

and charting, along with periapical radiographs using the

long-cone technique. Radiographic bone loss was recorded

dichotomously (presence or absence) to differentiate chronic

periodontitis patients from other groups. Furthermore, no

delineation was attempted within the chronic periodontitis

group based on the extent of alveolar bone loss.

Based on the gingival index (GI),21 probing pocket depth

(PPD), clinical attachment loss (CAL) and radiographic evi-

dence of bone loss, subjects were categorized into three

groups. Group I (healthy) consisted of 20 subjects with

clinically healthy periodontium, with a GI = 0, a PPD � 3 mm

and CAL = 0, with no evidence of bone loss on radiograph.

Group II (gingivitis) consisted of 20 subjects who showed

clinical signs of gingival inflammation, GI > 1, PPD � 3 mm

and had no attachment loss or radiographic bone loss. Group

III (chronic periodontitis) consisted of 20 subjects who had

signs of clinical inflammation, GI > 1, CAL > 1 in 30% of sites

with radiographic evidence of bone loss and PPD � 4 mm in

30% of sites. Patients with chronic periodontitis (group III)

were treated with a nonsurgical approach (i.e. SRP) and GCF

samples were collected from the same sites 6–8 week after the

treatment to constitute group IV (the after-treatment group).

2.1. Site selection and fluid collection

All the clinical and radiological examinations, group allocation

and sampling site selection were performed by one examiner

and the samples were collected on the subsequent day by a

second examiner. This was undertaken to prevent the

contamination of GCF with blood associated with the probing

of inflamed sites. Only one site per subject was selected as a

sampling site in group II (gingivitis) and group III (chronic

periodontitis), whereas, in the healthy group, multiple sites

with absence of inflammation were sampled to ensure the

collection of an adequate amount of GCF. In gingivitis patients,

the site with the highest clinical signs of inflammation (i.e.

redness, bleeding on probing and oedema), in the absence of

CAL, was selected. In chronic periodontitis patients, the site

showing the highest CAL [measured using a University of North

Carolina (UNC)-15 periodontal probe] and signs of inflamma-

tion, along with radiographic confirmation of bone loss, was

selected for sampling, and the same test site was selected for

sampling after treatment. On the subsequent day, after gently

drying the area, supragingival plaque was removed without

touching the marginal gingiva and the area was isolated using

cotton rolls to avoid saliva contamination. GCF was collected by

placing the microcapillary pipette at theentrance of the gingival

sulcus, gently touching the gingival margin. From each group, a

standardized volume of 1 ml was collected using the calibration

on white colour-coded 1–5 ml calibrated volumetric microca-

pillary pipettes (Sigma–Aldrich, St. Louis, MO, USA). Each

sample collection was allotted a maximum of 10 min and the

sites which did not express any GCF within the allotted time

were excluded. This was carried out to ensure atraumatism.

The micropipettes that weresuspectedtobecontaminated with

blood and saliva were also excluded. The collected GCF samples

were immediately transferred to airtight plastic vials and stored

at �70 8C until assayed.

2.2. CTSK assay

The GCF samples were expelled from the microcapillary

pipettes with a jet of air using a blower provided with the

pipettes and by further flushing them by a fixed amount of the

Table 1 – Descriptive statistics of the study population showing mean, standard deviation and range for the age, GI, CAL,PPD and GCF CTSK concentrations.

Groups Age (years) GI CAL (mm) PPD (mm) GCF CTSK (pmol/l)

Group I (n = 20)

Mean � SD 28.20 0 0 1.7 5.95

Range (minimum, maximum) (25, 39) (1, 2) (0, 11)

Group II (n = 20)

Mean � SD 26.90 1.87 0 2.6 6.90

Range (minimum, maximum) (25, 34) (1.1, 2.3) (2, 3) (4, 11)

Group III (n = 20)

Mean � SD 34.30 2.2 5.8 7.4 55.55

Range (minimum, maximum) (25, 42) (1.4, 2.9) (5, 8) (6, 10) (43.5, 69)

Group IV (n = 20)

Mean � SD 34.30 0.89 2.7 3.4 11.15

Range (minimum, maximum) (25, 42) (0.4, 1.5) (1, 5) (2, 6) (7.5, 14.5)

Table 2 – Results of ANOVA comparing the mean CTSKconcentrations in GCF between four groups.

Study groups Number of samples F p-Value

Group I 20

Group II 20 203.6251 <0.001*

Group III 20

Group IV 20

* Statistically significant.

Table 3 – Pair-wise comparison using Scheff’s test forGCF CTSK.

Study groups Meandifference

Std.error

p-Value

Group I and Group II �0.95 28.0104 0.9833

Group I and Group III �49.60 28.0104 <0.001*

Group I and Group IV �5.20 28.0104 0.2043

Group II and Group III �48.65 28.0104 <0.001*

Group II and Group IV �4.25 28.0104 0.3720

Group III and Group IV 44.40 28.0104 <0.001*

* Statistically significant.

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 0 4 6 – 1 0 5 11048

diluent to ensure that no GCF is lost by sticking to the walls of

the microcapillary pipette. After appropriate dilution of GCF

samples, the concentration of CTSK was determined by

enzyme linked immunoassay (ELISA) kit (Quantikine Human

CTSK immunoassay, Biomedica, Vienna, Austria, Catalogue

no. BI-20432), as instructed by the manufacturer.

Appropriately diluted samples were incubated in the wells

of a divided microplate that have been precoated with the

polyclonal sheep anti CTSK antibody. CTSK was detected in

the samples on incubation with horseradish peroxidase

conjugated polyclonal antibodies against CTSK. After final

incubation with tetramethylbenzidine substrate solution to

measure the amount of CTSK, the reaction was stopped by 2 M

sulphuric acid and absorbance read on ELISA reader using

450 nm as primary wavelength. The concentration of CTSK in

the tested samples was evaluated from the standard curve,

plotted using the absorbance value obtained for the standards

provided with the kit.

2.3. Statistical analysis

All data were analyzed using a software program (SPSS1

Version 10.5, SPSS Inc., Chicago, IL, USA). Test for the validity

of normality assumption using standardized range statistics

was carried out and it was found that the assumption is valid.

Accordingly, parametric tests were carried out for comparing

the means of CTSK concentration in different groups. Paired ‘t’

test was used to compare CTSK concentrations in GCF in

groups III and IV. Pair-wise comparison using Scheff’s test for

GCF CTSK was carried out to explore, which pair or pairs differ

significantly at 5% level of significance. Pearson’s correlation

test was used to observe any correlation between the GCF

CTSK concentration and clinical parameters. Based on the

pilot study including five subjects in each group, the sample

size was estimated at 20 subjects in each group to achieve 80%

power to detect a difference of 0.5 between the null hypothesis

and the alternative mean.

3. Results

The mean CTSK concentration in GCF was found to be the

highest in group III, i.e. 55.55 pmol/l (approximately 1.50 pg/ml).

The meanCTSKconcentration inGCF ingroup I and groupIIwas

5.95 pmol/l (approximately 0.16 pg/ml) and 6.90 pmol/l (approxi-

mately 0.19 pg/ml) respectively. The mean CTSK concentration

in GCF in group IV decreased to 11.15 pmol/l (approximately

0.30 pg/ml), slightly more than that in groups I and II. The mean

concentration and range of CTSK levels in all the groups along

with standard deviation is shown in Table 1.

To test the hypothesis of equality of means among the four

groups ANOVA was carried out, which indicated that the

means differ significantly among the groups ( p < 0.05)

(Table 2). Further multiple comparisons using Scheff’s test

was carried out to find out which pair or pairs differ

significantly. The results showed that the differences were

statistically significant only between groups I and III, groups II

and III, and groups III and IV (p < 0.05) (Table 3).

When group IV (after treatment group) and group III were

compared using paired ‘t’ test, the difference in the concen-

trations of CTSK was statistically significant suggesting that

after SRP, CTSK levels decreased considerably (Table 4).

Table 4 – Paired ‘t’ test to compare CTSK concentrations in GCF in group III and group IV.

Study groups N Mean Std. deviation Mean difference t-Value p-Value

GCF CTSK

Group III 20 55.55 9.4647 44.40 16.286 <0.001*

Group IV 20 11.15 2.3694

* Statistically significant.

Table 5 – Pearson’s correlation coefficient test comparingGCF CTSK with GI, PPD and CAL.

Groups CTSK and GI CTSK and PPD CTSK and CAL

Group I – 0.0582 –

Group II – 0.1919 –

Group III �0.6572* �0.8748* �0.7383*

Group IV �0.0116 �0.7662* �0.5036

* Statistically significant.

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 0 4 6 – 1 0 5 1 1049

Pearson’s correlation coefficient test was carried out to find

correlation between clinical parameters, i.e. GI, PPD, CAL and

CTSK concentration in GCF. It showed a significant negative

correlation between CTSK concentration and clinical para-

meters in groups III and IV (Table 5).

Confidence interval was calculated for differentiating the

limits of GCF CTSK values in different groups to consider CTSK

as a marker of osteoclastic activity in periodontal disease.

Differentiating value with probability 0.95 for chronic general-

ized periodontitis for GCF was found to be more than 37 pmol/l

(approximately 1.0 pg/ml). However, less than or equal to

12 pmol/l (approximately 0.32 pg/ml) was found to be differ-

entiating value with 0.95 probability for healthy or gingivitis

(Table 6).

4. Discussion

Periodontal diseases are initiated by Gram-negative tooth-

associated microbial biofilms that elicit a host response, with

resultant osseous and soft tissue destruction. In response to

endotoxins derived from periodontal pathogens, several

osteoclast-related mediators (matrix metalloproteinases,

cathepsins and other osteoclast-derived enzymes) target the

destruction of alveolar bone and supporting connective

tissues.1

The cysteine protease CTSK, which is capable of hydro-

lysing extracellular bone matrix proteins, is highly expressed

in osteoclasts, and is a well-known marker of osteoclast

activity.6

Mogi and Otogoto have demonstrated increased concen-

tration of CTSK in GCF of chronic periodontitis patients as

compared to that of healthy subjects.20 The present study was

Table 6 – Differentiating values for different groups for GCF CT

Study groups Mean Std. deviation (SD) Mean � 2

Group I 5.95 3.36 �0.77

Group II 6.90 2.35 2.19

Group III 55.55 9.46 36.62

thus designed with the additional groups of gingivitis and after

treatment, to evaluate the role of CTSK in different stages of

periodontal disease and to assess the effect of nonsurgical

periodontal therapy on CTSK concentrations in GCF from

patients with chronic periodontitis, which can further confirm

the role of CTSK in periodontal disease.

In the present study the influence of age and gender of the

subjects on the CTSK concentration was minimized by

including an equal number of males and females in each

group and selecting the subjects within the specified age group

of 25–42 years.

GCF was collected using microcapillary pipettes to avoid

nonspecific attachment of the analyte, which is seen with

filter paper fibers22 ensuing in false reduction in the detectable

CTSK levels that in turn can underestimate the correlation of

CTSK levels to disease severity/ progression. The disadvantage

of this method is the possibility of trauma to the marginal

gingiva, but utmost care was taken to avoid this during GCF

collection. Furthermore, loss of GCF due to sticking of the

sample to the capillary walls was avoided by flushing the

capillary with a fixed amount of diluent. A fixed amount of 1 ml

was collected from each site and the collection time was

limited to a maximum of 10 min to minimize the effect of

variability in the GCF flow rate at sites with periodontal health

and disease on the concentration of CTSK.

Although an alternative assay for CTSK is the measure-

ment of its activity by using a fluoro-substrate, such an assay

has problems in terms of substrate specificity and sensitivity.

ELISA (detection limit: 0 pmol/l + 3SD: 1.1 pmol/l) used in the

present study, similar to the previous study,20 thus allowed

accurate quantitative estimations of CTSK with high sensi-

tivity and specificity.

The results of our study are in accordance with those of

Mogi and Otogoto’s study, which also reported an increase in

the concentration of CTSK from health to disease, however,

the levels of CTSK were below the detection limit in the

healthy group in their study.20 The similar levels in healthy

and gingivitis groups in our study further confirm that

although CTSK is secreted by macrophages23 and fibroblasts,24

it is predominantly expressed in osteoclasts.6 This finding also

reveals that proteinases other than CTSK (such as matrix

metalloproteinase) may be involved in breakdown of collagen

network in gingivitis. The increased CTSK levels in GCF in

SK (pmol/l).

SD Differentiating values with probability 0.95

12.67 Less than or equal to 13 pmol/l

11.61 More than 2 pmol/l or less than or equal to 12 pmol/l

74.48 More than 37 pmol/l or less than or equal to 74 pmol/l

a r c h i v e s o f o r a l b i o l o g y 5 4 ( 2 0 0 9 ) 1 0 4 6 – 1 0 5 11050

patients with periodontitis indicate accumulation of osteo-

clasts at the diseased sites and amplification of resorptive

signals in the inflamed periodontium by pro-inflammatory

cytokines like TNF-a, IL-1 and IL-6, as periodontitis is a chronic

inflammatory disease.25

In the present study, the clinical parameters correlated

negatively with GCF CTSK concentrations in the periodontitis

group. Mogi and Otogoto also found decrease in concentration

of CTSK as the PPD increased.20 This negative correlation could

be attributed to the consumption of CTSK in degradation of

type I collagen at its noncollagenous termini (N- and C-

telopeptide regions) and release of cross-linked N- and C-

telopeptides.26,27

The mean concentration of CTSK in GCF in chronic

periodontitis group showed a significant reduction after

nonsurgical periodontal therapy (SRP) and strict oral hygiene

measures. The mean CTSK concentration in GCF after

treatment in periodontitis subjects was slightly more than

that in healthy and gingivitis groups. This could be due to the

individual variation in the resolution of periodontitis after

treatment.

Thus, this study showed that CTSK concentration in GCF

increases in periodontitis. However, CTSK concentration

decreases with increasing severity (increase in clinical para-

meters) of the periodontal disease. Further, the treatment

aimed at arresting periodontitis progression resulted in

statistically significant reduction in the levels of CTSK in

GCF. Thus, CTSK in GCF can be considered as a ‘marker of

osteoclastic activity’ in periodontal disease. Differentiating

values with probability 0.95 have shown that CTSK concen-

tration in GCF increasing to �37 pmol/l can be considered as

indicative of chronic periodontitis.

An increase in concentration of CTSK has been detected in

serum in various chronic inflammatory diseases such as RA10

and diffuse sclerosing osteomyelitis of mandible.8 Apart from

this, CTSK has been implicated in obesity16 and diseases like

diabetes mellitus,13 osteoporosis7 and atherosclerosis.12 Spil-

lover of GCF with increased CTSK concentration from the

diseased periodontal sites into serum, may increase the

severity of bone resorption associated with systemic diseases

like RA, osteoporosis, diabetes mellitus and may also increase

the severity of conditions like obesity and amyloidosis. Thus,

our study paves the way for future studies to correlate CTSK

levels in serum and GCF.

It has been shown that inhibition of CTSK by SB-357114,

CTSK inhibitor, results in inhibition of bone resorption in a

nonhuman primate model of postmenopausal bone loss.28

Exploring the use of CTSK as a novel therapeutic target in

periodontal disease can thus be an interesting field of research

in future.

The present study confirms the critical role of CTSK in bone

remodeling by degrading type I collagen at its noncollagenous

termini (N- and C-telopeptide regions) and release cross-

linked N- and C-telopeptides, which provide a responsive

measure of osteoclast-mediated bone resorption. Thus, within

the limits of the present study, the role of CTSK as a ‘marker of

osteoclastic activity’ in periodontal disease could be proposed.

At this point, further studies with larger sample size and

longer follow-up will be needed to confirm the findings of our

study.

Funding

The present study was partly funded by Colgate Research

Grant, Colgate-Palmolive India Ltd., Mumbai, India.

Conflict of interest

The authors report no conflict of interest.

Ethical approval

Ethical approval was obtained from the Ethical Committee,

Government Dental College and Research Institute, affiliated

to Rajiv Gandhi University of Health Sciences, Bangalore,

Karnataka, India, No. ACA/SYN/GDC-B/PG/2007-08.

Acknowledgement

The authors acknowledge Colgate-Palmolive India Ltd.,

Mumbai, India, for partly funding the project.

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