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. Author manuscript; available in PMC: 2014 Jul 7.
Published in final edited form as: Cell Stem Cell. 2010 Nov 5;7(5):571–580. doi: 10.1016/j.stem.2010.09.012

Inhibition of Sca-1-Positve Skeletal Stem Cell Recruitment by Alendronate Blunts the Anabolic Effects of Parathyroid Hormone on Bone Remodeling

Xiangwei Wu 1,2,3, Lijuan Pang 1,2,3, Weiqi Lei 1, William Lu 4, Jun Li 1,2,3, Zhaoyang Li 4, Frank J Frassica 1, Xueling Chen 1,3, Mei Wan 1, Xu Cao 1,*
PMCID: PMC4084813  NIHMSID: NIHMS242267  PMID: 21040899

SUMMARY

The anabolic effects of parathyroid hormone (PTH) on bone formation are impaired by concurrent use of anti-resorptive drugs. We found that the release of active transforming growth factor (TGF)-β1 during osteoclastic bone resorption is inhibited by alendronate. We showed that mouse Sca-1-positive (Sca-1+) bone marrow stromal cells are a skeletal stem cell subset, which are recruited to bone remodeling sites by active TGF-β1 in response to bone resorption. Alendronate inhibits the release of active TGF-β1 and the recruitment of Sca-1+ skeletal stem cells for the bone formation. The observation was validated in a Tgfb1−/− mouse model, in which the anabolic effects of PTH on bone formation are diminished. The PTH-stimulated recruitment of injected mouse Sca-1+ cells to the resorptive sites was inhibited by alendronate. Thus, inhibition of active TGF-β1 release by alendronate reduces the recruitment of Sca-1+ skeletal stem cells and impairs the anabolic action of PTH in bone.

INTRODUCTION

Current therapies for osteoporosis and the prevention of osteoporotic fractures include the inhibition of osteoclastic bone resorption by anti-resorptive drugs, such as alendronate and risedronate (Liberman et al., 1995; Cranney et al., 2002a; Cranney et al., 2002b), and the stimulation of osteoblastic bone formation by parathyroid hormone (PTH) (Neer et al., 2001; Kurland et al., 2000; Orwoll et al., 2003). Concurrent use of anti-resorptive agents and PTH was expected to be more effective because this approach would be expected to reduce bone loss and to stimulate new bone formation. In clinical trials of concurrent PTH and alendronate, however, the anabolic effects of PTH were impaired by the anti-resorptive agent alendronate (Finkelstein et al., 2010; Finkelstein et al., 2003; Black et al., 2003). This finding suggests that osteoclastic bone resorption is necessary for PTH-induced bone formation but the mechanisms underlying this effect are obscure. An improved understanding of the role that bone resorption plays in PTH-induced anabolic bone formation would provide a mechanistic rationale for the development of strategies that permit the effective use of both PTH and anti-resorptive drugs in the treatment of osteoporosis.

In the adult skeleton, bone is remodeled constantly via bone resorption by osteoclasts and bone formation by osteoblasts occurring throughout life (Bonnick, 2006; Iqbal, 2000; Raisz, 2005; Zaidi, 2007). Normally, these effects are balanced, but in some situations, such as aging or certain pathological conditions, bone resorption exceeds bone formation and there is net bone loss (Teitelbaum, 2000; Riggs, 1991; Parfitt, 1982). In the remodeling cycles, bone formation occurs at newly formed resorptive sites and maintains the bone microarchitecture and its mechanical properties (Hill, 1998). Bone marrow stroma is composed primarily of non-hematopoietic stromal cells (BMSCs), a subset of which is multipotent, able to differentiate into osteoblasts, chondrocytes, stromal cells that support hematopoiesis, and marrow adipocytes. The term “skeletal stem cells” has been suggested for bone marrow-derived, multipotent and self-renewing stromal cells capable of generating skeletal cell types in vivo (Bianco et al., 2008). The bone formation is achieved by murine Sca-1-positive (Sca-1+) BMSCs that are recruited to the bone resorptive sites by the release of factor(s) during osteoclastic bone resorption, e.g., the active form of transforming growth factor (TGF)-β1 (Tang et al., 2009). This TGF-β1-mediated coupling process is essential for balancing bone resorption and formation (Tang et al., 2009). In the current study, we investigated the role of the release of active TGF-β1 during osteoclastic bone resorption on the anabolic effects of PTH on bone formation.

RESULTS

The Effects of Combined Use of PTH and Alendronate on Bone Formation Are Not Additive

To investigate the cellular mechanism responsible for the impaired anabolic effects of PTH on bone formation during combined therapy with anti-resorptive drugs, we analyzed mice at an age when the bone mass is in decline but active bone remodeling is still occurring (Figure S1) (Cao et al., 2003; Beamer et al., 1996; Watanabe and Hishiya, 2005). The mice were injected with the vehicle, PTH, alendronate, or pretreatment with alendronate followed by concurrent use of PTH. The bone mass was estimated by microcomputed tomography (μCT) analysis of the proximal tibia trabecular bone (Figure 1A). Compared to treatment with the vehicle, treatment with PTH or alendronate alone stimulated an increase in trabecular bone mineral density (TBMD), but additive effects on TBMD were not observed in mice treated with both drugs (Figure 1B). The trabecular bone volume fraction (TBV/TV), thickness (Tb.Th) and number (Tb.N) were higher in mice treated with PTH or alendronate alone than those treated with the vehicle, but again additive effects were not seen in the mice treated with both drugs (Figures 1C–1E). These results suggest that the combined administration offers no benefit over and above that achieved by PTH alone.

Figure 1. Effects of PTH Combined with Alendronate on Trabecular Bone Formation during Bone Remodeling in Mice.

Figure 1

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(A) Representative three-dimensional μCT images of proximal tibiae from 8-month-old mice injected with vehicle (Veh), PTH (1-34), alendronate (Aln), and combination of PTH and alendronate (PTH+Aln). Scale bar represents 1 mm.

(B–E) Quantitative μCT analysis of the secondary spongiosa of proximal tibiae. Trabecular volumetric bone mineral density (TBMD) (B), trabecular bone volume fraction (TBV/TV) (C), trabecular thickness (Tb.Th) (D), and trabecular number (Tb.N) (E) were measured (n = 10 for each treatment group. *p < 0.05. n.s., not significant) Data are presented as mean ± SEM.

See also Figure S1.

PTH-induced Recruitment of BMSCs to Bone Remodeling Sites Is Inhibited by Alendronate

To investigate whether the PTH-induced osteogenic potential of BMSCs is affected by the combined treatment with alendronate, we isolated bone marrow cells from the mice treated as described above, plated them at clonal density with irradiated guinea pig marrow feeder cells to optimize the colony forming efficiency, and counted the number of colony forming unit-fibroblast (CFU-F). The isolated bone marrow cells were also induced in osteogenic medium, and the number of colony forming unit-osteoblast (CFU-Ob) was counted. There was no significant difference in the number of CFU-Fs in the bone marrow cells isolated from the different treatment groups. The number of CFU-Obs was higher in the mice treated with PTH alone than that treated with the vehicle, and alendronate did not inhibit the PTH-stimulated increase in the number of CFU-Obs (Figures 2A–2C), indicating that the PTH-induced osteoblast differentiation potential of BMSCs was not affected by alendronate. We then measured the effects of PTH and alendronate on the number of osteoblasts and osteoclasts associated with trabecular bone during remodeling. The number of osteoclasts was significantly higher in PTH-treated mice than in mice treated with the vehicle or alendronate. The number of osteoclasts in mice treated with the combination was lower than that in mice treated with PTH alone; the number was similar to that observed when alendronate was used alone (Figures 2D and 2F). Similarly, the number of osteoblasts in mice treated with PTH alone was higher than that in mice treated with the vehicle, alendronate alone, or both (Figures 2E and 2G). As a result, PTH-stimulated bone formation rate and osteoid volume were inhibited by alendronate (Figures 2H and 2I). These results suggest that the alendronate-associated reduction in the number of osteoblasts in the PTH-treated mice may result from an alendronate-associated interruption in the recruitment of BMSCs during bone remodeling because the PTH-induced osteoblast differentiation potential of BMSCs was not affected by alendronate.

Figure 2. Reduction of Osteoblast Number in Trabecular Bone of Mice with Combined Treatment of PTH(1-34) and Alendronate.

Figure 2

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(A) Colonies formed from harvested bone marrow of the mice as indicated in CFU-F and CFU-Ob assays (1×105 bone marrow nucleated cells were plated into six-well plates). The top panels show six-well plates containing CFU-Fs stained with methanol green. The bottom panels show six-well plates containing CFU-Obs stained with Alizarin Red.

(B, C) The colony-forming efficiency was determined by the number of colonies per 105 marrow cells plated. Data are represented as mean ± SEM of triplicate cultures of 0.1, 0.5, and 1×106 bone marrow nucleated cells from five individual mice (*p < 0.05. n.s., not significant).

(D, E) Light micrographs of tartrate-resistant acid phosphatase (TRAP) staining (D) and Goldner’s Trichrome-staining (E) performed on trabecular bone sections from distal femora of mice as indicated. Black arrowheads indicate the osteoid surface. Scale bars represent 50 μm.

(F–I) Bone histomorphometric analysis of trabecular bone of the secondary spongiosa from the mice as indicated. Number of osteoclasts per tissue area (N.Oc/T,Ar) (F), number of osteoblasts per tissue area (N.Ob/T.Ar) (G), bone formation rate per bone surface (BFR/BS) (H), and osteoid volume per bone volume (OV/BV) (I) were measured (n = 10 for each treatment group. *p < 0.05, **p < 0.01. n.s., not significant). Data are presented as mean ± SEM.

Alendronate Inhibits Release of Active TGF-β1 during Osteoclastic Bone Resorption

We have shown that active TGF-β1 released during osteoclastic bone resorption induces migration of BMSCs to the bone remodeling sites (Tang et al., 2009). We therefore examined whether the inhibition of osteoclastic bone resorption by alendronate affected the release of active TGF-β1. The levels of active TGF-β1, as measured by enzyme-linked immunosorbent assay (ELISA), were significantly higher in the bone marrow of mice treated with PTH than in those treated with the vehicle or alendronate alone (Figure 3A). The combined treatment resulted in a reduction in the levels of active TGF-β1, but not in the levels of total TGF-β1 (Figures 3A and 3B), suggesting that alendronate inhibited activation of TGF-β1. The levels of active and total TGF-β1 in the sera were similar in all the treatment groups (Figures 3C and 3D). To confirm the observation, we measured the levels of phosphorylated Smad2/3 (p-Smad2/3) in the bone marrow cells because TGF-β1 induces phosphorylation of Smad2/3 as its major signaling pathway (Macias-Silva et al., 1996; Eppert et al., 1996; Lagna et al., 1996; Zhang et al., 1996). We found that the PTH-induced enhancement of p-Smad2/3 in the bone marrow cells was inhibited by combined treatment with alendronate (Figures 3E and 3F). The levels of p-Smad2/3 in the alendronate-treated bone marrow cells were also lower relative than those in the vehicle-treated mice (Figures 3E and 3F). Histological analysis of mature osteoclasts and p-Smad2/3-positive (p-Smad2/3+) cells in the femur trabecular bone revealed a high number of p-Smad2/3+ cells in the microenvironment of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in mice treated with vehicle (Figure 3G), indicating the release of active TGF-β1 during osteoclastic bone resorption. The number of PTH-induced p-Smad2/3+ cells was reduced significantly on combined use of alendronate, and treatment with alendronate alone also resulted in a reduction in the number of p-Smad2/3+ cells as compared to treatment with vehicle (Figures 3G and 3H). The reduction in the number of p-Smad2/3+ cells showed a positive correlation with the reduction in the number of osteoclasts (Figures 3G and 3H). Notably, the levels of active TGF-β1 and the number of p-Smad2/3+ cells after the combined treatment were significantly increased compared to treatment with alendronate alone, most likely due to the stimulation of TGF-β1 expression by PTH, but the increased p-Smad2/3+ cells were not in the locale of osteoclastic resorption. Taken together, these results indicate that alendronate inhibits the release of active TGF-β1 and does so through inhibition of osteoclastic bone resorption.

Figure 3. PTH-induced Release of Active TGF-β1 during Bone Remodeling Is Inhibited in Mice with Combined Use of Alendronate.

Figure 3

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(A–D) The amount of active and total TGF-β1 in the bone marrow (A, B) and serum (C, D) were measured by ELISA (n = 5 for each treatment group. *p < 0.05, **p < 0.01. n.s., not significant).

(E, F) Western blot analysis of the levels of p-Smad2/3 and Smad2/3 in the bone marrow cells. Fold changes of mean pixel of p-Smad2/3 to Smad2/3 were measured (n = 3 for each treatment group. *p < 0.05).

(G) Images of distal femora sections from the mice, as indicated, were co-stained with TRAP (purple) and p-Smad2/3 antibody (brown). b, bone. Scale bar represents 100 μm.

(H) Counts of p-Smad2/3+ cells in total tissue area and on osteoclast surface (N.p-Smad2/3+ cells/T.Ar, Number of p-Smad2/3+ cells per tissue area) (n = 10 for each treatment group. *p < 0.05. n.s., not significant).

Data are presented as mean ± SEM.

PTH-induced Bone Formation during Bone Remodeling Is Inhibited in Tgfb1−/− Mice

To investigate whether the alendronate-induced inhibition of release of active TGF-β1 impairs the anabolic action of PTH in bone, Tgfb1−/− mice and their wild-type littermates (Tgfb1+/+) were injected intermittently with PTH, alendronate or the vehicle for 4 weeks, and the anabolic effects of PTH on bone remodeling were examined. As estimated by μCT analysis of the proximal tibia trabecular bone, the three-dimensional bone parameters (including TBMD, TBV/TV, Tb.Th, and Tb.N) were significantly increased in the Tgfb1+/+ mice injected with PTH or alendronate relative to the mice injected with the vehicle (Figures 4A–4E). However, the effects of PTH on bone remodeling were diminished in the Tgfb1−/− mice compared to their wild-type littermates, whereas the effects of alendronate on bone formation were not affected in the absence of TGF-β1 (Figure 4A). As shown, PTH failed to increase TBMD and even reduced TBV/TV, Tb.Th, and Tb.N in Tgfb1−/− mice. In contrast, alendronate increased TBMD, TBV/TV, Tb.Th, and Tb.N in Tgfb1−/− mice in a fashion similar to that observed in Tgfb1+/+ mice (Figures 4B–4E). This reduction in the effects of PTH on bone in Tgfb1−/− mice suggests that the release of active TGF-β1 during bone resorption is essential for the effects of PTH on bone formation during remodeling.

Figure 4. Anabolic Effects of PTH on Bone Formation during Bone Remodeling Are Diminished In the Tgfb1−/− Mice.

Figure 4

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(A) Representative images of three-dimensional μCT of proximal tibiae from Tgfb1+/+ and Tgfb1−/− mice treated with vehicle (Veh), PTH or alendronate alone. Scale bar represents 1 mm.

(B–E) Quantitative μCT analysis of the secondary spongiosa of proximal tibiae. TBMD (B), TBV/TV (C), Tb.Th (D), and Tb.N (E) were measured (n = 10 for each treatment group. *p < 0.05, **p < 0.01. n.s., not significant). Data are presented as mean ± SEM.

Osteoblast Recruitment Is Uncoupled from PTH-induced Bone Resorption in Tgfb1−/− Mice

In Tgfb1−/− mice, the levels of p-Smad2/3 in the remodeling microenvironment were significantly lower than that in their wild-type counterparts (Figure S2). On analysis of the osteoblast differentiation potential of bone marrow cells isolated from the Tgfb1−/− mice, we found that the number of CFU-Fs and CFU-Obs was similar in the bone marrow cells isolated from Tgfb1−/− mice and in those of their wild-type littermates; we also found that PTH did not change the number of CFU-Fs but increased the number of CFU-Obs, whereas while alendronate had no effect on either. Notably, the patterns of PTH or alendronate effects on CFU-Fs and CFU-Obs in Tgfb1−/− mice and their wild-type littermates were comparable (Figures 5A–5C), indicating that the effects of PTH and alendronate on CFU-Fs and CFU-Obs are TGF-β1-independent. Further measurement of the number of osteoblasts and osteoclasts during bone remodeling revealed that PTH stimulated both in Tgfb1+/+ mice compared to mice injected with the vehicle or alendronate (Figures 5D–5G), but that PTH failed to stimulate osteoblasts in Tgfb1−/− mice when osteoclasts were stimulated (Figures 5E and 5G), indicating uncoupled recruitment of BMSCs. As a result, bone formation rate and osteoid volume were not simulated by PTH in Tgfb1−/− mice compared to the control mice (Figures 5H and 5I). Thus, the recruitment of osteogenic BMSCs to the bone remodeling sites was interrupted in the Tgfb1−/− mice, leading to a reduction in the anabolic action of PTH on bone.

Figure 5. Osteoblast Number Is Decreased in Tgfb1−/− Mice with Intermittent Administration of PTH.

Figure 5

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(A) Colonies formed from harvested bone marrow of the mice as indicated in CFU-F and CFU-Ob assays (1×105 bone marrow nucleated cells were plated into six-well plates). The top panels show six-well plates containing CFU-Fs stained with methanol green. The bottom panels show six-well plates containing CFU-Obs stained with Alizarin Red.

(B, C) The colony-forming efficiency was determined by number of colonies per 105 marrow cells plated. Data are represented as mean ± SEM of triplicate cultures of 0.1, 0.5, and 1×106 bone marrow nucleated cells from five individual mice (*p < 0.05. n.s., not significant).

(D, E) Light micrographs of TRAP-staining (D) and Goldner’s Trichrome-staining (E) performed on trabecular bone sections of the secondary spongiosa from distal femora of indicated treatment groups. Black arrowheads indicate osteoid surface. Scale bars represent 100 μm.

(F–I) Histomorphometric analysis of remodeling trabecular bone. N.Oc/T,Ar (F), N.Ob/T.Ar (G), BFR/BS (H) and OV/BV (I) were measured (n = 10 for each treatment group. *p < 0.05, **p < 0.01. n.s., not significant).

Data are presented as mean ± SEM.

See also Figure S2.

Sca-1+CD29+CD45−CD11b− BMSCs Are A Skeletal Stem Cell Subset

Murine Sca-1+ BMSCs have been shown as bone marrow-derived multipotent mesenchymal stem cells (Belema-Bedada et al., 2008). We have shown that Sca-1+CD29+CD45−CD11b− cells are recruited to bone remodeling sites in response to osteoclastic bone resorption. We therefore examined the stem cell characteristics of the Sca-1+CD29+CD45−CD11b− BMSCs, which are more homogenous cell population than Sca-1+ BMSCs. Sca-1+ CD29+CD45−CD11b− BMSCs were cultured in single cell suspensions for single colony forming. Five individual, well-separated colonies were selected and the number of cells was individually expanded by passaging (Figure 6A). To assess the multilineage differentiation capacity of the five clonal strains, each clonal strain derived from a single CFU-F underwent osteogenic, adipogenic, and chondrogenic induction by different culture media (Figure 6B). All clones were capable of osteogenesis, 4 of the 5 clones were adipogenestic, and 3 clones were chondrogenetic, indicating the multilineage differentiation capacity of Sca-1+CD29+CD45−CD11b− BMSCs (Table S1).

Figure 6. Characterization of Murine Sca-1+CD29+CD45−CD11b− BMSCs.

Figure 6

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(A) Five single CFU-F derived cell strains were isolated and individually expended for characterization assays.

(B) In vitro differentiation of single CFU-F derived cell strains into multilineage cells. (i) Murine BMSCs were cultured in osteogenic media for 4 weeks, and the mineralized nodules were evaluated via Alizarin Red staining and visualized with light microcopy. (ii) Murine BMSCs were cultured in adipogenic media for 2 weeks, and the accumulation of numerous lipid vesicles within the cells was identified by Oil-red-O staining and visualized with light microscopy. (iii) Murine BMSCs were cultured in chondrogenic media for 3 weeks, and the micromass pellet was stained with toluidine blue and visualized with light microscopy. The inset shows a higher magnification of boxed area. Scale bars represent 100 μm.

(C) Illustration of bone marrow cavity transplantation of single CFU-F-derived cell strain.

(D) FACS analysis of the sorted GFP+ cells from the bone marrow cell suspension obtained from mice with single CFU-F transplantation.

(E) The sorted cells were replated to generate colonies. The expression levels of Sca-1, CD29, CD45 and CD11b of the secondary CFU-Fs were analyzed by FACS analysis.

(F) Illustration of subrenal capsule transplantation of single CFU-F derived cell strain.

(G) Formation of the heterotopic ossicle at 6 weeks after transplantation. (i,ii) Hematoxylin and eosin (H&E) staining of the heterotopic ossicle section (boxed area in i corresponds to ii). (iii) Immune-staining of the heterotopic ossicle section with specific GFP antibody. GFP+ cells are shown on bone surface and in bone matrix. b, bone. m, marrow. Black arrows indicate osteoblasts. Scale bars represent 50 μm.

See also Table S1.

The five green fluorescent protein (GFP)-positive (GFP+) clonal strains were then injected individually into the femur cavity of 8-week-old Rag2−/− mice with an immunodeficient background at a density of 1 × 106 per injection (Figure 6C). GFP+ cells from the injected bone were collected by fluorescence activated cell sorting (FACS) 8 weeks after injection (Figure 6D), resulting in 2.0 × 104–6 × 104 cells, which were plated in culture at a density of 1 ×103 cells per 100-mm dish. This procedure resulted in the generation of at least two secondary CFU-Fs in each clonal strain derived from five single CFU-Fs (Table S1). Importantly, all of the colonies generated by the secondary CFU-Fs were positive for Sca-1 and CD29 and negative for CD45 and CD11b (Figure 6E). The five clonal strains were then pelleted, resuspended in matrigel, and injected underneath the renal capsule of Rag2−/− mice to generate heterotopic ossicles for assessing their osteogenic differentiation capacity in vivo (Figure 6F). Complete ossicles, including bone, sinusoids, and hematopoietic components, were generated from individual clones (Figure 6G and Table S1). The lining cells on the bone surface and osteocytes in the bone matrix were GFP+, indicating that the donor cells induced osteogenesis (Figure 6G). These results show that the Sca-1+CD29+CD45−CD11b− BMSCs are a skeletal stem cell subset.

Release of Active TGF-β1 Is Essential for Recruitment of Sca-1+ Skeletal Stem Cells during PTH-induced Bone Remodeling

We then investigated whether the migration of Sca-1+CD29+CD45−CD11b− cells to the bone remodeling sites is interrupted in mice by the combined treatment of PTH and alendronate. The GFP-labeled CFU-F-derived clonal strain was injected into the femoral cavity of Tgfb1−/− or Tgfb1+/+ mice that had been treated with the vehicle, PTH, alendronate, or the combination of PTH and alendronate. The injected cells were detected by immunostaining the femoral sections with an antibody specific for GFP 1 week after injection. In Tgfb1+/+ mice, the GFP+ cells were largely found at the bone remodeling sites, as indicated by the presence of TRAP-positive osteoclasts, and PTH significantly enhanced the recruitment of the donor cells to the bone resorption sites (Figures 7A and 7B). In contrast, in Tgfb1−/− mice and Tgfb1+/+ mice treated with alendronate alone or in the combination of PTH and alendronate, the GFP+ cells were dispersed largely in the bone marrow, with a few at the bone surface (Figures 7A and 7B). The GFP+ cells of Tgfb1−/− or Tgfb1+/+ mice with different treatments were also examined by flow cytometry analysis to assess their viability in different bone marrow microenvironments. As shown in Figures 7C and 7D, the survival rate of the GFP+ cells did not differ significantly by treatment regimen. Together, these results show that recruitment of Sca-1+ skeletal stem cells to the remodeling sites by TGF-β1 is essential for the anabolic effects of PTH on bone formation during remodeling (Figures 7E).

Figure 7. PTH-induced Recruitment of Sca-1+ Skeletal Stem Cells to Bone Remodeling Sites Is Inhibited with Combined Use of Alendronate or In Tgfb1−/− Mice.

Figure 7

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(A) Femur sections from the indicated mice transplanted with single CFU-F derived GFP+Sca-1+CD29+CD45−CD11b− clonal strain were stained with TRAP and the antibody to GFP. b, bone. Scale bar represents 25 μm.

(B) Number of GFP+ cells on bone surface 1 week after transplantation was measured and presented as cells per millimeter of bone perimeter in sections (n = 5 for each treatment group. *p < 0.05 versus Tgfb1+/+ treated with Veh, #p < 0.01 versus Tgfb1+/+ treated with PTH. n.s., not significant).

(C) GFP+ cells from bone marrows and compact bones 1 week after transplantation were assessed by flow cytometry analysis.

(D) Percentage of GFP+ cells is shown (n = 3 for each treatment group. n.s., not significant).

(E) Model of blunting anabolic effects of PTH on bone formation by alendronate. PTH enhances recruitment of Sca-1+ skeletal stem cells by stimulating bone remodeling and releasing active TGF-β1 (left panel). Alendronate impairs PTH anabolic effects on bone formation by inhibiting the recruitment of Sca-1+ skeletal stem cells (right panel). Data are presented as mean ± SEM.

DISCUSSION

It is believed that bone resorption is essential for the anabolic effects of PTH on bone formation. In human adults, PTH enhances new bone formation through stimulation of bone remodeling, in each cycle of bone remodeling, new bone is formed precisely at the sites that have recently undergone resorption. The initial and critical step is to attract bone marrow multipotent osteogenic cells to the resorption sites. The second step is to promote the differentiation of the recruited cells into osteoblasts that can secrete matrix proteins and minerals, thus forming new bone. We have identified that active TGF-β1 is released during bone resorption and that it directs the migration of Sca-1+CD29+CD45−CD11b− BMSCs as the primary factor to couple bone resorption and formation (Tang et al., 2009). Therefore, inhibition of TGF-β1 activation by alendronate causes insufficient recruitment of the cells to the resorptive sites for the new bone formation during PTH-stimulated bone remodeling, leading to a reduction of bone remodeling, with no additive effects for the concurrent use of PTH. Similarly, when osteoclast activity was inhibited by osteoprotegrin (OPG), PTH anabolic action on bone was also reduced in oophorectomized mice. In this case, pretreatment with alendronate or OPG followed by either PTH(1-34) alone or concurrent use of PTH(1-34) reduced osteoblasts and serum levels of osteocalcin compared with PTH(1-34) treatment alone (Samadfam et al., 2007). Moreover, the anabolic action of PTH on bone is diminished in TGF-β1 knockout mice. Thus, release of active TGF-β1 during osteoclastic bone resorption is essential for PTH-induced anabolic bone formation.

Bone formation is a complex process coupled with angiogenesis (Schipani et al., 2009; Street et al., 2002; Towler, 2007; Wang et al., 2007), which involves the differentiation of many types of cells. Therefore, recruitment of skeletal stem cells to bone resorptive sites with potential differentiation into different types of cells is critical for bone remodeling. Typically, the microenvironment provides signals that direct the cell-lineage-specific differentiation of the stem cells. The elasticity of the matrix plays an important role, with a stiff matrix directing the differentiation of bone marrow mesenchymal stem cells into osteoblasts (Engler et al., 2006). At fresh resorptive sites, the bone mineral matrix is exposed and lacks a covering of lining cells, providing a stiff elastic microenvironment that is rich in bone matrix proteins for the differentiation of skeletal stem cells to osteoblasts. Soluble factors also contribute to the lineage specification of stem cells. The osteotropic factors found in the bone matrix (including BMPs, IGF-I, IGF-II, and PDGF) have been shown to induce differentiation of skeletal stem cells into osteoblasts. These factors are also released during osteoclastic bone resorption as important components of the osteogenic microenvironment at the resorptive sites. This mechanism warrants new bone formation always occurs at the freshly osteoclastic resorptive sites to maintain the micro-architecture of the bone for its mechanical property. Apparently, skeletal stem cells uncommitted before being recruited to the osteogenic microenvironment serve this purpose well.

Our recent study indicates that PTH induces endocytosis of PTH1R and TβRII, as a complex, and that this feature coordinates the signaling of both PTH and TGF-β in coupling bone formation and resorption during the process of bone remodeling (Atfi and Baron, 2010; Qiu et al., 2010). Interestingly, two clinical studies indicate that sequential treatment of osteoporosis with PTH followed by alendronate increase bone density more than does PTH or alendronate alone (Rittmaster et al., 2000; Black et al., 2005). Women who had received PTH (1–84) monotherapy for 1 year were randomly reassigned to an additional year with either a placebo or alendronate. Alendronate treatment after PTH therapy led to an increase of 31% in bone mineral density compared with a 14% increase in the PTH-placebo group (Black et al., 2005). Similar results were also obtained in another clinical trial (Rittmaster et al., 2000). The benefit of sequential use of PTH and alendronate is likely secondary to the fact that the release of active TGF-β1 and the subsequent recruitment of skeletal stem cells during each cycle of PTH-stimulated bone remodeling are not impaired. Taken together, this mechanism responsible for the alendronate-impaired anabolic effects of PTH on bone suggests that the use of PTH before treatment by an anti-resorptive drug could be an effective therapy.

EXPERIMENTAL PROCEDURES

Mice

C57BL/6J (wild-type) mice were purchased from Charles River and Tgfb1+/− were obtained from the Mouse Models of Human Cancers Consortium Repository, US National Cancer Institute. Tgfb1+/− mice were crossed with Rag2−/− mice to generate Tgfb1+/−Rag2−/− mice to prevent the death of homozygotes by autoimmune disease. Tgfb1+/−Rag2−/− mice were maintained as heterozygotes and were crossed to generate Tgfb1−/−Rag2−/− and Tgfb1+/+Rag2−/− mice.

In the combined PTH and alendronate therapy model, 8-month-old C57BL/6J male mice were subcutaneously administered alendronate (A4978, Sigma-Aldrich) or a vehicle (equivalent volume of sterile phosphate buffer saline [PBS]) at a dose of 50 μg/kg three times weekly for 8 weeks. For the last 4 weeks, the mice were subcutaneously injected PTH (1-34) (Bachem, Inc., 40 μg/kg/day) or vehicle (equivalent volume of 1mM acetic acid in PBS) five times per week. Sixty C57BL/6J mice were classified into four different groups, with15 mice per group. (1). Veh, the mice were injected with the vehicles of both PTH (1-34) and alendronate. (2). PTH, the mice were injected with both PTH (1-34) and vehicle of alendronate. (3). Aln, the mice were injected with both alendronate and vehicle of PTH (1-34). (4). PTH + Aln, the mice were injected with both alendronate and PTH (1-34).

In the model of Tgfb1−/−Rag2−/− mice injected with PTH and alendronate, 2-month-old male Tgfb1−/−Rag2−/− and Tgfb1+/+Rag2−/− mice were injected with PTH (1-34) (40 μg/kg/day, five times per week), alendronate (50 μg/kg three times weekly) or vehicle as described above for 4 weeks (15 per group).

All animals were maintained in the Animal Facility of the Johns Hopkins University School of Medicine. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University, Baltimore, MD, USA.

Isolation and Culture of Murine BMSCs

Bone marrow cells were collected from 6-week-old wild-type male C57BL/6 mice euthanized by cervical dislocation; the cells were cultured with Minimum Essential Medium [alpha] (α-MEM, Mediatech, Inc.) supplemented with penicillin (100 U/ml, Sigma-Aldrich), streptomycin sulfate (100 μg/ml, Sigma-Aldrich), and 20% lot-selected fetal bovine serum (FBS, Atlanta Biologicals) at 37°C in a 5% CO2 humidified incubator. After 72 hours of adhesion, nonadherent cells were removed and adherent cells were cultured an additional 7 days with a single media change. The adherent cells were then retrieved by trypsin digestion. Cells aliquots were incubated for 20 minutes at 4°C with phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)-, peridinin chlorophyll protein (Per CP)-, and allophycocyanin (APC)-conjugated antibodies against mouse Sca-1, CD29, CD45, and CD11b (Bio-legend). Acquisition was performed on a fluorescence-activated cell sorting (FACS) Aria model (BD Biosciences), and analysis was performed using a FACS DIVE software version 6.1.3 (BD Biosciences). The sorted CD29+Sca-1+CD45−CD11b− cells were enriched by further culture. To isolate individual clonal strains, the sorted cells were passed consecutively through 16- and 20-gauge needles to obtain single cell suspensions. 1×103 cells were cultured in 100-mm dishes for single colony forming. Individual, well-separated colonies were selected using cloning cylinders as reported (Kuznetsov et al., 1997; Sacchetti et al., 2007), and the number of cells was individually expanded by passaging and confirmed by flow cytometry.

In Vitro Differentiation Assays of BMSCs into Multilineage Cells

For osteogenic differentiation, cells were seeded at a density of 5 × 103/cm2 with α-MEM supplemented with 10% FBS, 10−7 M dexamethasone (Sigma-Aldrich), 10 mM β-glycerol phosphate (Sigma-Aldrich), and 50 μM ascorbate-2-phosphate (Sigma-Aldrich). Cultures in α-MEM supplemented with 10% FBS served as a negative control. After 3 weeks of differentiation, the mineralization capacity of the cells was evaluated by Alizarin Red staining (2% of Alizarin Red S (Sigma-Aldrich) dissolved in water with the pH adjusted to 4.2).

For adipogenic differentiation, cells were seeded at a density of 1 × 104/cm2 with α-MEM supplemented with 10% FBS, 10−6 M dexamethasone, 0.5 μM IBMX (Sigma-Aldrich), and 10 ng/ml of insulin (Sigma-Aldrich) for 2 weeks. Cultures of cells in α-MEM supplemented with 10% FBS served as a negative control. Lipid accumulation was identified by Oil-red-O staining (0.5 g of Oil-red-O [Sigma-Aldrich] were dissolved in 100 ml of isopropanol [Sigma-Aldrich], and diluted to 60% with distilled water).

For chondrogenic differentiation, cells (1 × 106) were seeded in polypropylene tubes with high-glucose D-MEM supplemented with 10−7 M dexamethasone, 1% ITS (Sigma-Aldrich), 50 μM ascorbate-2-phosphate, 1 mM sodium pyruvate (Sigma-Aldrich), 50 μg/ml of proline (Sigma-Aldrich), and 20 ng/ml of TGF-β3 (R&D Systems). Culture cells in high-glucose D-MEM supplemented with 10% FBS served as a negative control. After 3 weeks in culture, the pellets were fixed in 10% buffered formalin for 48 hours and embedded in paraffin. Then 4-μm-thick sections were processed for toluidine blue staining (1 g of toluidine blue [Sigma-Aldrich] was dissolved in 100 ml of 70% alcohol and diluted to 10% with 1% sodium chloride, pH adjusted to 2.3).

CFU-F and CFU-Ob Assays

At the time of euthanasia, bone marrow from femoral, tibial, and humeral medullary cavities were collected, and cell numbers were determined after removal of red blood cells with Zapoglobin (Coulter Corp.). The numbers of CFU-Fs and CFU-Obs in murine bone marrow isolates and in cultures of bone marrow cells were determined in cocultures with irradiated guinea pig marrow cells, as reported (Kuznetsov and Gehron, 1996). Briefly, marrow cells were obtained from the femurs and tibiae of 2- month-old female Hartley guinea pigs by flushing with a 22-gauge needle and then were resuspended. Cells were γ-irradiated with a Co57 source for 50 minutes at 1.2 Gy/minute, as reported (Di Gregorio et al., 2001). After rinsing by centrifugation, cells were resuspended in αMEM medium with 20% FBS, counted and cultured at 2.5 × 106 per well of a six-well plate.

For assay of CFU-F and CFU-Ob number, 0.1, 0.5, or 1×106 murine marrow cells were plated into six well plates in 3 ml of α-MEM supplemented with glutamine (2 mM), penicillin (100 U/ml), streptomycin sulfate (100 μg/ml), and 20% lot-selected FBS. Duplicate cultures were established. After 2 to 3 hr of adhesion, unattached cells were removed, and 2.5 × 106 irradiated guinea pig feeder cells (provided by Dr. Brendan J. Canning) were added to cultivation medium of adherent cultures just after washing. On day 14, cultures were fixed and stained with 1 mg/ml of methanol green. The colonies containing 50 or more cells were counted. For CFU-Ob assay, the cells were cultured with osteogenic medium as described above for 21 days and analyzed with Alizarin Red staining. The colony-forming efficiency was determined by counting the number of colonies per 105 marrow cells plated. Secondary passage of CFU-Fs assay was performed as reported (Sacchetti et al., 2007).

Subrenal Capsule Transplantation

We pelleted 2 × 103 cells, resuspended them in 2 μl of matrigel, and then injected them underneath the renal capsule of 8-week-old Rag2−/− mice with an immuno-deficient background to generate heterotopic ossicles for histological study, as reported (Chan et al., 2009). The mice were euthanized 6 weeks after transplantation, and the transplants were processed for staining.

Bone Marrow Cavity Transplantation

Age-matched Tgfb1−/− or wild-type littermates with an immunodeficient background (Tgfb1−/−Rag2−/− and Tgfb1+/+Rag2−/− mice, male) were used as recipients. The mice were classified into different treatment groups (five in each group) as described above. GFP-labeled cells in 10 μl of α-MEM were injected into the bone marrow cavity of the left femora, as previously described (Tang et al., 2009). The mice were euthanized 1 week after transplantation, and the distal femora were processed for staining. In some cases, cells were collected from bone marrows and compact bones by collagenase digestion as reported (Zhu et al., 2010), and the total number of GFP positive cells was assessed by flow cytometry analysis.

Microcomputed Tomography (μCT) Analysis

Trabecular bone in metaphyseal secondary spongiosa of tibiae were analyzed by μCT. Procedures are described in detail in the Supplemental Methods.

Histochemistry, Immunohistochemistry, and Histomorphometric Analysis

Standard protocols were followed. Procedures are described in detail in the Supplemental Methods.

Biochemical and Cellular Assays

Details about ELISA, and Western blot analysis are provided in the Supplemental Methods.

Statistical Analysis

Analyses of significant differences were performed using an analysis of variance. Each experiment was performed in at least three independent cultures/animals per genotype, treatment and condition, and data are presented as mean ± SEM. A probability value of less than 5% was considered significant.

Supplementary Material

01

Acknowledgments

The authors thank Elaine P. Henze (Editorial Services Department of Orthopaedic Surgery, Johns Hopkins University) for editing the manuscript and Dr. Brendan J. Canning (Asthma and Allergy Center, Johns Hopkins University) for providing guinea pig marrow cells. This research was supported by US National Institutes of Health grants AR 053973 and DK05750 to Xu Cao.

Footnotes

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Supplementary Materials

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NIHMS242267-supplement-01.doc (1.7MB, doc)

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