Teriparatide increases the maturation of circulating osteoblast precursors
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- D’Amelio, P., Tamone, C., Sassi, F. et al. Osteoporos Int (2012) 23: 1245. doi:10.1007/s00198-011-1666-2
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This study shows that teriparatide promotes the circulating osteoblast (OB) precursor degree of maturation in patients affected by postmenopausal osteoporosis.
Anabolic treatment with teriparatide has proven effective for the therapy of postmenopausal osteoporosis and significantly reduces the risk of non-vertebral fragility fractures. The aim of this study was to investigate the effect of teriparatide on circulating OB precursors.
We evaluated by flow cytometry and real-time PCR the expression of OBs typical markers in peripheral blood mononuclear cells during treatment with teriparatide plus calcium and vitamin D, raloxifene plus calcium and vitamin D or calcium and vitamin D alone at various time points. Serum bone alkaline phosphatase and osteocalcin (OC) were measured as markers of bone turnover.
Our results show that circulating OB precursors are more numerous and more immature in patients affected by fragility fractures than in osteoporotic patients without fractures. We also show that teriparatide treatment increases the expression of alkaline phosphatase and of OC in OB precursors; thus, it increases their degree of maturation.
We suggest that teriparatide acts as anabolic agents also by promoting the maturation of OB precursors.
The number of osteoblast (OB) precursor cells circulating in the peripheral human blood has been for longer underestimated due to peculiarity of the designed assays employed by researchers . Recent papers reported that circulating OB precursor cells are present in peripheral human blood in significant number, correlate with typical OB markers, such as osteocalcin (OC) and alkaline phosphatase (AP) and can form nodules in vitro and bone tissue in vivo [1, 2]. The role of these cells during skeletal growth and fracture repair has also been proposed by Eghbali-Fatourechi et al. . These authors observed that OB precursors were over five times more numerous in the circulation of adolescent boys compared to adult men and that they increased after fracture. Otherwise, we recently demonstrated that during fracture healing, there is an important individual variation in the amount of OB and osteoclast precursors; hence, it is not possible to foresee a role for these cells in the callus formation in humans . Kumagai et al. elegantly demonstrated the homing ability of circulating osteogenic cells during fracture healing by a parabiotic mice model . They showed that these cells are physiologically mobilized to contribute to callus formation and fracture repair in mice. Reduction of the circulating OB precursor pool has been described in postmenopausal osteoporosis , whereas our previous study described an increased maturation of these cells during fracture healing ; however, their physiological role in bone metabolism remains to be clarified.
Recent data suggest that postmenopausal osteoporosis may be due not only to an increased osteoclast formation and activity, but also to a reduced OB activity; in particular, ours and other workgroups demonstrated that OB inhibitors are increased in patients affected by fragility fractures [6, 7].
Over the last years, anabolic treatment with teriparatide [rhPTH(1–34)] or with 1–84 PTH has been proposed as therapy for postmenopausal osteoporosis: these drugs significantly reduce the risk of non-vertebral fragility fractures [8, 9]. Anabolic treatment stimulates both trabecular and cortical bone formation, resulting in the improvement of the bone microarchitecture. These changes represent a reversal of osteoporotic bone loss and may explain the vertebral and non-vertebral skeletal effects, including reduction in fracture rates. Furthermore, in contrast to anti-resorptive agents, anabolic treatment preferentially increases bone formation through direct early stimulation of OBs . Previous studies demonstrated that PTH acts directly on OBs by binding the PTH receptor I [11–14]. A recent paper demonstrates that the PTH/PTHrP receptor expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of murine OBs . PTH receptor was also found to be expressed in AP-positive cells in human bone marrow . PTH signaling pathway involves the activation of genes important for the functions of the mature OBs [17–20], increases the number of OBs, decreases their apoptotic rate, and increases their bone-forming activity. Some literature data suggest that among anti-resorptive drugs, the selective estrogen receptor modulator, raloxifene, has some effects on mature OB and, in particular, is able to stimulate OB activity and enhance bone formation in vitro [21, 22] and in vivo .
Nowadays, in literature, there are no available data on the possible effects of teriparatide on the bone marrow output of circulating osteoblast-lineage cells; thus, the present study aims to evaluate the effect of teriparatide in stimulating bone marrow release of osteoblast-lineage cells as compared to calcium and vitamin D supplements and to raloxifene.
The study was approved by the “Clinical Study Review Committee” of the San Giovanni Battista Hospital, Torino, and all patients signed an informed consent statement prior to recruitment. A total of 25 women (65 ± 11 years) were enrolled in this study: 15 women affected by postmenopausal osteoporosis without fractures and 10 with fractures. The presence of diseases that influence bone metabolism was ruled out based on medical history and physical examination. All the women had been in spontaneous menopause for at least 1 year. Bone mineral density (BMD) was measured by double-emission X-ray absorptiometry with a Hologic QDR 4500 (Hologic Inc.) bone densitometer. Secondary osteoporosis was ruled out by means of medical history, physical examination, and blood tests (serum calcium and phosphorus, bone AP, serum protein electrophoresis, and 25-OH vitamin D).
To be included into the study, women have to be after at least 1 year of spontaneous menopause with a BMD T-score value of −2.5 SD or less according to the WHO criteria, with or without fractures. Male subjects, patients with mental incapacity to give the informed consent, patients with secondary osteoporosis, or taking calcium and vitamin D, thyroid hormones, corticosteroids, estrogen, bisphosphonates, parathyroid hormone, fluoride, strontium ranelate, and raloxifene in the previous 6 months or treated with an immunosuppressant (e.g., cyclosporine, azathyoprine) within the previous year, subjects with hypersensitivity or serious adverse experience with raloxifene, teriparatide, or calcium and vitamin D or with a history of cancer were excluded from the study.
Patients without fractures were randomly assigned to treatment with 60 mg/day raloxifene (Evista ®, kindly provided by Ely Lilly SpA Italy, five patients) plus calcium 1,200 mg/day and vitamin D 800 UI/day, or with Teriparatide 20 μg/day (Forsteo ®, kindly provided by Ely Lilly SpA Italy, five patients) plus calcium 1,200 mg/day and vitamin D 800 UI/day, or with calcium 1,200 mg/day, and vitamin D 800 UI/day alone (five patients). Patients with fractures were treated with teriparatide plus calcium and vitamin D. Presence of osteoporotic fracture was defined as the presence of at least a major (hip, vertebral, wrist) fracture due to low-energy trauma. The existence of a fracture was demonstrated by a physician evaluation of X-ray. Calcium and vitamin D supplements (Natecal D3®) were kindly provided by Italfarmaco S.p.A.
Blood was drawn from an antecubital vein after an overnight fast of ten or more hours at baseline and after 15 days, 1, 6, 12, and 18 months of treatment. All measurements were taken from a single sample at a single time point: spine and femoral neck BMD were measured at the end of the study.
Markers of bone turnover
ELISA kits were used to measure serum bone AP (BAP, Instant ELISA; Bender), and serum OC (Instant ELISA; Bender) in duplicate according to standard procedures .
Effect of treatment on OB-circulating precursor
Peripheral blood mononuclear cells (PBMCs) were obtained with the Ficoll-Paque method from 30 ml of blood in lithium heparin, as previously described [24–26]. The serum was stored at −80°C until the measurements were done.
To evaluate the effect of treatment on circulating OB precursors, we measured these cells in the PBMCs at baseline and at different time points after treatment. Briefly, PBMCs were stained with FITC-conjugated anti-CD15 antibody (in order to exclude granulocytes that expressed AP, supplied by e-Bioscience) or with the corresponding isotype control and then incubated for 30 min at 4°C. The cells were fixed for 15 min at 4°C with 1 ml cold 0.1% paraformaldehyde in PBS and then incubated for 15 min at RT with PBS supplemented with 5% saponin (Sigma Aldrich), APC-conjugated anti-AP antibody (supplied by R&D System, Inc), PE-conjugated anti-OC antibody (supplied by R&D System Inc), or with the corresponding isotype control, in accordance to manufacturer’s instruction. Then cells were washed with PBS supplemented with 0.5% saponin. CD15−/AP+/OC+ cells were regarded as OB precursors according to the literature [1–3]. The degree of maturation of OB precursors was evaluated by the mean fluorescence intensity (MIF) of OC. The MIF express the number of molecule present on each cell . The flow cytometry analysis is shown in the supplemental Fig. 1.
To assess the possible effect of teriparatide on stem cell compartment , we evaluated the expression of CD34 and AP on PBMCs according to previous studies [2, 28]. Briefly, freshly isolated PBMCs from five patients were stained with FITC-conjugated anti-CD34 antibody (eBioscience), PE-conjugated anti-OC antibody (R&D System Inc), and APC-conjugated anti-AP antibody (R&D System Inc), or with the corresponding isotype control, and incubated at 4°C for 30 min; the cells were analyzed at baseline after 15 and 30 days.
Membrane antigen expression was analyzed with CellQuest software (Becton Dickinson & Co), and displayed as bivariate dot plots or histograms. The MIF was also recorded. Each plot depicts the results from 100,000 events representing viable cells gated by cell size and granularity.
Gene expression in OB cells
To characterize the expression of OB genes in OC+ cells, we isolated these cells from buffy coats obtained from five healthy donors from the blood bank. Briefly, PBMCs were incubated with normal donkey serum 10% (Jackson ImmunoResearch) and human IgG 10% (FcR Blocking Reagent, Miltenyi Biotec) at RT for 30 min in order to block non-specific binding. Cells were stained with PE-conjugated anti-OC antibody (R&D System Inc.) and then processed with Easy Sep PE selection kit (supplied by StemCell Technology) as described . The cells were processed for RNA extraction and a real-time PCR was carried on.
Expression level of OB genes in OC+ cells were measured performing RT-PCR assay on RUNX-2, Osterix, OC, and alkaline Phosphatase (TNAP). In order to evaluate whether teriparatide affect these genes, their expression was analyzed also in PBMCs from treated patients at each time point.
Total cellular RNA was extracted using TRIzol reagent (Ambion, Huntingdon, UK), chloroform extraction, and subsequent isopropanol precipitation according to the manufacturer’s protocol. One microgram of total RNA was reverse-transcripted to single-stranded cDNA using the Promega ImProm-II™Reverse Transcription System (Promega). cDNA was stored at −20°C until use. RT-PCR was performed with IQ SYBR Green Supermix (BIORAD), gene expression was quantified by using β-actin amplification as housekeeping control and 2−∆∆Ct method for the quantitative analysis. The primers used are reported in the supplemental Table 1. All the genes were quantified by considering signals under 33 Ct.
The normal distribution of each parameter was determined by kurtosis; all except OC MIF were normally distributed. One-way ANOVA was used to compare baseline characteristics among patients with and without fractures, OC MIF was compared by the Mann–Whitney U test; the Wilcoxon’s test was used to compare OC MIF values at baseline and after treatment, whereas the other parameters were compared by means of Student’s paired t test. As patients with fractures were older than patients without fractures, OB precursors amongst these cohorts were compared after weighting cases for age with the specific function of the statistical software used. Correlations between parameters were performed by means of Spearman’s coefficient or with partial correlation after correction for age. The IBM SPSS 17.0 software package was used to process the data with p < 0.05 as the significance cut off.
Mean and standard deviation of baseline features of patients divided according to treatment
Calcium and vitamin D
77 ± 7
60 ± 4
58 ± 5
59 ± 3
Menopausal period (years)
28 ± 12
9 ± 5
9 ± 3
9 ± 4
Body mass index
22.6 ± 2.2
22.9 ± 1.8
22 ± 1.3
23.5 ± 2.1
Lumbar BMD (g/cm2)
0.671 ± 0.08
0.699 ± 0.08
0.718 ± 0.07
0.761 ± 0.25
Femoral neck BMD (g/cm2)
0.520 ± 0.07
0.563 ± 0.1
0.551 ± 0.07
0.555 ± 0.1
29.9 ± 7.5
26.7 ± 6.62
27.1 ± 1.27
27.07 ± 1.8
3.92 ± 1.84
4.39 ± 1.84
4.2 ± 1.34
4.3 ± 1.93
12.21 ± 2.7
8.41 ± 2.6
7.5 ± 1.8
7.52 ± 1.9
1.36 ± 0.5
0.44 ± 0.17
0.36 ± 0.17
0.33 ± 0.17
56.93 ± 9.99
85.8 ± 6.12
84.9 ± 10
83.82 ± 8.55
77.82 ± 2.3
38.79 ± 2.81
39.2 ± 15.2
38.1 ± 16.1
Circulating OB precursors are more immature in fractured than in non-fractured patients
Patients with fractures were older (77 ± 7 years ± SD vs. 58 ± 4 years ± SD, p = 0.000) and with longer postmenopausal period (28 ± 12 years ± SD vs. 9 ± 3 years ± SD, p = 0.000) than patients without fractures; hence, OB precursors were compared between the two groups after weighting cases for age.
Teriparatide increases the maturation of circulating OB precursors
The increase in stem cells compartment does not account for the increase in AP MIF
It has been reported that teriparatide is able to increase stem cells release from bone marrow into the circulation (see  for a review); hence, we evaluated if the early increase in AP MIF could be due to an increase in stem cells. To address this issue, we performed FACS analyses on five patients with fractures treated with teriparatide to evaluate the expression of CD34, AP, and OC in the monocyte compartment as previously described [2, 28]; the baseline characteristics of these patients were comparable to those of the overall teriparatide-treated group. We found no increase in CD15−/CD34+/AP+ and CD15−/CD34+/AP− cell number or in CD34 MIF despite an increase in AP MIF, during the first month of treatment (data not shown). These data confirms that the effect of teriparatide is direct on OB precursor cells, whereas we found no effect on stem cell compartment.
Calcium plus vitamin D and raloxifene treatment do not influence bone formation markers and circulating OB precursors
It is well-known that intermittent treatment with teriparatide is able to reduce fracture risk in postmenopausal osteoporosis acting as a bone anabolic agent , inducing new bone formation and improving bone microarchitecture . Teriparatide promotes bone formation by increasing the number of OBs through multiple effects, including activation of quiescent lining cells , increased OB proliferation [31, 32], differentiation [31, 33], and attenuation of pre-OB and OB apoptosis [10, 34, 35]. However, the specific contribution of each of these effects to the overall anabolic activity of teriparatide remains controversial. A recent study by Rubin MR et al.  suggests that PTH 1–84 stimulates bone formation by increasing the number and the degree of maturation of circulating osteogenic cells in patients affected by hypoparathyroidism. Today, there are no evidences on the possible effect of teriparatide on circulating OB precursors in osteoporosis.
In this study, we showed that OB precursor cells are increased in patients affected by severe form of osteoporosis complicated with fractures and that in these patients, the degree of maturation of the precursors is lower than in osteoporotic patients without fragility fractures. The OC MIF reduction accounts for more immature OB precursors in the circulation of patients with severe osteoporosis and possibly a reduction of their recruitment in bone [3, 5]. The observation of an increased number of circulating OB precursors with low expression of OC corroborates the previous data from Pirro et al. .
After teriparatide therapy, we observed an early increase in the MIF of AP, after only 30 days, whereas the increase in the OC MIF was observed after 6 months of therapy. To investigate whether the early increase in AP was due to a generic-increased release of stem cells from the bone marrow, we evaluated CD34 expression. Our data show that the observed increased AP MIF is not due to a generic increase in stem cell compartment following teriparatide treatment, as previously suggested .
We did not detect an increase in the circulating OB precursor cells, but we observed an increased maturation rate of these cells as shown by an increase in the MIF of AP and of OC. In the recent study by Rubin et al. , an increase in the number and in the maturation of OB precursors in patients with hypoparathyroidism treated with 1–84 PTH has been demonstrated. Our study confirms Rubin’s observation of increased maturation of OB precursors after treatment, but does not show increased OB precursors number. This last result may be due to the different kinds of population enrolled (osteoporotic patients with normal parathyroid function as respect to patients with hypoparathyroidism) and for the treatment used (1–34 PTH vs. 1–84 PTH). These differences may account for the different finding of increased OB precursors in Rubin’s model; on the other hand, our study confirms Rubin’s observation of increased maturation of OB precursors following treatment . As regards to the two control groups, we do not observe significant variation in any of the considered parameters in calcium and vitamin D group nor in the raloxifene group, thus confirming the effect seen in the teriparatide-treated patients.
In order to address the question whether the observed increased expression of OC in OB precursor is accompanied by a general increase in OB gene expression, we performed a quantitative RT-PCR. In the whole PBMCs, we did not find an upregulation of OB gene expression profile following teriparatide treatment. Otherwise, PCR confirmed that OC+ cells in the circulation expressed from 1.5 to 2.0-fold increase in OB genes as respect to OC− cells. These data further confirm that OC+ cells may be interpreted as early OB precursors. The lack of a general increase in OB genes within the PBMCs may be due to the lower and variable number of these cells in peripheral blood.
The increase in bone formation markers in the serum of patients treated with teriparatide is in line with literature data  and is due to the increased bone apposition by OBs present on the bone surface. In our study, the increase in these markers confirms that the drug works and also warrants patient’s compliance to treatment.
Despite some previous data in literature that suggest an effect of raloxifene on bone formation [21–23], we did not observe any significant effect of this treatment on bone formation markers or on circulating OB precursors. The main limitation of the study is the small size of the observed cohort; nevertheless, our results suggest that the increased maturation of OB precursors is one of the anabolic effects of teriparatide.
This work was supported by an unrestricted grant from Ely Lilly and the Fondazione Internazionale Ricerche Medicina Sperimentale (FIRMS) Compagnia San Paolo. SP was supported by a fellowship from the Italian Association for Cancer Research (AIRC); FS was supported by a fellowship of the Regione Piemonte. Teriparatide and Raloxifene were kindly provided by Ely Lilly; calcium and vitamin D supplements were kindly provided by Italfarmaco SpA.
Conflicts of interest