ARTÍCULOS ORIGINALES
Effect of bisphosphonates on the levels of RANKL and OPG in gingival crevicular fluid of patients with periodontal disease and post-menopausal osteoporosis
María E. Verde1, Daniela Bermejo2, Adriana Gruppi2, Miriam Grenón1
¹ Periodontology Department, School of Dentistry, National University of Córdoba, Córdoba, Argentina.
² Center of Biochemistry and Immunology Research. School of Chemistry Sciences. National University
of Cordoba, Córdoba, Argentina.
CORRESPONDENCE Dr. Maria Eugenia Verde Fray Mocho 2242 - B° Parque Corema. Cordoba Capital. C.P 5009 Argentina eugenia.verde@unc.edu.ar
ABSTRACT
The Receptor activator of nuclear factor-kappa B ligand (RANKL)/RANK/Osteoprotegerine (OPG) system has been proposed as essential for osteoclast biology and identified as key part in regulating the physiology and pathology of the skeletal system. The study of the RANKL/RANK/OPG system has increased the understanding of the mechanisms involved in the bone remodeling process, especially in postmenopausal osteoporosis and periodontal disease. Bisphosphonates have become the mainstay of the treatment and prevention of post-menopausal osteoporosis. They inhibit the formation and dissolution of calcium phosphate crystals in bone and also osteoclasts, thus reducing bone turnover.Current investigations relate osteoporosis with the appearance and progression of periodontal disease. Although the etiology of both is different, the bone loss present in both shares several characteristics. Thus, therapy used for osteoporosis can be considered of value in the treatment of periodontal disease. The aim of this study was to evaluate the levels of RANKL, OPG and their relationship in gingival crevicular fluid (GCF) in patients with periodontal disease and postmenopausal osteoporosis/ osteopenia in relation to consumption of bisphosphonates. We studied 66 periodontal active sites obtained from 17 postmenopausal women patients aged between 45-70 years old with osteoporosis/osteopenia and periodontal disease. GCF samples were collected using sterile filter paper strips. To determine the concentration of RANKL and OPG, a commercial ELISA assay was used. The values of RANKL, OPG and their ratio (RANKL/ OPG) were compared with Mann-Whitney U Test. The values of RANKL, OPG and their ratio obtained in patients with osteoporosis/osteopenia and periodontal disease with or without bisphosphonates treatment showed no differences. Bisphosphonates do not alter the concentration of RANKL and OPG and their ratio in the GCF of patients with osteoporosis/ osteopenia and periodontal disease, probably because these cytokines may not be the main target of bisphosphonates to inhibit bone resorption in periodontal disease.
Key words: Periodontal disease; RANKL; OPG; Bisphosphonates; Osteoporosis.
RESUMEN
Efecto del consumo de bifosfonatos en los niveles del ligando del receptor activador del factor nuclear kappa-B y osteoprotegerina en fluido crevicular de pacientes con enfermedad periodontal y osteoporosis post-menopáusica
El sistema: Receptor activador del factor nuclear kappa-B ligando (RANKL)/RANK/Osteoprotegerina (OPG) ha sido propuestos como esencial para la biologia osteoclastica, ya que ha sido identificado como participante clave en la regulacion fisiologica y patologica del sistema oseo. El estudio del sistema RANKL-RANK-OPG ha facilitado la comprension de los mecanismos intervinientes en el proceso de remodela - cion osea, especialmente en la osteoporosis post-menopausica y la enfermedad periodontal. Los bisfosfonatos se han convertido en el pilar principal del tratamiento y prevencion de la osteoporosis post-menopausica. Ellos inhiben la formacion y disolucion de los cristales de fosfato de calcio en el hueso y tambien inhiben a los osteoclastos reduciendo el recambio oseo. Actualmente, varios trabajos de investigacion asocian la osteoporosis con el inicio y la progresion de la enfermedad periodontal. Aunque la etiologia de ambas es diferente, la perdida de masa osea comparte varias caracteristicas y la terapeutica utilizada para la osteoporosis puede ser considera de valor para el tratamiento de la enfermedad periodontal. El objetivo de este estudio fue evaluar el efecto del consumo de bifosfonatos en fluido crevicular (FC) sobre los niveles de RANKL, OPG y la relacion RANKL/OPG en pacientes post-menopausicas con enfermedad periodontal y osteoporosis/ osteopenia. Se estudiaron 66 sitios periodontalmente activos obtenidos de pacientes mujeres post-menopausicas con edades entre 45-70 anos de edad con enfermedad periodontal y osteoporosis/ osteopenia. La toma del FC se realizo mediante tiras de papel de filtro esteriles. Para determinar la concentracion de RANKL y OPG se utilizo el ensayo de ELISA comercial siguiendo las instrucciones del fabricante. Los valores obtenidos de las citoquinas y su relacion fueron comparados con el Test U de Mann-Whitney. No se observaron diferencias en las concentraciones de RANKL y OPG encontradas, ni en su relacion, en pacientes con enfermedad periodontal y osteoporosis/osteopenia con y sin tratamiento de bifosfonatos. Esto sugiere que probablemente estas citoquinas no serian el blanco principal de los bifosfonatos para inhibir la resorcion osea en la enfermedad periodontal.
Palabras clave: Enfermedad periodontal; RANKL; OPG; Bifosfonatos; Osteoporosis.
INTRODUCTION
Periodontal disease is an inflammatory condition
caused by multiple factors where the most
significant environmental risk factor is the bacteria
that reside in the biofilm. According to the report
by Page and Kornman1, these bacteria are essential
but not enough to cause periodontal disease, while
factors related to the host, such as heredity and
environmental factors like smoking or consumption
of certain drugs, are important determinants of the
occurrence and severity of this condition.
In the appearance and progression of periodontal
disease there is an influence of a wide range
of determinants and risk factors, among them
osteoporosis/osteopenia and estrogen deficiency are
included. Immune and skeletal systems share a
variety of regulatory molecules such as cytokines.
In the bone marrow, immune cells interact with
bone cells. Consequently, the physiology and
pathology of one system may affect the other;
indeed, abnormal activation of the immune system
leads to bone destruction 2.
Members of the tumor necrosis factor alpha
superfamily have been proposed as essential for
osteoclast biology because they were identified as
key parts in the physiological and pathological
regulation of the bone system 3. The members
RANKL/RANK/OPG form a system involved in
osteoclast formation and activation. The binding of
RANKL to its receptor RANK, in the presence of
macrophage-colony stimulating factor (M-CSF),
provides the fundamental signal to drive the
development of osteoclasts from hematopoietic
progenitor cells, as well as to activate osteoclasts.
OPG binds to RANKL and inhibits bone resorption,
preventing both cellular differentiation and function
of osteoclasts 4. Osteoclast recruitment depends on
the balance between the RANKL and OPG 5.
The RANK-RANKL-OPG axis is involved in the
regulation of bone metabolism in periodontitis and
osteoporosis/osteopenia, in which an increase in the
relative expression of RANKL and a decrease in
OPG can favor the balance to osteoclastogenesis
and bone reabsorption 6,7. Increased RANKL and
decreased OPG have been observed in various
inflammatory and bone diseases such as osteoporosis,
rheumatoid arthritis, periodontal disease, and
multiple myeloma 8-10. Local deregulation of RANKLOPG
levels may lead to alveolar bone reabsorption
as has been demonstrated in experimental models of
periodontitis 11,12. Many studies show that, compared
to healthy subjects, patients with periodontitis
exhibit higher RANKL expression and a reduction
of OPG levels in GCF 10,13,14, or gingival tissue 14-17.
Periodontitis and osteoporosis represent main
health problems, especially in elderly women.
Although the etiology of postmenopausal osteoporosis
and periodontal disease is different, the bone loss
that occurs in both diseases shares several features 6.
Thus, therapy used for osteoporosis can be
considered of value in the treatment of periodontal
disease. Among the many drugs for osteoporosis
treatment, bisphosphonates are the most commonly
prescribed and first-line drugs in most cases 18.
Bisphosphonates have been characterized as
modulators of osteoclast function and bone
metabolism 19. Particularly they act on bone tissue,
decreasing bone turnover, reducing bone resorption
and the number of new bone multicellular units.
At cellular level, they reduce the recruitment
of osteoblasts and osteoclasts, the adhesion of
osteoclasts to the bone as well as the release of
cytokines by macrophages. Considering RANKL
expression is essential for osteoclast differentiation;
the effect of bisphosphonates on osteoclast
differentiation could be related to a decrease in the
expression of RANKL or by an increase of OPG.
On the basis of these properties, several generations
of bisphosphonates have been successfully developed
in the treatment of post-menopausal osteoporosis,
osteopenia and Paget’s disease 20.
Considering that RANKL/RANK/OPG system
mediates periodontal disease and osteoporosis/
osteopenia and that bisphosphonates are established
as one of the effective drugs for osteoporosis
treatment and that they can act on the RANKL/
RANK/OPG mentioned, the aim of this study was
to investigate the effect of bisphosphonate treatment
on the levels of RANKL and OPG, and their ratio in
GFC of post-menopausal women patients with
periodontal disease and osteoporosis/osteopenia.
MATERIALS AND METHODS
Study population and clinical examination
A total of 66 samples of GFC were obtained from
active periodontal sites of 17 post-menopausal
women aged between 45-70 years old with and
without bisphosphonate treatment (risedronate or
ibandronate: 150 mg/ 1 tablet a month for at least
3 months, prior to the study), all with chronic
periodontitis and osteoporosis/osteopenia. All women
were recruited from the Cimateric Department,
University Hospital of Maternity and Neonatology,
National University of Cordoba, Argentina. The
study protocol was approved by a Bioethical
Institutional Committee and explained to all
participants who signed the informed consent
forms.
Complete medical and dental histories from all
patients were considered. None of them had had any
systemic illness nor taken therapeutic medication
such as antibiotics or anti-inflammatory drugs that
could affect the periodontal status for at least 6
months before the study, and also they did not have
a history of aggressive periodontitis and they had
not received periodontal treatment before they
entered the study. The selection of the patients was
made according to criteria proposed by the World
Health Organization 21. To determine the clinical
periodontal status, all subjects were subject to a
clinical periodontal examination including the
measurement of probing pocket depth (PPD) and
clinical attachment level (CAL) at six sites around
each tooth with a manual probe, using a Marquis
probe. Dichotomous measurement of supragingival
plaque index (PI) and bleeding index (BI) were also
recorded.
Diagnosis of osseous and periodontal disease
The presence of 1 or more sites with PPD ≥ 4 mm
and ≥ 4 mm CAL 22 in a patient was defined as/
considered to be periodontitis. Sites with bleeding
on probing were initially defined as active sites 8.
Dual X-ray absorptiometry (DXA) was used to
evaluate the bone mineral density, since this method
is considered “gold standard” for the diagnosisprognosis
of osteoporosis, monitoring the natural
history of the disorder and response to treatment 21.
The diagnosis of osteoporosis/osteopenia was
verified from the densitometry reports from
proximal femur and lumbar column, following the
criteria established by the WHO at the Consensus
Development Conference 23.
Osteopenia was defined as a bone mineral density
T score (difference between the measured bone
mineral density and the mean value for young white
women in standard deviations [SDs]) of less than
–1 SD or at least –2.5 SD. Osteoporosis was defined
by a bone mineral density T score of less than –
2.5 SD 23. The individuals with osteopenia were
grouped with those with osteoporosis to form an
osteopenia/osteoporosis group.
Sampled sites were then categorized into two
groups: a group of 12 post-menopausal women with
osteoporosis/osteopenia treated with bisphosphonates
(n=55 samples) and a group of 5 post-menopausal
women with osteoporosis/osteopenia without
bisphosphonates therapy (n = 11 samples).
Collection of Gingival Crevicular Fluid
GCF was obtained using sterile filter paper strips
(Periopaper; Oraflow, New York, NY, USA). The
selected sites were cleared of supragingival plaque,
isolated with cotton rolls and dried with a gentle
stream of air to prevent saliva contamination. A
sterile Periopaper™ strip (ProFlow Inc., Amityville,
NY, USA) was gently inserted into the periodontal
pocket until mild resistance was felt and it was left
in place for 30 seconds. Mechanical irritation was
avoided and strips contaminated with blood were
discarded.
After GCF collection, strips were placed in an
Eppendorf tube and samples were stored at -80°C
before laboratory analysis.
4-5 GCF samples were obtained from postmenopausal
women from the group with osteoporosis/
osteopenia under bisphosphonate treatment and 2-3
GCF samples were obtained from post-menopausal
women from the group with osteoporosis/
osteopenia without bisphosphonate therapy.
Paper strips for each patient were pooled, and the
GCF was extracted and assayed for the content of
RANKL and OPG. GCF was extracted from the
paper strips with buffer (50 mM phosphate buffer,
pH 7.2, containing protease inhibitors), and
collected after centrifugation at 15,000 g and 4oC
for 10 min.
Quantification of RANKL and OPG
Concentrations of RANKL and OPG from GCF
samples were determined by ELISA, following
manufacturer’s instructions (Antigenix America
Inc., New York, NY, USA) and the ratio of both
cytokines was established.
Briefly, diluted GCF samples were loaded into
single wells of a 96-well plate coated with 20 ng/ml
of anti-human RANKL antibody or with 4 ng/ml of
anti-human-OPG antibody, and incubated overnight
at 4°C. Plates were washed four times. After the last
wash, 0.2 ug/ml of biotin labelled anti-human
RANKL or OPG were added and incubated for
60 minutes at room temperature.
The reaction was developed with the addition of
streptavidin coupled to peroxidase and visualized
with the addition of 3.3’-5.5’-tetramethylbenzidine
which was transformed into a colored product in the
presence of the enzyme. The reaction was stopped by
the addition of sulfuric acid and color was measured
in a microplate spectrophotometer at 450 nm.
The concentration was measured with reference to
standard curves using known amounts of recombinant
RANKL and OPG.
Data Analysis
Data were expressed as means ± standard deviation.
Taking into account the size of samples, a nonparametric
analysis (Mann-Whitney Test U) was
used. GraphPad Software (version 5.00 for Windows,
San Diego California USA) was used to process the
data. P-values ≤0.05 were considered statistically
significant.
RESULTS
Clinical parameters of the patients
Fifty five periodontally active sites from twelve
patients were analyzed in the study group of postmenopausal
women with osteoporosis/osteopenia
treated with bisphosphonates (age: 56.21 ± 9.24
years old) and eleven periodontal active sites from
five patients were studied in the control group of
postmenopausal women without treatment with
bisphosphonates (age: 56.55 ± 4.68 years old).
Table 1 summarizes the clinical characteristics of
subjects included in this study. No statistically
significant differences in clinical characteristics
(age, PPD, CAL, PI and BI) were observed between
groups. In the case of clinical records, PPD and
CAL were obtained from an average of measured
active sites, while the BI and PI were an average of
the entire oral cavity.
Table 1: Baseline characteristics of the study groups (mean ± SD).
Levels of RANKL, OPG and RANKL/OPG
ratio in GCF from patients treated or
not with bisphosphonates
GCF samples obtained from subjects under bis -
phosphonate treatment (n= 55) showed no significant
differences in the levels of RANKL in comparison
with the group without bisphosphona tes treatment
(n = 11), p = 0.88. The values were 26.81 ± 18.66
and 27.78 ± 19.50 pg/ml, respectively (Fig. 1).
Fig. 1: Distribution levels of RANKL in GCF from active
periodontal sites of women with bone disease under
bisphosphonate treatment –WT- (n = 55) or without
treatment–NT- (n = 11). The individual values represent the
concentration of RANKL in GCF [Total RANKL (pg) / volume
(ml)] in each patient.
OPG showed similar behavior to RANKL. OPG concentrations showed no statistically significant difference (p = 0.62) between both group of patients, whose values were 0.05 ± 0.02 pg/ml in GCF samples from subjects under treatment and 0.04 ± 0.01 pg/ml in GCF samples from patients without treatment (Fig. 2).
Fig. 2: Distribution levels of OPG in GCF from active
periodontal sites of women with bone disease under
treatment–WT-(n = 55) or without treatment –NT-(n=11). The
individual values represent the concentration of GCF in OPG
[Total OPG (pg) / volume (ml)] in each subject..
As the RANKL/OPG ratio is considered indicative of bone resorption, we investigated if changes in RANKL and OPG levels reflected changes in this ratio. The group under bisphosphonate treatment exhibited a RANKL/OPG ratio of 693.5 ± 561.3 pg/ml, while the group without bisphosphonate treatment showed similar values, 677.7 ± 437.6 pg/ml. There were no significant differences among these groups (Fig. 3).
Fig. 3: Distribution levels of RANKL / OPG in GCF from active
periodontal sites of women with bone disease under treatment
-WT-(n = 55) or without treatment –NT-(n=11). The individual
values represent the concentration in GCF of the RANKL /
OPG [Total RANKL (pg) / volume (ml) / total OPG (pg) /
volume (ml)] in each subject.
DISCUSSION
Bisphosphonates have been shown to inhibit the
progression of osteoporosis as a result of the reduction
of bone loss and they have been characterized
as modulators of osteoclast function and bone
metabolism 19.
Previous reports suggest that
bisphosphonates can reduce
not only the maturation and
osteoclast function directly,
but they can also act indirectly
through the synthesis of
mediators that interfere with
osteoclastogenesis 24-26, further
regulating essential signaling
molecules involved in osteoclastogenesis
such as RANKL 27.
The system RANKL/RANK/
OPG acts as a final effector
molecular system. RANKL, RANK, and the decoy receptor OPG, are three key
molecules that regulate the recruitment and function
of osteoclasts 28. The binding of RANKL to RANK
provides the fundamental signal to drive the
development of osteoclasts from hematopoietic
progenitor cells, as well as to activate mature
osteoclasts. OPG negatively regulates by binding to
RANKL, thus inhibits bone reabsorption by
preventing both cellular differentiation and function
of osteoclasts 4,28. The recruitment of osteoclasts
depends on the balance between RANKL and OPG 5.
To our knowledge, this is the first clinical trial to
study the effect of risedronate/ibandronate treatment
on RANKL and OPG levels in GCF and their relative
ratio in post-menopausal women with osteoporosis/
osteopenia and periodontal disease.
Several studies evaluated the effect of bis phosphonates on the levels of RANKL and OPG; however, the
results have been controversial. Tipton et al. 29 demonstrated that the action of alendronate and
pamidronate on human gingival fibroblasts, through
altering the production of RANKL and OPG, appears
to contribute to a microenvironment that favors the
inhibition of bone reabsorption due to an increase
or no change in the levels of OPG, and a decrease
in the production of RANKL. Viereck et al. 30 have demonstrated that bis phosphonates such as
pamidronate and zoledronate increase the expression
of OPG mRNA in primary human osteoblasts. Pan
et al. 31 demonstrated that zoledronic acid may inhibit
bone reabsorption by reducing the expression
of RANKL and increasing transmembrane OPG
secretion in cells such as osteoblasts. However, it does
not affect gene expression of RANKL and OPG.
Dobnig et al. 32 showed a significant increase in serum
levels of OPG in patients treated with alendronate and
risedronate, whereas serum levels of RANKL were
unchanged throughout the treatment period; on the
other hand, a positive correlation between changes in
serum levels of OPG and BMD was found.
On the contrary, Kim et al. 33 demonstrated that
nitrogen-containing bisphosphonates, such as
alendronate and pamidronate, do not alter the
RANKL and OPG mRNA expression in favor of the
inhibition of the osteoclast formation 30. Alvarez et
al. 34 demonstrated that serum OPG decreases after
treatment with tiludronate. In contrast, RANKL
serum levels and RANKL/OPG ratio are unmodified
in patients with Paget’s disease. D’Amelio et al. 35
demonstrated that risedronate inhibits the in vitro
formation of osteoclast by reducing the number and
degree of differentiation of osteoclast precursors
and by reducing the half-life and the inhibition of
the production of pro-osteoclastogenics cytokines.
Moreover, risedronate is able to reduce the recruitment
of osteoclasts from peripheral blood and this could
be the effect of the reduction in cytokine production.
Besides, it was observed a reduction in the levels of
OPG and RANKL (soluble) in serum during treatment
with bisphosphona tes. A study by Kwak et al. 36
concluded that risedronate inhibitory effects on
osteoclast differentiation are related to decreased
expression of RANKL.
According to previous studies, osteotropic agents
modulate osteoclast formation through the regulation
of RANKL/OPG ratio instead of increasing or decreasing
levels of RANKL or OPG alone 37,38. The increase
in the RANKL/OPG ratio results in a favorable condition
for osteoclast formation and vice versa.
In this study it was observed that risedronate and
ibandronate consumption did not change the GCF
RANKL and OPG levels, and RANKL/OPG ratio
in patients suffering osteoporosis/osteopenia and
periodontal disease. So it could be suggested that
RANKL and OPG might not be the main target
of bisphosphonates to inhibit bone resorption in
periodontal disease, reaffirming the results of Kim
et al. 33 and Pan et al. 31.
The discrepancy in the results about the effects of
bisphosphonates on the above cytokines can be
attributed to several reasons. Firstly, the research
design used was different to previous reports.
Experimental studies were carried out with mice
33,36 and with human cells in vitro 29-31; however, in
this study, the effect of bisphosphonates was
evaluated on patients, as in other researches 32,34,35.
Secondly, all drugs analyzed were aminobisphosphonates and they had a different chemical
structure. Thirdly, the methods of analysis of the
cytokines varied if the gene expression or the
presence of proteins was studied.
In conclusion, the present study showed that
nitrogen-containing bisphosphonates, ibandronate
and risedronate, did not alter the GCF RANKL and
OPG levels and their relative ratio in periodontal
disease. It could be suggested that RANKL and OPG
might not be the main target of bisphosphonates to
inhibit bone resorption in periodontal disease.
ACKNOWLEDGMENTS
E.Verde acknowledges the Secretary of Science and Technology – Universidad Nacional de Cordoba (SECyT-UNC) for the PhD scholarship awarded and the Secretary of Science and Technology - School of Dentistry (SECyT-FO) for the financial support for this publication. The authors acknowledge the assistance of Dr. Monica Nanez de Lucino from the University Hospital of Maternity and Neonatology.
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