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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 20, 2004 as doi:10.1096/fj.04-2214fje. |
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* Department of Internal Medicine,
Department of Genetics, Biology and Biochemistry Research, and
Department of Medical Oncology, Center on Experimental Medicine (CeRMS), University of Torino, Turin, Italy
1Correspondence: Dipartimento di Medicina Interna, Corso Dogliotti 14, Torino 10126, Italy. E-mail: patriziadamelio{at}netscape.net
SPECIFIC AIMS
Postmenopausal osteoporosis is a common disorder characterized by decreased bone density and increased fracture risk. An imbalance between bone formation and bone resorption is thought to underlie the pathogenesis of reduced bone mass in osteoporosis.
1. Osteoclastogenesis: the aim of the present study is to evaluate a possible increase in spontaneous (i.e., without the addition of M-CSF, TNF-
, or RANKL in the medium) osteoclast formation in postmenopausal osteoporotic women with respect to healthy subjects (age, sex, and BMI matched).
2. Sensitivity to vitamin D: we tested the sensitivity of the osteoclast precursors to 1,25-OH vitamin D3 in patients with respect to controls.
3. Osteoclast activity: we evaluated possible differences in osteoclast activity between patients and controls
4. Clinical parameters: we investigated the relationship between osteoclastogenesis and bone mineral density and the common markers of bone turnover used in clinical practice.
5. Cytokines: we analyzed the role of the production of RANKL and TNF- in an in vitro osteoclast formation.
6. Addition of M-CSF and RANKL: we compared spontaneous osteoclastogenesis with osteoclastogenesis obtained after cytokines stimulus, in order to verify data obtained in previous studies.
PRINCIPAL FINDINGS
1. Osteoclastogenesis
By the TRAP and VNR staining on day 10 of culture it was possible to detect the formation of large multinucleated TRAP and VNR positive cells (defined osteoclasts according to previous literature). In the patients, the average number of osteoclasts was 48.23 ± 25 (without 1,25-OH vitamin D3) per 105 cells. In the controls, the number of osteoclasts was 10.41 ± 13 (without 1,25-OH vitamin D3 per 105 cells) (Table 1
).
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2. Sensitivity to vitamin D
In the patients, the average number of osteoclasts was 28.5 ± 24.6 (with 1,25-OH vitamin D3). In the controls, the number of osteoclasts was 4.4 ± 6.6 (with 1,25-OH vitamin D3) per 105 cells (P=0.001) (Table 1)
. Considering patients and controls together, the reduction in the number of osteoclasts in wells treated with 1,25-OH vitamin D3 was significant as shown by the paired Student’s t test (P=0.01). Osteoclast bone reabsorbing activity in wells treated with 1,25-OH vitamin D3 was higher both in patients and controls with respect to osteoclast reabsorbing activity in wells without 1,25-OH vitamin D3 (P=0.001).
3. Osteoclast activity
Osteoclast activity was evaluated by measuring the percentage of lacunar bone resorption area on dentine slices. Bone resorption was significantly higher in patients with respect to controls (9.5% in patients and 0.9% in controls, Table 1
).
4. Clinical parameters
Comparisons between patients and controls for age, postmenopausal period, BMI, and markers of bone metabolism demonstrated a greater increase in the level of urinary cross laps for patients vs. controls (Table1). Our data showed significant inverse correlations between BMD measured at lumbar spine and femoral neck with number of osteoclasts, and level of RANKL and TNF-. There was a significant inverse correlation between the level of urinary cross laps and lumbar BMD, while the correlation between level of urinary cross laps and RANKL and TNF- is strictly direct. Stepwise regression demonstrated that lumbar BMD is a predictor of the number of osteoclasts formed without addition of 1,25-OH vitamin D3 (r2=0.56) whereas RANKL alone is a predictor of the number of osteoclasts formed with the addition of 1,25-OH vitamin D3 (r2=0.63).
5. Cytokines
Our data demonstrate an increase in the production of RANKL and TNF- by PBMC of osteoporotic women with respect to controls (Table 1)
.
6. Addiction of M-CSF and RANKL
We observed a significant increase in osteoclast number and percentage of lacunar resorption in controls but no significant differences after the addition of these factors to patients. There were no differences between patients and controls or between wells treated or untreated with 1,25-OH vitamin D3 in the number of osteoclast or percentage of lacunar resorption in the presence of M-CSF and RANKL.
CONCLUSIONS AND SIGNIFICANCE
To our knowledge there are no data in literature that correlate clinical features with spontaneous osteoclast formation in vitro and with the level of cytokines in PBMC cultures in postmenopausal osteoporosis. In the present study, we analyzed the relationship between osteoclast formation and clinical parameters such as BMD and common markers of bone metabolism (BGP, BAP, and cross laps) and their relationship with the cytokines most involved in osteoclast formation: TNF- and RANKL.
In contrast to a recent study by Jevon et al., our data demonstrate an increase in osteoclast formation in postmenopausal osteoporotic women compared with healthy controls (age, sex, and BMI matched). The study by Jevon et al. demonstrated increased osteoclast activity in osteoporotic patients with respect to controls without an increase in the number of osteoclasts formed. Their data were obtained in cultures of PBMC with addition of M-CSF and RANKL. It was our opinion that the addition of exogenous cytokines could alter the endogenous differences in production of these cytokines in patients with respect to controls and consequently a possible increase in spontaneous osteoclastogenesis in osteoporotic patients. Our experiments in the presence of M-CSF and RANKL, an analogous condition, confirm the data presented by Jevon et al. and reinforce this hypothesis.
Our data on cytokine production may explain the increase in osteoclast formation and function in PBMC cultures. An increase in TNF- and M-CSF production in PBMC cultures of women after oophorectomy has been previously demonstrated.
We evaluated osteoclast formation in an in vitro culture of PBMC, adding only 1,25-OH vitamin D3 [10–8 M] to determine a possible difference in the sensitivity of osteoclast precursors to this metabolite in patients and controls. We found a significant reduction in osteoclast formation, while there was a significant increase in osteoclast reabsorbing activity adding 1,25-OH vitamin D3. There is a great deal of literature about the use of 1,25-OH vitamin D3 in culture of PBMC. These studies have different results: some authors showed an increased osteoclast formation in PBMC treated with 1,25-OH vitamin D3, while others showed a reduction in osteoclasts activity adding only 1,25-OH vitamin D3. This contrast may be due to different methods of culture, as some authors use coculture between PBMC and an osteoblast line, or addition of cytokines such as M-CSF or RANKL.
The significant inverse correlations between these parameters and BMD are interesting. These findings confirm our hypothesis that there is an increased spontaneous osteoclastogenesis in osteoporotic women with respect to controls; also the level of TNF- and RANKL is inversely related to BMD at lumbar spine and femoral neck, whereas the correlation between these cytokines and cross laps is strictly direct. This datum confirms the role of the production of TNF- and RANKL in PBMC cultures in determining postmenopausal bone loss by increasing osteoclast number and activity. The linear regression models reinforce this hypothesis, lumbar BMD being a predictor of the number of osteoclasts. The lumbar BMD explained 
60% of spontaneous osteoclastogenesis in wells not treated with vitamin D, while in wells treated with vitamin D, the level of RANKL on day 6 explains 60% of osteoclastogenesis. This datum could suggest a mechanism of action of vitamin D that is RANKL dependent.
Our data demonstrate that 1) spontaneous osteoclast formation and activity is increased in osteoporotic patients with respect to healthy controls; 2) there is no difference in PBMC sensitivity to 1,25-OH vitamin D3 in osteoporotic patients compared to controls. Vitamin D in vitro acted as a promoter of osteoclast activity; 3) osteoclast formation in the presence of 1,25-OH vitamin D is explained for 60% by the level of RANKL; this datum could suggest a mechanism of action of vitamin D that is RANKL dependent; 4) the number of osteoclasts is inversely correlated with BMD, and BMD is a predictor of osteoclast formation; and 5) levels of TNF- and RANKL are significantly higher in patients than in controls and are inversely correlated with BMD, while directly correlated with level of cross laps. These data suggest a physiopathological mechanism for postmenopausal bone loss.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2214fje;
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