Osteoporosis International

, Volume 21, Issue 2, pp 297–306

Association between circulating osteoprogenitor cell numbers and bone mineral density in postmenopausal osteoporosis

Authors

    • Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental MedicineUniversity of Perugia, Hospital “Santa Maria della Misericordia”
  • C. Leli
    • Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental MedicineUniversity of Perugia, Hospital “Santa Maria della Misericordia”
  • G. Fabbriciani
    • Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental MedicineUniversity of Perugia, Hospital “Santa Maria della Misericordia”
  • M. R. Manfredelli
    • Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental MedicineUniversity of Perugia, Hospital “Santa Maria della Misericordia”
  • L. Callarelli
    • Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental MedicineUniversity of Perugia, Hospital “Santa Maria della Misericordia”
  • F. Bagaglia
    • Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental MedicineUniversity of Perugia, Hospital “Santa Maria della Misericordia”
  • A. M. Scarponi
    • Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental MedicineUniversity of Perugia, Hospital “Santa Maria della Misericordia”
  • E. Mannarino
    • Unit of Internal Medicine, Angiology and Arteriosclerosis Diseases, Department of Clinical and Experimental MedicineUniversity of Perugia, Hospital “Santa Maria della Misericordia”
Original Article

DOI: 10.1007/s00198-009-0968-0

Cite this article as:
Pirro, M., Leli, C., Fabbriciani, G. et al. Osteoporos Int (2010) 21: 297. doi:10.1007/s00198-009-0968-0

Abstract

Summary

The role of circulating osteoprogenitor cells in postmenopausal osteoporosis is unknown. We found that alkaline-phosphatase-positive (AP+) cells are the lacking cells in osteoporosis, whose reduction is related to bone loss. Conversely, the increased number of alkaline phosphatase/CD34-positive cells may reflect the reactive bone marrow contribution to bone formation.

Introduction

Circulating osteoprogenitor cells mineralize in vitro and in vivo. Loss of osteogenic cells may account for bone loss in osteoporosis. We studied whether there is an association between the number of circulating osteoprogenitor cells and bone mineral density (BMD) in postmenopausal women with and without osteoporosis.

Methods

The number of circulating AP+, osteocalcin-positive (OCN+), AP+/CD34+, and OCN+/CD34+ cells was quantified in 54 postmenopausal osteoporotic women and 36 age-matched nonosteoporotic controls.

Results

The number of AP+ cells was lower in osteoporotic women than in controls (127 ± 16 vs 234 ± 23 per microliter; p < 0.001); higher levels of AP+/CD34+, OCN+, and OCN+/CD34+ cells were found in osteoporotic than controls (p < 0.01 for all). The number of AP+ cells was correlated with lumbar BMD (rho = 0.29; p = 0.008) and proximal femur BMD (rho = 0.31; p = 0.005) whereas inverse correlations were found between AP+/CD34+ cells, OCN+, OCN+/CD34+, and BMD. Reduced AP+ cells and increased AP+/CD34 +, OCN+, and OCN+/CD34+ cells were predictors of low BMD, independent of traditional risk factors for osteoporosis.

Conclusion

In postmenopausal osteoporotic women, a reduced number of circulating AP+ cells and increased levels of AP+/CD34+, OCN+, and OCN+/CD34+ cells are associated with reduced bone mineral density, the interpretation of such a cellular imbalance needing exploration.

Keywords

Alkaline phosphataseBMDOsteoblastOsteoporosisOsteoprogenitor cell

Introduction

Bone marrow contains osteoprogenitor cells (OPCs) which contribute to new bone tissue formation [1, 2] and fracture repair [3]. Although most studies have confirmed the contribution of bone resident OPCs to bone formation [13], recent focus has been directed at the possible contribution of osteogenic cells which gain access to bone formation sites through the systemic circulation [4, 5]. In peripheral blood of adult human subjects, 1% to 2% of mononuclear cells express surface antigens that are typical of the osteogenic lineage, like alkaline phosphatase (AP) and osteocalcin (OCN) [4]. Interestingly, these cells form mineralized bone in vitro and in vivo, thus confirming their bone-forming capacity [4]. The prevalence of cells expressing osteocalcin was fivefold greater in adolescent boys than in adults, further suggesting a role of these cells in the process of bone growth [4]. Circulating OPCs have been found to be mobilized to fracture sites and to contribute to osteogenesis in the early stages of fracture healing [6]. Thus, both OPCs resident in bone and circulating OPCs play a relevant role in bone tissue formation and repair.

Bone-related proteins, like alkaline phosphatase or osteocalcin, are currently used to stain circulating OPCs [4, 5, 7]. Recently, CD34, a surface antigen expressed by hematopoietic and endothelial progenitors, has been found to be expressed by both circulating AP-positive and OCN-positive cells [7, 8]. Human peripheral blood CD34-positive cells are recruited to the fracture sites via the systemic circulation where they enhance vasculogenesis and osteogenesis and lead to functional recovery following fracture [9, 10]. Hence, coexpression of bone-related antigens and CD34 may suggest the presence of immature circulating cells which may differentiate both into endothelial and osteogenic cells. Accordingly, bone marrow primitive adult hematopoietic stem cells regenerate the hematopoietic compartment and can differentiate to osteoblasts [11].

Bone loss in women begins before menopause and is accelerated in old age [12]. Osteoporosis, whose prevalence is especially high among elderly postmenopausal women, increases the risk of fractures at any site, thus exposing this population to particularly high morbidity and mortality [13, 14]. It is recommended that all postmenopausal women be assessed for the presence of osteoporosis risk factors [1517]; in addition, it is recommended that all women who possess at least one risk factor for osteoporosis be imaged with a dual-energy X-ray absorptiometry (DXA) to determine those who have low bone density and who are thus at increased risk for fractures [1517]. Though evaluating for traditional risk factors does increase the number of women who are correctly diagnosed with osteoporosis by DXA, it does not capture all women who indeed have osteoporosis leading to undiagnosed and untreated women who are at great risk of fractures [1820]. Thus, there is a need for novel markers which will improve the traditional methods we currently have for predicting who is at risk for osteoporosis.

Pathophysiologically, osteoporosis is defined by an imbalance between bone resorption and bone formation. Accelerated bone resorption by osteoclasts has been established as a key mechanism in osteoporosis; however, recent experimental evidence suggests that inappropriate apoptosis of osteoblasts/osteocytes accounts for, at least in part, the imbalance in bone remodeling which occurs in osteoporosis [2123].

The purpose of the present study was to investigate whether the number of circulating OPCs, expressing the bone-related proteins alkaline phosphatase and osteocalcin and the hematopoietic–endothelial surface antigen CD34, is related to the presence of osteoporosis in postmenopausal women and whether this number correlates with bone mineral density independently of traditional risk factors for reduced bone density.

Materials and methods

Study subjects

The study population consisted of 54 consecutive postmenopausal female outpatients with newly diagnosed and never-treated osteoporosis and 36 age- and sex-matched nonosteoporotic (bone mineral density (BMD) T-score > −1) postmenopausal controls selected among women independent in daily living activities who attended our Unit of Bone and Mineral Metabolism for screening of postmenopausal osteoporosis. Women were considered postmenopausal if they had not been menstruating for at least 1 year. Diagnosis of postmenopausal osteoporosis was based on the presence of a T-score ≤ −2.5 SD at either the lumbar spine, femoral neck, or proximal femur with either the absence or presence of ≥1 self-reported or radiologically documented fragility fracture having occurred at least 6 months prior to study recruitment. Fragility fractures were defined when they occurred without trauma or falling from a standing height or less. Exclusion criteria included history of chronic diseases, such as renal, hepatic, cardiac, and rheumatic diseases, current or prior use of drugs that could interfere with bone mass (i.e., glucocorticoids, antiresorptive drugs, and hormonal replacement therapy), and history of traumatic fractures. A trained interviewer conducted a questionnaire with each participant asking questions regarding their age, age of menopause and menarche, smoking habits, family history of hip fractures, personal history of fragility fractures, daily calcium intake, alcohol consumption, pattern of habitual physical activity, medical history, comorbid diseases, and medication use. Information was also obtained by review of medical records and laboratory data. Calcium intake was quantified according to an Italian validated food frequency questionnaire [24]. Alcohol consumption was documented for wine, spirits, and beer and the weekly consumption of drinks was recorded [25]. Patterns of habitual physical activity were measured according to the modified Baecke questionnaire validated for the elderly [26, 27]. The study was approved by the local Ethics Committee and all participants gave their informed consent.

Clinical evaluation and bone mineral density

All the determinations were made at the medical center at 8:00 h, with a room temperature between 21°C and 23°C, after a 13-h overnight fast. Height and weight were measured to the nearest 0.1 cm and 0.1 kg, respectively; subjects were wearing hospital gowns and had bare feet. Body mass index (BMI) was calculated as weight in kilograms divided by height squared in meters. Brachial blood pressure was measured by a physician with a mercury sphygmomanometer after patients sat for 10 min or longer. The average of three measurements was considered for the analysis.

Areal BMD (g/cm2; bone mineral content relative to projection area) was measured by DXA (Hologic Discovery W, Hologic Inc., Bedford, MA, USA) at the lumbar spine (L1–L4) and the proximal femur. At these measurement sites, the coefficient of variation at our laboratory was 0.49% for the lumbar spine and 0.51% for the proximal femur. Results for areal BMD were transformed to T-scores, calculated as the difference between the actual measurement and the mean value of healthy gender-matched adult controls divided by their standard deviation.

Biochemical assays

Radioimmunoassay was used to measure serum 25-OH vitamin D (DiaSorin Inc., MN, USA). Serum intact parathyroid hormone and estradiol levels were measured by an immunoenzymatic method (Access, Beckman Coulter Inc., CA, USA). The bone-specific isoenzyme of alkaline phosphatase was measured by immunoradiometric assay (Tandem R Ostase, Pantec srl, Torino, Italy). Serum CTX was measured by enzyme-linked immunosorbent assay (Pantec srl, Torino, Italy). Serum osteocalcin was assayed by immunometric method (DPC Immunolite, CA, USA). Total cholesterol, triglycerides, and high-density lipoprotein cholesterol were determined by enzymatic–colorimetric method (Dimension Autoanalyzer; DADE Inc. Newark, NJ); LDL cholesterol was calculated by the Friedewald equation in all participants.

Assay of circulating osteoprogenitor cells

Mononuclear cells were isolated from platelet-depleted peripheral venous blood by density centrifugation (Lymphoprep, Axis-Shield PoC AS, Oslo, Norway). Exclusion of nonviable mononuclear cells was performed by staining with 7-aminoactinomycin D (Beckman Coulter, Inc., Fullerton, CA, USA). Freshly isolated mononuclear cells were incubated for 30 min at 4°C in the dark with biotinylated antibody against human AP (R&D Systems, Minneapolis, USA), PC5-conjugated streptavidin (Beckman Coulter, Inc., Fullerton, CA, USA), fluorescein isothiocyanate (FITC)-conjugated antibody against human CD34 (Beckman Coulter, Inc., Fullerton, CA, USA), ECD-conjugated antibody against CD15 (Beckman Coulter, Inc., Fullerton, CA, USA), and PE-conjugated antibody against intracellular OCN (R&D Systems, Minneapolis, USA), according to manufacturer’s instructions. Anti-CD15 antibody was used to exclude contamination of isolated mononuclear cells with granulocytes. Isotype-identical antibodies at a concentration matched with specific antibodies served as controls (Beckman Coulter, Inc., Fullerton, CA, USA). All the antibodies were titrated to achieve working concentrations. After incubation, quantitative analysis was performed on a Coulter Epics XL measuring 100,000 cells per sample. OPCs were defined by negative staining for CD15 and positive staining for anti-AP, anti-CD34, and anti-OCN. Representative traces of flow cytometry analysis of OPCs stained with isotype and specific antibodies are shown in Figs. 1 and 2. The number of circulating OPCs was calculated by multiplying the frequency of fluorescent-positive events in the gate of lymphomonocytes by the total lymphomonocyte count. Levels of AP+/CD15−/CD34− cells strictly paralleled those of AP+/CD15− cells (rho = 0.99, p < 0.001); thus, only results for AP+/CD15− cells were presented. OPC count in two separate blood samples for each participant (subsample of 30 subjects) was highly reproducible (r = 0.93; p < 0.001).
Table 1

Clinical and biological characteristics of 90 study participants

 

Osteoporotic (N = 54)

Nonosteoporotic (N = 36)

Age, years

66 ± 8

65 ± 6

Age of menarche, years

13 ± 2

13 ± 2

Age of menopause, years

49 ± 5

50 ± 5

Family history of hip fractures, %

27

6*

History of fragility fractures, %

39

0*

Body mass index, kg/m2

24.7 ± 6.7

27.8 ± 6.9*

Smokers, %

26

25

Daily calcium intake, mg

595 ± 261

688 ± 302

Alcohol drinks/week ≥7, %

16

18

Physical activity score

6.5 ± 4.0

7.7 ± 2.8

LDL cholesterol, mg/dL

130 ± 31

109 ± 30*

bAP, μg/L

15.7 ± 6.1

13.2 ± 3.6*

25-OH vitamin D, ng/mL

13.8 ± 9.7

15.0 ± 6.0

sCTx, ng/mL

0.89 ± 0.6

0.65 ± 0.3

Parathormone, pg/mL

65.1 ± 39.8

59.2 ± 27.4

Osteocalcin, ng/mL

7.97 ± 5.8

5.50 ± 3.7*

Estradiol, pg/mL

8.7 ± 9.4

24.8 ± 37.1*

Proximal femur BMD, g/cm2

0.70 ± 0.11

0.98 ± 0.09*

Lumbar spine BMD, g/cm2

0.71 ± 0.13

0.95 ± 0.09*

Proximal femur T-score, SD

−1.9 ± 0.9

0.3 ± 0.7*

Lumbar spine T-score, SD

−3.09 ± 1.1

−0.8 ± 0.6*

Values are mean ± SD

LDL low-density lipoprotein, bAP bone-specific alkaline phosphatase, sCTX serum C-terminal telopeptide of type I collagen, BMD bone mineral density

*p < 0.05 for comparison between osteoporotic and non osteoporotic postmenopausal women

Table 2

Effects of staurosporine 1 μM on occurrence of annexin-V immunofluorescence staining among different osteoprogenitor cells

 

Control

Stauro (2 h)

Stauro (4 h)

Stauro (6 h)

Stauro (8 h)

Stauro (24 h)

AP+/CD34−/annexin+a

0.8 ± 0.07

11.3 ± 0.9

14.8 ± 0.8

16.3 ± 0.8

17.0 ± 0.7

32.7 ± 1.9

AP+/CD34+/annexin+b

0.08 ± 0.01

0.09 ± 0.01

0.09 ± 0.01

0.1 ± 0.01

0.09 ± 0.01

19.6 ± 2.0

OCN+/CD34−/annexin+c

0.1 ± 0.02

0.9 ± 0.07

1.0 ± 0.07

1.1 ± 0.07

1.2 ± 0.02

9.1 ± 1.2

OCN+/CD34+/annexin+d

0.4 ± 0.1

0.8 ± 0.1

1.0 ± 0.1

1.2 ± 0.08

1.1 ± 0.07

8.0 ± 0.9

PBMCs/annexin+

0.06 ± 0.01

1.4 ± 0.7

2.9 ± 0.7

6.0 ± 0.7

15.7 ± 2.1

46.4 ± 4.1

Values are mean ± SEM (results are representative of three individual experiments performed in duplicate)

AP alkaline phosphatase, OCN osteocalcin, Stauro staurosporine, PBMCs peripheral blood mononuclear cells

a Percent of AP+/CD34− cells

bPercent of AP+/CD34+ cells

cPercent of OCN+/CD34− cells

dPercent of OCN+/CD34+ cells

Assay of in vitro apoptosis of osteoprogenitor cells

After the mononuclear cell fraction was isolated from three healthy volunteers on a Lymphoprep gradient (Axis-Shield PoC AS, Oslo, Norway), AP+ cell selection was performed by immunomagnetic sorting with the miniMACS system (Miltenyi Biotec, Germany) after two consecutive elutions, according to the manufacturer’s instructions. Viability of the eluted AP+ cells was measured by propidium iodide staining and trypan blue exclusion test, showing that 98% of the AP+ cells were viable. Phenotypic characterization of the AP+ cell suspension was assessed by measuring the coexpression of CD34, by using PE-conjugated antibody against human CD34 (Beckman Coulter, Inc., Fullerton, CA, USA), and annexin-V, by using a FITC-conjugated-specific antibody (Beckman Coulter, Inc., Fullerton, CA, USA). Immediately after selection, aliquots from each volunteer of 10 × 105 AP+ cells were plated with Roswell Park Memorial Institute (RPMI) medium on 24-well plates. AP+ cells were plated either with or without adding to the culture medium aliquots of staurosporine (1 µM) and the occurrence of annexin-V immunofluorescence staining after 2-, 4-, 6-, 8-, and 24-h treatment among AP+/CD34− and AP+/CD34+ cells was evaluated. The effect of staurosporine was also tested on OCN+/CD34− and OCN+/CD34+ cells. For this purpose, the mononuclear cell fraction was isolated from the same three healthy volunteers on a Lymphoprep gradient after platelet depletion; 10 × 106 peripheral blood mononuclear cells were placed on 24-well plates with RPMI with either the addition or not of staurosporine 1 µM. The occurrence of annexin-V immunofluorescence staining after 2-, 4-, 6-, and 8-h treatment among OCN+/CD34− and OCN+/CD34+ cells was evaluated. The 1-µM staurosporine concentration was selected after testing several working concentrations (0.05, 0.1, 0.2, 0.5, 1 µM). The dose response (0.1, 0.2, 0.5, 1 µM) of 8-h staurosporine-induced apoptosis was determined by counting the percentage of annexin-V-positive events among different OPC subclasses (AP+/CD34−, AP+/CD34+, OCN+/CD34−, OCN+/CD34+) and peripheral blood mononuclear cells. Results are representative of three individual experiments performed in duplicate.

Statistical analysis

SPSS statistical package, release 10.0 (SPSS Inc, Chicago, IL, USA) was used for all statistical analyses. Values are expressed as the mean ± SD or SEM. Independent-sample t test and Wilcoxon rank sum test were used to compare the study variables between osteoporotic patients and control subjects. Correlation analyses were performed using the Pearson’s and Spearman’s coefficients of correlations (r and rho, respectively) in the merged groups of osteoporotic and nonosteoporotic participants. Logarithmic transformation to base e was used for nonparametric variables. Multiple linear regression analysis was used to estimate prediction of BMD by including the following independent variables in the model: age, ages related to menstrual history (menopause and menarche), smoking status, body mass index, familial history of hip fractures, personal history of fragility fractures, calcium intake, alcohol consumption, pattern of habitual physical activity (low, moderate, high), and OPCs. Standardized coefficients were calculated as a measure for the relative predictive value. Logistic regression analysis was performed for prediction of the osteoporotic status (osteoporotic vs nonosteoporotic) by including those independent variables indicated in the multiple-regression analysis. Statistical significance was assumed if a null hypothesis could be rejected at p = 0.05.

Results

The characteristics of 90 postmenopausal women, 54 with newly diagnosed untreated osteoporosis and 36 age-matched nonosteoporotic controls, are summarized in Table 1. Osteoporotic patients differed from control subjects with respect to family history of hip fractures and personal history of fragility fractures, which were both more frequent among osteoporotic subjects. Subjects with osteoporosis also had lower BMI, higher LDL cholesterol, bone-specific AP, and lower serum estradiol levels and BMD at lumbar spine and femur. Figure 3 illustrates the number of circulating AP+/CD15− and AP+/CD15−/CD34+ cells among osteoporotic patients and controls; osteoporotic women had a significant lower number per microliter of circulating AP+/CD15− cells than nonosteoporotic controls (127 ± 16 vs 234 ± 23; p < 0.001), while an increased number per milliliter of AP+/CD15−/CD34+ cells (2,728 ± 764 vs 656 ± 117; p = 0.004). Similarly, after exclusion of women with a personal history of fragility fractures, osteoporotic women still had a lower number per microliter of circulating AP+/CD15− (136 ± 24 vs 234 ± 23; p = 0.004) and a higher number per milliliter of AP+/CD15−/CD34+ cells (2,364 ± 994 vs 656 ± 117; p = 0.04) than nonosteoporotic controls. Figure 4 illustrates the number of circulating OCN+ and OCN+/CD34+ cells among osteoporotic patients and controls. Figure 5 shows the direct correlation of AP+/CD15− cells with lumbar spine BMD (rho = 0.29; p = 0.008, panel A) and proximal femur BMD (rho = 0.31; p = 0.005, panel B). Significant inverse correlates of proximal femur BMD included AP+/CD15−/CD34+ (rho = −0.33; p = 0.003), OCN+ (rho = −0.39; p < 0.001), and OCN+/CD34+ cells (rho = −0.39; p < 0.001). AP+/CD15−/CD34+ cells were also positively associated with serum osteocalcin (rho = 0.36; p = 0.002) and bone alkaline phosphatase (rho = 0.26; p = 0.02) and negatively with serum estradiol levels (rho = −0.44; p < 0.001). OCN+ and OCN+/CD34+ cells were positively correlated with serum osteocalcin (rho = 0.30 and 0.23, respectively; p < 0.05 for both). Participants were then divided into two groups according to a personal history of fragility fractures (yes vs no); we found a significantly lower number per microliter of circulating AP+/CD15− and a higher number per milliliter of AP+/CD15−/CD34+ cells in women with fragility fractures (113 ± 20 and 3,394 ± 1,260, respectively) compared to nonfractured postmenopausal women (187 ± 18 and 1,473 ± 486, respectively; p < 0.05 for both comparisons). Women with fragility fractures also had higher numbers of OCN+ and OCN+/CD34+ cells than nonfractured participants (71 ± 28 per microliter and 16,257 ± 4,557 per milliliter vs 33 ± 7 per microliter and 3,288 ± 657 per milliliter).
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Fig. 1

Representative traces of flow cytometry analysis of osteoprogenitor cells stained with isotype (upper panels) and specific antibodies anti-CD15, anti-AP, and anti-CD34 (lower panels). Lower panels show, from left to right, the exclusion of CD15-positive events (left panel), the selection of AP-positive events among the gated CD15 negative events (middle panel), and the results of double staining with anti-AP and anti-CD34 antibodies (right panel). In the latter panel, the AP-positive events are those resulting from the previous AP-positive selection, whereas the AP-negative events were also shown to appreciate the population of CD34-positive events that are AP negative

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Fig. 2

Representative traces of flow cytometry analysis of osteoprogenitor cells stained with isotype (upper panel) and specific antibodies anti-OCN and anti-CD34 (middle and lower panels)

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Fig. 3

Number of circulating AP+/CD15− and AP+/CD15−/CD34+ osteoprogenitor cells among osteoporotic patients and nonosteoporotic controls

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Fig. 4

Number of circulating OCN+ and OCN+/CD34+ osteoprogenitor cells among osteoporotic patients and nonosteoporotic controls

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Fig. 5

Correlations of AP+/CD15− osteoprogenitor cells with lumbar spine BMD (a) and proximal femur BMD (b)

In order to identify variables independently associated with BMD, we performed a multivariate linear regression analysis including the following independent variables in the model: age, age of menopause and menarche, smoking status, body mass index, family history of hip fractures, personal history of fragility fractures, calcium intake, alcohol consumption, pattern of habitual physical activity, and log-OPCs. BMI (beta = 0.57; p < 0.001), personal history of fragility fractures (beta = −0.36; p = 0.006), and log-number of AP+/CD15− cells (beta = 0.27; p = 0.008) were independently associated with proximal femur BMD, with a model R-squared of 0.62. The multivariate model R-squared was 0.55 after exclusion of the AP+/CD15− cell count. The multivariate model R-squared was 0.65 after inclusion of both the AP+/CD15− and AP+/CD15−/CD34+ cell count; interestingly, the numbers of both AP+/CD15− cells (beta = 0.29; p = 0.003) and AP+/CD15−/CD34+ cells (beta = −0.19; p = 0.04) were still associated with BMD independently from all the other risk factors of osteoporosis. Similarly, log-OCN+ and log-OCN+/CD34+ were negatively associated with proximal femur BMD (beta = −0.32, p = 0.002 and beta = −0.30, p = 0.005, respectively), but the power of this association was lessened to a level of nonsignificance by forcing in the multivariate model the log-AP+/CD15− count. Inclusion of humoral markers of bone turnover, time since menopause, and estradiol levels in the multiple-regression model did not affect the above-mentioned results. In the logistic regression model with osteoporotic status as categorical dependent variable (yes vs no), reduced BMI and low number of AP+/CD15− cells were still significantly associated with the presence of osteoporosis (p < 0.05 for both), and there was a suggestion for an independent positive association for the AP+/CD15−/CD34+ (p = 0.07) and the OCN+ (p = 0.054) cell count.

The results of the in vitro substudy are presented in Table 2. Time course of apoptosis induction by 1 μM staurosporine showed that AP+/CD34− cells underwent extensive early apoptosis in vitro after exposure to staurosporine, with higher annexin-V immunofluorescence staining of AP+/CD34− cells than AP+/CD34+ cells at all time intervals. Conversely, neither OCN+/CD34− nor OCN+/CD34+ cells stained significantly with annexin-V even after 8-h exposure to staurosporine; comparable levels of apoptosis were observed among OCN+/CD34− and OCN+/CD34+ cells after 24-h exposure to staurosporine. Dose response of apoptosis induction determined 8 h after addition of 0.1, 0.2, and 0.5 μM staurosporine showed extremely low annexin-V immunofluorescence staining of all OPC subpopulations (<1.5%); at 1-μM staurosporine concentration, significant annexin-V immunofluorescence staining was observed among AP+/CD34− cells (17.0 ± 0.7%), with low annexin-V immunofluorescence staining of the other OPC subpopulations (<1.5% for AP+/CD34+, OCN+/CD34−, and OCN+/CD34+ cells). Among peripheral blood mononuclear cells, dose response of apoptosis induction by staurosporine showed a progressive increase in the percentage of annexin-V-positive events (0.06 ± 0.02, 1.4 ± 0.04, 4.9 ± 0.07, and 14.3 ± 1.4 at 0.1-, 0.2-, 0.5-, and 1-μM staurosporine concentrations, respectively).

Discussion

Current guidelines underline the importance of assessing for risk factors for osteoporosis in all postmenopausal women and recommend BMD measurements with DXA in subjects at increased risk [1517]. However, only a limited percentage of subjects with risk factors for osteoporosis have a bone density test performed [20]. Moreover, assessment of traditional risk factors alone often results in a large number of unnecessary DXA referrals; it results in a high fraction of subjects who are tested because they have one or more traditional risk factors and have normal BMD [28, 29]. Thus, novel markers aimed at identifying patients who would benefit from BMD measurement are needed. Accumulating evidence suggests that circulating OPCs contribute to osteogenesis [46] and that inappropriate apoptosis of osteoblasts accounts for, at least in part, the imbalance in bone remodeling as occurs in the osteoporotic animal model [22, 23]. We observed that circulating OPCs, expressing the bone-related protein alkaline phosphatase, are markedly reduced in women with postmenopausal osteoporosis and are positively correlated with BMD. Interestingly, this association is independent of the presence of traditional risk factors for reduced BMD. To our knowledge, this is the first observation of a positive relationship between circulating AP-positive OPCs and bone density in humans, and of a possible role of a deficiency of these cells in the pathophysiology of osteoporosis. Accordingly, defects in telomere maintenance molecules impair osteoblast differentiation and promote osteoporosis in mice [30]. Hence, the results of the present study might support the role of circulating AP-positive cells in maintaining bone density. This hypothesis is in line with previous reports of a contribution in vitro to mineralization of bone marrow [31], periosteal [32], and circulating AP-positive cells [4].

In contrast to AP-positive OPCs, those OPCs expressing both AP and CD34 surface antigens as well as those expressing OCN were higher in postmenopausal osteoporosis than in control subjects and correlated negatively with bone mineral density. The reason for the increased number of AP/CD34-double-positive cells and OCN-positive cells is not inferable from the results of the present study. However, resistance to apoptosis of these OPCs might be hypothesized according to the results of the in vitro substudy, showing that annexin-V staining among AP+/CD34+ and OCN+ cells occurs only at a limited extent. Information for the understanding of the role of AP/CD34-positive cells in osteoporosis may be deduced also from current literature. Firstly, previous studies have demonstrated the ability of CD34+ cells to differentiate into osteoblasts [33] and the ability of CD34+ cells expressing bone-related proteins to participate in mineral deposition and bone repair after fracture [9, 33]. Secondly, the existence of a primitive cell which is able to generate both hematopoietic and osteocytic lineages has been demonstrated [11, 34]. Finally, CD34 expression is lost during osteoblast differentiating conditions [33]. Overall, these data suggest that AP+/CD34+ cells represent a more primitive population than AP+ cells, contributing to bone formation and vasculogenesis, both processes which are necessary for bone remodeling. Thus, the increase in the number of AP+/CD34+ in postmenopausal osteoporotic women might indicate an attempt of the bone marrow to mobilize immature bone-marrow-derived cells with hemopoietic–endothelial–osteogenic potential. However, increased availability of more immature OPCs (AP+/CD34+) is not paralleled by a concomitant increase of AP+/CD34− cells, possibly as a consequence of either an insufficient maturation of AP+/CD34+ cells into AP+/CD34− cells or a selective loss of circulating AP+/CD34− cells (Fig. 6). The latter hypothesis might be supported by the in vitro results of the present study, showing that AP+/CD34− are more prone in vitro to apoptosis than AP+/CD34+ cells.
https://static-content.springer.com/image/art%3A10.1007%2Fs00198-009-0968-0/MediaObjects/198_2009_968_Fig6_HTML.gif
Fig. 6

Hypothetical mechanism leading to reduced bone mineral density in postmenopausal osteoporotic women

Although simply quantifying peripheral OPCs does not explain the reason and the mechanism of their number in osteoporosis, our findings suggest that AP+/CD15− cells are the reduced cell lineage in osteoporosis, whose quantitative reduction is closely related to bone mineral loss. Interestingly, this marker is poorly associated with traditional risk factors for osteoporosis and provides additional and independent predictive power for reduced bone mineral density. Accordingly, multiple regression and logistic regression confirmed a reduced number of AP+/CD15− cells as an independent covariate of both BMD and osteoporotic status. Moreover, the independent association of AP+ cells with BMD seems to be stronger than that of OCN+ cells; accordingly, in the multivariate model including both AP+/CD15− and OCN+ cells, only AP+/CD15− continued to significantly predict BMD and osteoporotic status.

Recent traumatic bone fractures have been associated with a progressive increase in the number of circulating OPCs both in human and animal models [36]. Thus, the presence in our study of osteoporotic women with fragility fractures may have had an influence on the difference in the number of OPCs between osteoporotic subjects and healthy controls. However, after excluding osteoporotic women with fragility fractures, osteoporotic women without fractures still had a lower number of AP+/CD15− cells and a greater number of AP+/CD15−/CD34+ and OCN+ cells than nonosteoporotic controls. The presence of osteoporotic women with nonrecent fragility fractures allowed us to show that women with osteoporotic fractures have lower number of AP+/CD15− cells in comparison to both osteoporotic women without fractures and nonosteoporotic women. Thus, a suggestion for a possible role of AP+/CD15− cells in prediction of fragility fractures may be hypothesized, although higher statistical power is needed to answer this relevant issue.

Limitations of our study have to be acknowledged. Firstly, although univariate and multivariate correlation analyses provide a suggestion regarding the possible role of OPCs in BMD reduction, our approach is only hypothesis generating, and markers of BMD need to be validated in specific in vitro and prospective studies. Particularly, the divergent association between either AP+ or OCN+ and AP+/CD34+ cells and BMD was not supported by an in vitro demonstration of a different osteoblastogenic potential of all these cell subpopulations. Hence, our results must be considered preliminary and need to be confirmed. Secondly, the observational design of this study does not allow us to reach conclusions on the mechanism of perturbed OPC number in postmenopausal osteoporotic women, although different susceptibility to apoptosis might in part explain the imbalance of OPC subpopulations. Clearly, it would be desirable to demonstrate in a larger trial that low AP+/CD15− cell number may be reversed with consequent BMD increase. Thirdly, any inference on the potential relationship between OPCs and fragility fractures should be taken with caution, given that the latter finding has been obtained in a small underpowered subgroup of patients. Finally, examination of more primitive osteoblastic progenitors and mesenchymal stem cells done by staining for CD133 and STRO-1 would certainly give additional information on the relationship between sources and stages of differentiation of different OPCs and bone health.

In conclusion, alkaline-phosphatase-positive cells may represent a reduced cell lineage in osteoporosis, whose quantitative reduction is closely related to bone mineral loss; alkaline-phosphatase/CD34-double-positive cells may represent more immature cells that are mobilized from the bone marrow in an attempt to contribute to bone formation. A further understanding of the balance between different OPCs in osteoporosis is of extreme importance for the development of more effective therapies aimed towards restoring a physiological bone mineral density.

Acknowledgments

The authors thank Iliana Lega, MD, for her assistance in the preparation of the manuscript.

Conflicts of interest

None.

Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2009