Influence of Hormone Replacement Therapy on Osteoprotegerin and Receptor Activator of Nuclear Factor Kappa-B Ligand Concentrations in Menopausal Women

Abstract

The study was designed to compare the levels of the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in the serum and saliva of menopausal women and verify whether there is a correlation between concentrations of these cytokines and bone mineral density (BMD) of the femur and mandible. Blood and saliva samples were collected from a group of 60 women during menopause—30 untreated (control group) and 30 treated with hormone replacement therapy (HRT) (research group). Densitometry was conducted on the femoral bone and mandible. BMD in the research group was significantly higher than in the control group. The OPG concentration was slightly higher in the research group. The concentration of RANKL was slightly higher in the control group. Differences in RANKL/OPG between groups were not high enough to be statistically significant. Analysis of the RANKL in saliva revealed a significantly higher concentration of this factor in the control group. Observed beneficial effects of HRT on bone tissue may be exerted through the RANKL/OPG ratio, but further research is required to clearly confirm this thesis.

Introduction

B ones, like other tissues, are composed of cells and intercellular substance. The bone tissue undergoes continuous remodeling. Osteocytes are bone-forming cells surrounded by the matrix. Osteocytes are derived from osteoblasts and play an important role in bone metabolism. Mentioned cells affect both mass and shape of bones. Osteocytes control the activity of osteoblasts and osteoclasts in a basic multicellular unit and take part in the process of formation and remodeling of bone tissue. Osteoclasts remove old bone tissue and in its place, osteoblasts produce a new bone matrix. The balance between these opposite processes is essential for maintaining a proper macro- and microstructure of bones, and consequently, the appropriate strength of a skeleton.

Discovery of mechanisms regulating the metabolic processes, which take place in bone tissue led to understanding of skeletal diseases' pathomechanism. This enabled introduction of new treatment methods for diseases affecting bone tissue. The formation of bone tissue can be divided into several processes: activation of osteoclasts, bone resorption, osteoblast activation combined with inhibition of osteoclasts, and the process of final bone formation (Trouvin and Goëb 2002). Function and maturation of osteoclasts depend on many factors, among which, osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B (RANK), and RANK ligand (RANKL) play a key role (Lerner 2004; Rogers and Eastell 2005). RANKL is the primary cytokine initiating osteoclast formation and osteolytic function. OPG, an inhibitor of osteoclastogenesis, is its endogenic antagonist.

OPG, also known as the osteoclastogenesis inhibitor factor, is a protein that belongs to the family of tumor necrosis factor receptors (TNFR) (Simonet and others 1997). OPG, by binding RANKL, leads to inhibition of function of this cytokine. The OPG gene expression level depends on many factors. Cytokines [TNF-α, interleukin (IL)-1α, IL-18, and TNF-β], bone morphogenetic proteins, 17β-estradiol, and mechanical stress can increase its expression. Factors that reduce the expression of OPG include medications (corticosteroids, immunosuppressants), parathyroid hormone (PTH), prostaglandin E2, and basic fibroblast growing factor (FGF) (Yano and others 1999).

RANKL is a transmembrane protein, which belongs to the TNF family. Its concentrations in the body are lower than OPG. Recent studies have shown that osteocytes are a major source of RANKL. Osteocyte apoptosis generates signals that lead to increased production of RANKL and bone resorption in the part of bone, which contains dying cells (Xiong and O'Brien 2012). Expression of RANKL is determined by other cytokines (IL-1, IL-6, IL-11, and TNF-α), glucocorticoids and PTH and 1,25(OH)2D3. RANKL works through the receptor RANK located on the surface of target cells, by stimulating them to differentiation, function, and programmed death (Hofbauer and others 1999; Von Tirpitz and others 2003). The first step of osteoclast formation is the stimulation of osteoclast precursors to proliferation by the transcription factor PU.1 and the growth factor colony-stimulating factor. The next step is the binding of RANKL with the cell-surface receptor. This leads to the sequence of cellular changes within the osteoclast progenitors, resulting in the formation of active osteoclasts capable of performing osteolytic processes. RANKL cannot only stimulate resorption by osteoclasts, but together with IL-1, it can prolong the life span of mature osteoclasts (Lacey and others 1998; Kryśkiewicz and Lorens 2006).

Knowing the route RANKL/OPG, as well as factors affecting the expression of RANKL and OPG genes, may explain the pathogenesis of many diseases of bone tissue (Samelson and others 2008). Estrogens increase gene expression of OPG and inhibit the expression of the RANKL gene. Therefore, in postmenopausal women who have sex hormone deficiency, levels of RANKL in bone marrow cells and lymphocytes should be elevated. This dependence may explain the mechanisms of postmenopausal osteoporosis (Lorenz and others 2004).

Aim of the Study

The study was designed to investigate and compare levels of RANKL and OPG in serum and saliva of menopausal women treated and not treated with hormone replacement therapy (HRT). In addition, the study was designed to verify whether there is a correlation between the concentrations of these cytokines and bone mineral density (BMD) of the femur and mandible and to verify the local impact of salivary OPG/RANKL on the BMD of the mandible.

Materials and Methods

The study was conducted on a group of 60 patients in the menopausal period. The research group consisted of 30 women undergoing menopause HRT for 6–12 months before the study (age range 49–59 years, mean age 53.0 years). The control group consisted of 30 postmenopausal women, at least 12 months after the last period, not treated with HRT (age range 53–59 years, mean age—55.4 years). Patients in the control group have never been receiving HRT. Examined patients were treated in the out-patients gynecological clinic of Public Hospital No. 4 in Lublin. All patients gave their consent for the study and procedures received approval of the Local Ethics Committee in Lublin (number of decision KE-0254/246/2005 and KE-0254/47/2010). The study was carried out in accordance with the ethical principles contained in the Declaration of Helsinki.

The control group was divided into 2 subgroups: M—women after menopause and OV—women after ovariectomy. The study group was divided according to the same criteria: M+HRT—menopausal women on HRT, OV+HRT—a group of women after surgical removal of ovaries, using HRT. Patients from groups OV and OV+HRT (mean age 54.2 years) underwent surgery at least 36 months before the study was conducted. Surgeries were performed as treatment of diseases of the reproductive system.

An individual anamnesis chart was developed. The questions in the survey included age, date of last menstrual period, duration of HRT, diseases, number of births, medication, occupation, addictions, physical activity, and surgeries. Women qualified for the study did not suffer from any severe general diseases, had no addictions, and have not been taking medications continuously. None of the patients qualified for the study had any fractures during the menopausal period before the study or during the study. Women qualified for the control and study groups were matched in terms of age. Women from groups M+HRT and OV+HRT were supplemented with estrogen and progesterone in combination (combination therapy). Patients received Femoston in tablets (2 mg of Estradiol hemihydrate and 10 mg of dydrogesterone). Estrogen was taken on a continuous basis and progesterone was added for the last 2 weeks of each course (course lasted 28 days).

A clinical examination was carried out using a dental mirror, dental and periodontal probe. During the oral examination, attention was paid to the state of dentition, especially the number of extracted teeth, the state of oral mucosa, periodontal status, the presence of dentures, and other possible problems that occur in the mouth. Patients who were qualified for further tests did not have any aggressive inflammations of oral mucosa and the depth of periodontal pockets was lower than 5 mm. Patients did not need any extractions of teeth and had average or good oral hygiene. Women qualified for the study did not use any removable dentures. Follow-up visits were arranged every 3 months to monitor the periodontal and dental status, perform nonsurgical scaling, root planning, and caries treatment. The examination of patients' oral status, during check-ups in the 5-year period between tests in 2005 and 2010, indicated that no surgical procedures were needed.

Tests were carried out on samples obtained in 2005, and then follow-up research was carried out in 2010 on samples from the same cohort. Research on blood samples was carried out 2 times: in 2005 and 2010. Tests on saliva collected from the same group of patients were carried out in 2010. During the 5-year period between tests, women from groups treated with HRT received a fixed dose of hormone drugs. During the time between testing procedures, no severe diseases were diagnosed and no fractures have occurred. Unstimulated saliva and venous blood from a cubital vein were collected from women in the fasting state. Blood and saliva samples were collected in the morning hours (7–9 a.m.). Chewing gum was prohibited for 2 h before collection of diagnostic material. Patients before collection of saliva were asked to rinse mouth with distilled water and relax for 5 min. After this time, patients were asked to lean their head forward and keep mouth open for 5 min to allow saliva drain into the testing tube. At the end of time of collection, patients were asked to spit the remaining saliva into the tube. After centrifugation of the material, the obtained supernatant (saliva) and serum (blood) were stored until biochemical tests at −70°C. The concentration of OPG and RANKL in saliva and serum were determined by enzyme-linked immunosorbent assay (ELISA), using a kit from Biomedica (Anti-Human CD254 RANK Ligand Purified, Human Osteoprotegerin ELISA) and ELx800 Absorbance Microplate Reader with a wavelength 450 nm (Bio-Tek) (Moschen and others 2005; Li and others 2012). All procedures were performed according to the indications given by the manufacturer. The detection limit for OPG was 0.14 pmol/l and for RANKL, 0.08 pmol/l.

The study of femoral BMD was performed in 2010 in the Densitometric Laboratory of Institute of Agricultural Medicine in Lublin, by means of the DPX-A Lunar equipment and absorptiometry of X-ray beams of 2 energies, using the method of DXA (General Electric Healthcare Technologies Lunar) Prodigy Advance with use of the Femur option and absorptiometry of X-ray beams of 2 energies. The scan time was 30 s. The BMD was measured at the upper femoral neck. The area of interest was determined by analysis software. Images of the mandible were scanned and the area of foramen mentalis was marked and the computer calculated the average bone density of the area. The bone density was specified in g/cm².

Results were statistically analyzed with use of program STATISTICA. The arithmetic mean and standard deviation were calculated. The significance of differences between groups is based on confidence intervals determined from the analysis of variance. The interdependence between selected traits was expressed by the Pearson's correlation. The paired t-test was used to determine whether results from 2005 have changed significantly in 2010. The risk of error of inference of the study is 5%, which means that the results were significant, if the P was equal or less than 0.05.

Results

The content of OPG in the serum of women in 2005 was slightly higher in the groups receiving HRT, but this difference was not high enough to be statistically significant. Differences in levels of serum OPG in 2010 between groups (M) and (M+HRT) and groups (OV) and (OV+HRT) were statistically insignificant (Table 1).

Table 1.

Concentration of Osteoprotegerin and RANK Ligand in Serum and Saliva

Cytokine	Research group	Sample quantity	Serum concentration (2005) pmol/L (M±SD)	Control group	Sample quantity	Serum concentration (2005) pmol/l (M±SD)	Statistically significant differences between results in control and research group (P<0.05)
OPG	M+HRT	15	6.66±0.82	M	15	6.27±0.80	No
	OV+HRT	15	6.56±0.70	OV	15	6.39±0.19	No

Cytokine	Research group	Sample quantity	Serum concentration (2005) pmol/l (M±SD)	Control group	Sample quantity	Serum concentration (2005) pmol/l (M±SD)	Statistically significant differences between results in control and research group (P<0.05)
RANKL	M+HRT	15	0.26±0.05	M	15	0.31±0.16	No
	OV+HRT	15	0.27±0.05	OV	15	0.28±0.02	No

Cytokine	Research group	Sample quantity	Serum concentration (2010) pmol/l (M±SD)	Control group	Sample quantity	Serum concentration (2010) pmol/l (M±SD)	Statistically significant differences between results in control and research group (P<0.05)
OPG	M+HRT	15	5.09±0.72	M	15	4.62±0.37	No
	OV+HRT	15	5.33±0.64	OV	15	5.51±0.54	No

Cytokine	Research group	Sample quantity	Serum concentration (2010) pmol/l (M±SD)	Control group	Sample quantity	Serum concentration (2010) pmol/l (M±SD)	Statistically significant differences between results in control and research group (P<0.05)
RANKL	M+HRT	15	0.17±0.03	M	15	0.24±0.05	Yes
	OV+HRT	15	0.26±0.05	OV	15	0.21±0.05	Yes

Cytokine	Research group	Sample quantity	Salivary concentration (2010) pmol/l (M±SD)	Control group	Sample quantity	Salivary concentration (2010) pmol/l (M±SD)	Statistically significant differences between results in control and research group (P<0.05)
OPG	M+HRT	15	1.84±0.45	M	15	2.14±0.47	No
	OV+HRT	15	1.94±0.33	OV	15	1.89±0.06	No

Cytokine	Research group	Sample quantity	Salivary concentration pmol/l (M±SD)	Control group	Sample quantity	Salivary concentration pmol/l (M±SD)	Statistically significant differences between results in control and research group (P<0.05)
RANKL	M+HRT	15	0.052±0.018	M	15	0.069±0.019	No
	OV+HRT	15	0.051±0.027	OV	15	0.072±0.029	Yes

SD, standard deviation; OPG, osteoprotegerin; M, women after menopause; OV, women after ovariectomy; HRT, hormone replacement therapy; OV, RANKL, receptor activator of nuclear factor kappa-B ligand.

The concentration of RANKL in 2005 in the serum of women from groups (M) and (OV) was higher than in the groups receiving HRT, but the difference was not statistically significant. Analysis of the results obtained in 2010 indicates that in the group (M+HRT), the RANKL content was the lowest and in the group (OV+HRT), the highest. The differences between research and control groups were statistically significant (Table 1).

In all groups, the differences between the levels of serum OPG in 2005 and 2010 were significant. The results indicate that the level of OPG in serum decreased significantly in 2010. Analysis of the concentration of RANKL in serum in 2005 and 2010 revealed that the differences between the values were significant. In 2010, the level of RANKL in serum decreased significantly (Table 1).

Differences in the content of OPG in saliva between the control and research groups showed no statistical significance. Statistical analysis of the RANKL level in the saliva showed that the concentration of this cytokine was higher in the control groups, when compared to research groups. Differences in the RANKL content between groups (OV+HRT) and (OV) were statistically significant (Table 1).

The Pearson's correlation indicates the level of linear dependence between the variables. The value of (+1) stands for a perfect positive (increasing) linear relationship, (−1) stands for a perfect negative (decreasing) correlation. Values approaching (0) show lack of correlation between variables. The closer the coefficient is to values (−1) or (+1), the stronger the relationship between the results. An assessment of the Pearson's correlation between the concentration of OPG in serum and saliva showed that in groups (OV) and (OV+HRT), the correlation was significant and very high. Based on the determined regression line, it can be considered that an increase in the serum OPG concentration resulted in an increase in OPG levels in the saliva (Table 2) Analysis of the Pearson's correlation for RANKL in serum and saliva showed no significant relationship.

Table 2.

Correlation Between Concentrations of RANK Ligand and Osteoprotegerin

Group	Sample quantity	Pearson's correlation between concentration of RANKL and OPG in serum in 2005	Pearson's correlation between concentration of RANKL and OPG in serum in 2010	Pearson's correlation between concentration of RANKL and OPG in saliva in 2010	Pearson's correlation for OPG in serum and saliva in 2010	Pearson's correlation for RANKL in serum and saliva in 2010
M	15	−0.6050	0.2135	0.2183	−0.0751	−0.3714
OV	15	−0.3340	−0.0538	−0.0840	0.8371	0.3957
M+HRT	15	−0.0317	−0.1700	0.0034	0.0755	0.0967
OV+HRT	15	−0.0201	0.1182	−0.0674	0.6054	0.4007

Analysis of the Pearson's correlation between the serum OPG and RANKL in 2005 showed that there is a slight negative correlation between these 2 factors. In the group of (M), a correlation was significant and it was high (r=−0.6050). Based on the determined regression line, it can be considered that the increase in serum RANKL by 0.1 U caused a reduction in OPG levels in the blood serum of 0.30952 U (Table 2). Results for OPG and RANKL in serum in 2010 did not show a significant correlation. Analysis of the relation between these cytokines in the saliva also did not show a significant correlation (Table 2).

BMD in the groups using HRT was higher than in control groups, although differences in the BMD of the mandible between the control and research group were not high enough to be statistically significant (Table 3).

Table 3.

Bone Mineral Density Levels of the Femur and Mandible in the Tested Groups of Women (g/cm ²)

Research group	Sample quantity	BMD of mandible–area of teeth 35–45 g/cm² (M±SD)	Control group	Sample quantity	BMD of mandible–area of teeth 35–45 g/cm² (M±SD)	Statistically significant differences between results in control and research group (P<0.05)
M+HRT	15	1.20±0.15	M	15	0.98±0.54	No
OV+HRT	15	1.04±0.36	OV	15	0.87±0.29	No

Research group	Sample quantity	BMD of femoral bone, g/cm² (M±SD)	Control group	Sample quantity	BMD of femoral bone, g/cm² (M±SD)	Statistically significant differences between results in control and research group (P<0.05)
M+HRT	15	0.92±0.05	M	15	0.77±0.12	Yes
OV+HRT	15	0.91±0.05	OV	15	0.80±0.07	Yes

BMD, bone mineral density.

Analysis of correlation was designed to verify whether the osteoprotective influence of HRT on the mandible was caused by local function of OPG in saliva. The Pearson's correlation between OPG in the saliva and BMD of the mandible indicates that in the group (M+HRT), the relationship between the measurements was significant (r=0.7931). Based on the determined regression line, it can be considered that the increase in the OPG level in the saliva of 0.1 U caused an increase in the BMD of the mandible by 0.02583 U. An equally important correlation can be observed in the group (OV+HRT) (r=0.7535) (Table 4). Based on the determined regression line, it can be considered that the increase in the OPG level in the saliva of 0.1 U caused an increase in the BMD of the mandible of 0.0823 U. The result of Pearson's correlation clearly indicates a positive relationship between these 2 variables.

Table 4.

Correlation Between Osteoprotegerin Concentration and Bone Mineral Density of the Femur and Mandible in Year 2010

Group	Sample quantity	Pearson's correlation between concentration of OPG in serum and BMD of femoral bone	Pearson's correlation between concentration of OPG in serum and BMD of mandible	Pearson's correlation between concentration of OPG in saliva and BMD of femoral bone	Pearson's correlation between concentration of OPG in saliva and BMD of mandible
M	15	0.3981	0.3810	0.0964	−0.1519
OV	15	0.4063	0.2216	0.5186	−0.1442
M+HRT	15	0.1216	0.3558	0.1812	0.7931
OV+HRT	15	0.3060	0.6082	0.5793	0.7535

Analysis of Pearson's correlation between serum OPG and femur BMD levels showed no significant dependence between these metrics (Table 4). Analysis of Pearson's correlation between the serum OPG concentration and mandible BMD levels showed a significant positive correlation between these metrics only in group (OV+HRT) (r=0.6082). Analysis of Pearson's correlation between saliva OPG and femur BMD levels showed a significant correlation between these metrics in groups (OV) (r=0.5186) and (OV+HRT) (r=0.5793).

Discussion

HRT is considered to be the most effective way of countering the effects of sex hormone deficiency appearing after the expiration of ovarian function. This statement has been confirmed by numerous researches. The results presented in this study clearly indicate that HRT has a positive effect on maintaining a high level of BMD. In the present study, BMD was measured at the femoral neck and mandible. Monitoring of bone changes in the femur has similar sensitivity as monitoring of changes in the lumbar spine (Xu and others 2007). Analysis of mandible BMD made it possible to investigate the local function of salivary OPG/RANKL. Densitometric studies performed on both the mandible and femoral bone revealed that groups receiving HRT showed a higher level of BMD than control groups. These findings are consistent with the results of a study carried out by Stanosz and others, who examined the influence of HRT on the concentration of hormones, growth factors, and BMD of women with diagnosed osteopeny (Stanosz and others 2009). On the basis of obtained data, researchers suggested that hormone supplementation of women in the menopausal age leads to normalization of sex hormone levels and an increase in bone mineralization. This thesis was confirmed by the work of Miller and others, who studied the influence of HRT on the markers of bone metabolism and BMD of the femur and vertebral column (Miller and others 2000) The results of the study showed that in groups treated with HRT, there was an increase in the mineralization of bone tissue.

Diseases, which lead to the loss of bone mass, have a common pathogenesis at the molecular level. Excessive induction of cytokine production may be relevant in the pathogenesis of some diseases. Knowledge of the role of cytokines in the state of homeostasis and specific pathological processes may be used in the advancing of the awareness of their biological role. The factor that has a significant impact on bone metabolism is a cytokine system belonging to the TNF family—an OPG/RANKL system. It is a signaling pathway through which osteoblasts regulate the pool of active osteoclasts (Boyle and others 2003). Hsu and others (2006), in their work, studied the influence of the RANKL gene polymorphism on BMD of the hip bone. The study was conducted on a group of 1,120 patients with very high and very low BMD. Obtained results made it possible to postulate the thesis that patients having the genotype TC/CC for rs9594782 RANKL gene polymorphism (TNFRSF11 A) are much more vulnerable to lowering skeletal mineral density. Bai and others in their work obtained the results, which supported the thesis that there is a clear correlation between the expression of RANKL and bone structure (Bai and others 2011). Authors carried out tests on a group of 80 patients. Obtained results demonstrated that in patients with low BMD, the concentrations of RANKL were higher than in patients with normal bone density. Determined by these researchers, the Pearson's correlation showed a negative relationship between the level of RANKL (r=−0.256, P=0.015) and BMD of the vertebral column. These findings are partially consistent with the data obtained in the present study. Results from 2005 concerning the concentration of RANKL in the serum, showed that the use of HRT had a slight impact on reducing the level of this cytokine. Data obtained after repeating tests of blood serum in the year 2010 showed a significant decrease in serum RANKL in the group (M+HRT). A clear difference in the concentration of RANKL was obtained in saliva. Analysis of RANKL levels in the salivary gland secretion showed that the highest concentration of this cytokine was found in groups not receiving HRT. These findings indicate the positive effect of hormone therapy on reducing the concentration of osteolytic cytokines.

OPG has a protective effect on bone tissue, which was confirmed in studies carried out on animals and humans. The administration of OPG inhibited bone resorption in rats and confirmed the vital importance of OPG as an inhibitor of bone resorption (Price and others 2001). Stern and others (2007) demonstrated that high OPG levels positively correlated with bone density of the spine and hip in women receiving estrogen supplementation. Bekker and others (2001) studied the effect of administration of a single dose of OPG in postmenopausal women, in a dose of 3.0 mg/kg body weight. Researchers obtained a significant reduction in levels of bone resorption markers after 4 days, after administration of OPG. In the present study, higher levels of OPG were found in the serum of menopausal women who used HRT, than among women not treated with estrogen therapy. Comparison of data from 2005 and 2010 showed a significant decrease in serum OPG concentrations in all groups, but the decrease was the highest in the group (M), which did not receive HRT. A study carried out by Flórez-Moreno showed that median values of salivary RANKL significantly increased and median OPG salivary values significantly decreased over time after the activation of orthodontic forces on teeth and maxillary and mandibular bones (Flórez-Moreno and others 2013). These results clearly support the thesis that local bone remodeling leads to changes in the salivary RANKL/OPG ratio. In the present study, there were no significant differences in the concentrations of OPG in the saliva between the control and research groups. Analysis of correlation between the concentration of OPG and levels of bone mineral density showed that in some groups, which were treated with HRT, this relationship was positive and statistically significant. A positive correlation was found between salivary OPG and BMD of the mandible. These results indicate that salivary OPG has an important local osteoprotective effect. The osteoprotective effect of HRT may manifest by an increase in the secretion of OPG. Lactoferrin (LF) uses OPG as a pathway that mediates its anabolic action on the bone tissue. A study by Hou and others (2012) performed on ovariectomized rats showed that LF treatment significantly increased the OPG mRNA levels, and suppressed the RANKL mRNA levels.

Analysis of the OPG/RANKL ratio in serum in 2005 revealed a negative r-Pearson's correlation (r=−0.6050) between the levels of these cytokines. The increase in the level of RANKL leads to a decrease in the concentration of OPG. This relationship is important in maintaining the delicate balance between these cytokines, which are necessary for normal bone metabolism. When the concentration of RANKL increases, bone resorption may be intensified, while the predominance of OPG may result in the pathological reduction of resorption.

An important part of the study preparation was the selection of patients who have been qualified for examination. It should be stressed that there are many factors that have a significant impact on the levels of cytokines in serum and in saliva. A study by Lappin and others has shown that a significant impact on the ratio of OPG/RANKL is smoking (Lappin and others 2007). There is a general agreement that periodontal disease has an important impact on the levels of cytokines (Cochran 2008). Research conducted by Giganti and others (2012) has shown that the OPG concentration is significantly higher in the group of patients with fractures when compared to the control group. Research conducted by these authors also proved that even the type of fracture has an impact on cytokine concentrations. Systemic diseases also show a significant effect on the levels of cytokines. Multiplicity of factors affecting the OPG/RANKL ratio requires the use of very strict qualifying criteria for the study. Exclusion of patients with advanced periodontal disease, addictions, recent fractures, or serious systemic diseases was designed to allow an examination of the impact of hormone supplementation on cytokine concentrations and indicate whether the ratio of OPG/RANKL is one of the pathways by which HRT affects the bone tissue. Reports on the relationship between the OPG/RANKL, metabolic bone turnover, and hormone levels of healthy men and women are ambiguous. The study of Khosla and others (2002) has revealed that the concentration of OPG increases with age, regardless of the gender. Elevated levels of OPG and RANKL in serum were found in various pathologies, for example, certain types of cancers (Grimaud and others 2003). These factors could have influenced some of the results obtained in this study, which made it difficult to make clear conclusions.

Saliva is increasingly used as a research material in medical diagnostics, because of the simple and nontraumatic process of collection of this material. Secretion of salivary glands can be used to assess the level of hormones, drugs, antibodies, or inflammatory factors. It is preferred to use mixed saliva, because it contains serum components (Cyprysiak and Tadeusiak 2001). For this reason, it is advisable to use unstimulated saliva for the examination. Unstimulated saliva in 70% is derived from the submandibular salivary glands, which produce the serous-mucous secretion. In recent years, diagnostic difficulties associated with the use of this material, mainly related to different levels of biological agents in the saliva than in serum, have been overcome. Currently, diagnostic tests performed on saliva provide results as reliable as those of blood tests (Wong 2006). Systemic deficiency of sex hormones influences the whole skeleton, including bones of the stomatognatic system. Data obtained in the present study indicate that the concentration of the cytokines OPG/RANKL in saliva was lower than in serum. The analysis of the level of correlation showed that there was a significant relationship between concentrations of OPG in saliva and serum in some groups. What is more, analysis of the Pearson's correlation between the concentration of OPG in saliva and BMD of the mandible revealed that the local concentration of OPG has a positive influence on the mandible. What is more, the content of RANKL in saliva was significantly higher in groups not treated with HRT. Those results confirm the positive effect of HRT on bone tissue and suggest that this effect may be expressed by the influence of sex hormones on the regulation of levels of cytokines. Saliva may prove to be a reliable diagnostic sample in the evaluation of levels of cytokines and a useful method of monitoring of treatment. It has to be stated, that salivary concentrations of OPG and RANKL are influenced by local inflammations. The periodontal disease increases the level of RANKL and decreases the concentration of OPG in saliva (Belibasakis and Bostanci 2012). Periodontitis by OPG/RANKL system leads to demineralization and bone loss. A study carried out by El-Sharkawy and others revealed that periodontal treatment combined with supplementation with Omega-3 polyunsaturated fatty acids and low-dose aspirin leads to significant reduction of salivary RANKL and matrix metalloproteinase-8 levels (El-Sharkawy and others 2010). Status of oral health has a significant influence on salivary levels of cytokines and should be taken in consideration during the process of qualification of patients for tests. It should be emphasized that the results obtained in the present study from saliva, were more significant than the results obtained from serum. This may be associated with the fact, that in case of saliva, local factors have a significant impact on the fluctuations in the levels of cytokines. A careful stomatological examination of patients and the possibility of conducting regular checkups of the oral cavity allowed to qualify for testing procedures only patients without severe inflammations or advanced periodontal disease. In the present study, patients who where qualified for further tests did not have any aggressive inflammations of oral mucosa and the depth of periodontal sockets was lower than 5 mm. Patients did not need any extractions of teeth and had average or good oral hygiene. Women qualified for the study did not use any removable dentures. This may explain why the results of tests on saliva were less significantly disturbed by the influence of factors other than HRT.

Conclusions

HRT has a clearly beneficial effect on BMD of patients after menopause. The study showed that there may be a correlation between the concentrations of RANKL/OPG and BMD of patients. The study has shown some trends that need to be clarified. Results suggest that the observed beneficial effects of HRT on bone tissue may be expressed through these cytokines. Obtained results indicate that there is a slight negative correlation between the concentration of serum OPG and RANKL. Saliva may prove to be a reliable diagnostic material for the examination of cytokine levels. There is a necessity for further research performed on a broader group of patients to clearly confirm these observations.

Future Perspective

HRT has a beneficial effect on BMD. OPG and RANKL seem to be one of the most important systems by which HRT affects the bone tissue. Assessment of the OPG/RANKL ratio may prove to be an important indicator of bone metabolism and pathological processes, which occur in bone tissue. What is more, saliva may be an equally valuable diagnostic material for analysis of cytokine concentrations as blood serum. Saidenberg-Kermanach proved, in research carried out on animals, the effectiveness of systemic administration of OPG and anti-TNF-α (Saidenberg-Kermanach and others 2004). Treatment with these substances leads to significant osteoprotection in inflammatory diseases. Further research on menopausal women could lead to the development of medicines, which would affect directly the OPG/RANKL ratio and could be included in the therapy of menopause.

Executive summary

1. HRT has a clearly positive impact on the state of mineralization of bone tissue.

2. Preliminary studies of levels of RANKL/OPG in postmenopausal women suggest that the observed beneficial effects of HRT on bone tissue may be exerted through these cytokines.

3. The results indicate that there may be a negative correlation between the serum OPG and RANKL.

4. Saliva seems to be a promising diagnostic material, which is easier to obtain than blood serum.

5. Saliva may be a reliable material for the examination of cytokines levels and further research is needed to unequivocally confirm this hypothesis.

6. Further research on the concentration of cytokines in serum and saliva carried out on a broader group is required to confirm observations made in this article.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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