Abstract
Aging of the general population has led to a substantial increase in the prevalence of osteoporosis over the past decades. While there are effective pharmacological agents that increase bone formation, decrease bone resorption, and decrease fracture risk, they do not uniformly cure osteoporosis. This has prompted investigations to examine whether combination therapy (COMBO) with these agents can result in an additive benefit. Since concomitant therapy with denosumab and teriparatide has shown promise in this respect, investigations were undertaken to explore whether the changes in osteogenic phenotype could provide insight into the cellular and molecular mechanism of this effect.
Investigations were performed in postmenopausal women receiving denosumab, teriparatide, or both for 3 months. Histomorphometric parameters were the primary outcome, while exploratory studies examined RNA expression in bone biopsies as well as in sorted and cultured bone marrow stromal cells (BMSCs). Osteogenic colony forming units of BMSCs were also evaluated. The studies demonstrated that COMBO results in an increase in osteoprogenitors, evidenced by an increase in osteoblastic colony-forming units. This was associated with an increased in BMSC expression of LGR6 (leucine-rich repeat containing G protein–coupled receptor 6), a stem cell marker and activator of the canonical Wnt signaling pathway. These data suggest that enhancement of canonical Wnt signaling contributes to the increase in osteoprogenitors and consequently an increase in bone density in postmenopausal women receiving COMBO for osteoporosis.
Keywords: osteoporosis, anabolic, antiresorptive, bone marrow stromal cells, LGR6
Osteoporosis is the most common disease affecting bone and is characterized by a reduction in bone matrix volume and microarchitectural deterioration, predisposing to fragility fractures. The pathophysiology of postmenopausal osteoporosis is multifactorial, including hormonal and environmental factors, with postmenopausal estrogen deficiency being the most common underlying cause. Regardless of the underlying etiology, the decrease in bone matrix volume observed is a consequence of imbalances in bone turnover, with increased bone resorption relative to bone formation.
Pharmacotherapies for osteoporosis can be divided into two categories, antiresorptive agents and anabolic agents (1). The goal of antiresorptive agents is to prevent osteoclast-mediated bone resorption, shifting the balance of bone turnover from a baseline where bone resorption exceeds bone formation, to a state of net bone formation since resorption is blocked. Treatment with antiresorptives, which include bisphosphonates and the receptor activator of nuclear factor κ β ligand (RANKL) inhibitor denosumab (DMAB), is also associated with a decrease in bone formation (2, 3). The parathyroid hormone (PTH) and parathyroid hormone–related protein analogues teriparatide (TPTD) and abaloparatide are anabolic agents that stimulate bone formation when administered intermittently (4). However, they also stimulate RANKL expression, which promotes osteoclast differentiation and activity, thus they increase both bone formation and bone resorption, with a net balance favoring bone formation. Clinical studies have sought to exploit the basis for these two approaches to osteoporosis treatment by combining anabolic agents with antiresorptive agents (5, 6). Studies using bisphosphonates as inhibitors of bone resorption have generally not demonstrated statistically significant additive effects of combination therapy (COMBO) on bone density; however, concomitant administration of TPTD and DMAB has shown a significant benefit compared to monotherapy with either agent (7).
While murine models have been used to investigate the molecular actions of both antiresorptive and anabolic osteoporosis therapies, the models most commonly studied are ovariectomized mice, which recapitulate the hormone-deficient postmenopausal state, but not the effects of aging and skeletal senescence (8). Furthermore, few if any of these studies sought to define the molecular basis for the resultant skeletal phenotypes. Thus, investigations were undertaken to address the molecular and cellular basis for the additive increase in bone density in postmenopausal women treated both with TPTD and DMAB.
Materials and Methods
Study Population
A description of the DATA-Biopsy study design and participant characteristics are described in detail elsewhere (ClinicalTrials.gov No. NCT00926380). Briefly, analyses were performed on samples from 26 postmenopausal women at high risk for fracture who were randomly assigned to treatment with either TPTD 20 mcg subcutaneous daily (N = 9), denosumab 60 mg subcutaneous once (N = 8), or both (N = 9) for a period of 3 months. Fluorochromes were administered to allow analyses of bone formation at baseline and 3 months in a single biopsy of the iliac crest (2). These results will be reported in a separate manuscript. In addition to the 8-mm iliac crest biopsy obtained for histomorphometry, an adjacent 2-mm bone core biopsy and a marrow aspirate were obtained to permit evaluation of bone RNA expression, characterization of osteoblast precursors, and osteoblast differentiation.
Bone RNA Extraction
Cores were dissected free of muscle, rinsed in phosphate-buffered saline (PBS) and immediately homogenized in Trizol (15596018, Thermo Fisher Scientific). After chloroform extraction (Sigma C2432) and isopropanol (Thermo Scientific 327272500) precipitation, RNA was resuspended in RNAse-free water and stored at −80 °C.
Bone Marrow Stromal Cell Isolation
Marrow aspirates (12-20 mL) were transferred into EDTA-coated tubes (Becton Dickinson 366643) and rocked to prevent clotting. PBS (Thermo Scientific AAJ67802AP) was added to bring the volume up to 35 mL, after which the diluted aspirate was filtered and layered onto 14 mL of Ficoll (1.074 g/mL) (Thermo Scientific 45-001-756). Samples were spun at 400g for 30 minutes at 22 °C, following which the bone marrow stromal cells (BMSCs) at the Ficoll/marrow interface were collected. BMSCs were rinsed twice with fluorescence-activated cell sorting (FACS) buffer (PBS with 0.5% bovine serum albumin and 2 mM EDTA), following which they were plated or subjected to FACS.
Fluorescence Activated Cell Sorting
Cells were incubated in Red Blood Cell lysis buffer (BioLegend 420301) on ice for 5 minutes, after which 3.3 volumes of cell-staining buffer (BioLegend 420201) was added. Cells were washed again in cell-staining buffer and resuspended in this buffer at a concentration of 5 to 106 cells/mL. Following incubation with human TruStain FC blocker (BioLegend 422301), cells were incubated on ice in the dark for 20 minutes with primary antibodies, including Pacific Blue Anti-CD235 (BioLegend 306612; RRID: http://antibodyregistry.org/AB_2116241), Pacific Blue antihuman lineage cocktail (BioLegend 348805; RRID: http://antibodyregistry.org/AB_2889063), APC anti-CD31 (BioLegend 303116; RRID: http://antibodyregistry.org/AB_1877151), and APC anti-CD34 (BioLegend 343608; RRID: http://antibodyregistry.org/AB_2074356). Cells were then rinsed with cell staining buffer 3 times and incubated in Zombie green viability dye (BioLegend 423111) in the dark at 22 °C for 15 minutes. Cells were rinsed and resuspended in cell-staining buffer and subjected to FACS to isolate lineage-negative/CD31 and CD34 dim cells as previously described (9). Controls for FACS included peripheral mononuclear cells incubated in a similar fashion, as well as OneComp beads for PacificBlue and APC (Thermo Fisher Scientific).
Bone Marrow Stromal Cell Culture
Cells were plated in aMEM (Gibco 41061069) with 10% fetal bovine serum (Gibco A31604) and penicillin and streptomycin (Gibco 15140122) at a density of 8 × 104 cells/cm2 to evaluate osteoblast differentiation and 2 × 104 cells/cm2 to evaluate colony-forming units (CFUs). Nonadherent cells were removed and cells were rinsed with PBS after 48 hours prior to adding fresh media. Twice weekly for the next 7 days of culture, half the media was replaced with fresh media containing dexamethasone (Sigma D2915) and ascorbic acid (Sigma A8960) to promote cell differentiation (2× differentiation media: 100 nM and 0.05 mM, respectively). Following this time period, fresh media supplemented with 0.025-mM ascorbic acid was added twice weekly. A subset of cells was initially cultured in regular media until 70% to 80% confluent, and then replated at a concentration of 3 × 103 to examine differentiation of a more homogenous population of passage 1 cells.
RNA Analysis
Total RNA was isolated using the Qiagen RNA Mini Kit (No. 74104, Qiagen) and reverse-transcribed using the PrimeScript RT Reagent kit with genomic DNA eraser (RR047, TakaRa) following the manufacturer's instructions. Complementary DNA expression was quantitated by quantitative polymerase chain reaction, and target gene expression was normalized for actin in the same sample using the method of Schmittgen and Livak (10).
Colony-Forming Unit Evaluation
Cells were fixed in neutral buffered formalin (Thermo Scientific 033314.M1) for 30 minutes, rinsed with water, and incubated for an additional 30 minutes with alkaline phosphatase staining solution to permit quantitation of osteoblastic CFUs. After image capture, colonies were stained with 0.05% crystal violet for total CFU (CFU-F) quantitation.
Statistical Analysis
Statistical analysis of between-group differences were analyzed by analysis of variance. A P value of less than .05 was considered indicative of statistical significance. No adjustments for multiple testing were made.
Results
Bone Core Gene Expression
RNA was isolated from the 2-mm bone core to evaluate osteoblast gene expression. Genes associated with bone formation and mineralization were selected based on their messenger RNA detection threshold in the bone RNA and for their reported regulation by PTH. A statistically significant increase in the expression of the bone matrix protein, type I collagen (Col I), was observed in samples obtained from participants on TPTD monotherapy, relative to those randomly assigned to DMAB or COMBO (P = .049); however, no difference was observed in the expression of osteocalcin (OCN), bone sialoprotein (BSP) or RUNX family transcription factor 2 (Runx2) (Fig. 1 and data not shown). Similarly, no between group differences were observed for sclerostin (SOST), rank ligand (RANKL), insulin-like growth factor 1 (IGF1), T-cell factor 1 (TCF-1), lymphoid enhancer binding factor 1 (Lef1), or secreted frizzled-related protein 1 (SFRP1). Although a trend to an increase in the canonical Wingless and Int-1 (cWnt) pathway ligand, Wnt10b was seen in the cores from participants randomly assigned to combination treatment, it did not reach statistical significance (P = .084) (see Fig. 1).
Figure 1.
Bone core messenger RNA expression. RNA expression of Col I was increased in the biopsies of participants receiving teriparatide (TPTD) relative to that of those receiving denosumab (DMAB) or combination (COMBO) therapy (*P = .049). There was no difference in expression of OCN among the groups (P = .718), and the increase in Wnt10b in those receiving combination therapy did not achieve statistical significance (P = .084).
Bone Marrow Stromal Cells Gene Expression
A fraction of the harvested BMSCs was subjected to flow cytometry to evaluate osteoprogenitor gene expression. While several markers have recently been identified for human osteogenic precursors, there is no uniform agreement as to which marker is most sensitive and specific for human BMSCs or the target of anabolic therapies. Thus, flow cytometry was performed to isolate lineage negative/CD34 and CD31 “dim” cells, which have previously been shown to comprise all cells capable of forming mineralized matrix when cultured under osteogenic conditions and lack hematopoietic and endothelial progenitors/cells (9). There was no difference in the expression of the osteoblast markers Col I, Runx2, or OCN in the cells isolated from participants in the 3 treatment groups. Evaluation of osteoprogenitor markers revealed a significant increase in expression of LGR6 (P = .042) in BMSCs of participants receiving COMBO vs those receiving monotherapy, but no difference was seen in the expression of EBF transcription factor 3 (EBF3) or C-type lectin domain containing 11A (Clec11a) (Fig. 2).
Figure 2.
Messenger RNA expression by bone marrow stromal cells (BMSCs) subjected to fluorescence-activated cell sorting. No difference in expression of osteoblast markers (Col I, Runx2, OCN) was observed among the 3 treatment groups. Similarly, no difference in expression of ostegenic stem cell markers Clec11a and EBF3 was observed; however, a statistically significant increase in expression of LGR6 was observed in the cells from participants receiving combination therapy (*P = .042). COMBO, combination; DMAB, denosumab; TPTD, teriparatide.
Evaluation of Colony-Forming Units
To determine the osteogenic potential of BMSC, cells were plated at low density (2.4 × 104 cells/cm2) to permit quantitation of CFUs. Osteogenic colonies were detected by evaluating alkaline phosphatase activity (CFU-AP) after 25 days in culture. Crystal violet staining was performed to permit quantitation of the CFU-F. COMBO led to an increase in the number of colonies with osteogenic potential (CFU-AP) relative to monotherapy (P = .01). No statistically significant increase in CFU-F colonies was observed in cells isolated from those treated with COMBO (P = .09) (Fig. 3).
Figure 3.
Evaluation of osteogenic colony forming units. Bone marrow stromal cells (BMSCs) were plated at low density to permit evaluation of total (CFU-F) and osteogenic colony-forming (CFU-AP) units. Treatment did not alter the total number of colonies formed (P = .085); however, a statistically significant increase was observed in the number of osteogenic colonies with COMBO therapy, relative to monotherapy with either TPT or DMAB (*P = .007). COMBO, combination; DMAB, denosumab; TPTD, teriparatide.
Osteoblast Differentiation
BMSCs were plated at a density of 8 × 104 cells/cm2 to evaluate the effects of COMBO on osteoblast differentiation. A time-dependent increase in genes associated with osteoblast differentiation, including Col I, Runx2, BSP, and OCN (P < .03 for all) was observed in cells from all treatment groups. However, a difference in gene expression among the treatment groups was observed only for the mature osteoblast differentiation marker OCN, whose expression was increased in cells from those receiving COMBO relative to TPT and DMAB alone (P = .018) (Fig. 4).
Figure 4.
Evaluation of osteoblast differentiation. The differentiation of bone marrow stromal cells (BMSCs) was examined from 14 to 35 days in culture. While all groups exhibited an increase in Col I (P < .001), Runx2 (P < .001), BSP P = .026), and OCN P = .001) over time, only OCN expression differed among the groups, being more highly expressed in cells from those who received COMBO therapy, relative to monotherapy with either TPT or DMAB (*P = .017 in COMBO vs TPT or DMAB). COMBO, combination; DMAB, denosumab; TPTD, teriparatide.
Discussion
These exploratory studies focused on defining cellular changes in bone and BMSCs that underlie the increase in bone density observed in postmenopausal women receiving combined anabolic therapy with TPTD and antiresorptive therapy with DMAB. Consistent with the anabolic actions of TPTD, an increase in Col I was observed in the bone cores from participants receiving TPTD monotherapy, suggesting an action on mature osteoblasts. The absence of this increase in those receiving COMBO likely reflects the decrease in bone formation previously reported in association with antiresorptive therapy (2, 3), suggesting that DMAB is able to inhibit the remodeling-based osteoblast-stimulating effect of TPTD.
No difference in the expression of genes associated with osteoblast maturation was observed in the enriched osteogenic BMSCs isolated by FACS from the 3 treatment groups, consistent with the fact that these mature cells are expected to be found in the bone matrix rather than the soluble marrow fraction. While no single marker of human trabecular osteoprogenitors has been defined, several genes have been shown to be preferentially expressed in this population. One of these genes, LGR6, was significantly increased in individuals who received COMBO with TPTD and DMAB. LGR6 gene variants have been associated with bone density in postmenopausal women (11), and overexpression of LGR6 in murine osteoblastic cells increases proliferation and osteogenic differentiation. Consistent with the role of LGR6 in osteogenesis, BMSCs from LGR6 knockout mice exhibit a reduction in trabecular bone, accompanied by impaired osteogenic colony formation and osteoblast differentiation due to attenuated cWnt signaling (12, 13). Interestingly EBF3, which is expressed in self-renewing BMSCs, preferentially in the osteogenic CAR (CXCL12-abundant reticular) cell niche (14), did not differ among treatment groups, nor did the expression of Clec11a, which is required for cWnt pathway activation by PTH in BMSCs (15).
Cell culture studies examining the osteogenic potential of BMSCs isolated from participants receiving COMBO exhibited an increase in CFU with osteogenic potential; however, there was no significant increase observed in CFU-F formation, suggesting that COMBO leads to an increase in osteogenic potential of precursors. The hypothesis that COMBO leads to an increase in osteogenic precursors is further supported by the increase in OCN gene expression observed in the cultured osteoblasts of participants who received COMBO. This increase in OCN gene expression was observed only in the cultures that were immediately seeded on BMSC isolation, not on those from first-passage cells. The absence of effects of TPTD and DMAB in the BMSC passaged in culture further supports the hypothesis that COMBO increases the number of osteogenic progenitors, and that this effect is lost when the cells are cultured and passaged prior to evaluation. This increase in osteoprogenitors may underlie the increase in modeling based bone formation observed in the participants who received COMBO. Notable in this respect, serum osteocalcin levels were significantly higher in the TPTD women than in women receiving DMAB or COMBO, suggesting that the actions of TPTD alone target primarily mature osteoblasts and that osteoprogenitors are the primary target of COMBO.
These studies have several limitations. Because the primary end point of these studies was histomorphometric evaluation using quadruple labels, the 3-month intervention required to do so was of relatively short duration. Second, tissue and cells were obtained at a single time point, due to the invasive nature of the sampling methods required. This limits evaluation of the short-term molecular changes that are expected to be observed with daily injections of TPTD, given its short pharmacologic half-life. Notable in this respect is the absence of significant changes in IGF-1 and SOST observed in cells and biopsies of those receiving either TPTD or COMBO, presumably because these samples were obtained 18 to 24 hours after the last injection of TPTD. Due to the limited tissue obtained for RNA analyses, not all potential transcripts of interest could be analyzed. Therefore, transcripts involved in bone formation and mineralization were prioritized; however, those expressed at very low levels were excluded. Last, the closure of research laboratories during the COVID-19 pandemic and the extended closure of core facilities for FACS of uncharacterized (with respect to infectious agents) and unfixed cells led to a decrease in sample size for data from cultured and FACS BMSCs. Nevertheless, these investigations demonstrate an increase in osteoprogenitors in postmenopausal women receiving COMBO with TPTD and DMAB, compared to those receiving monotherapy with either agent, and suggest that an increase in cWnt signaling contributes to this effect.
Acknowledgments
We are grateful to Dr Sundeep Khosla for advice on isolating and sorting BMSCs.
Abbreviations
- BMSCs
bone marrow stromal cells
- BSP
bone sialoprotein
- CFUs
colony-forming units
- CFU-AP
colony-forming units with alkaline phosphatase activity
- CFU-F
total colony-forming units
- Clec11a
C-type lectin domain containing 11A
- Col I
type I collagen
- COMBO
combination
- DMAB
denosumab
- EBF3
EBF transcription factor 3
- FACS
fluorescence-activated cell sorting
- LGR6
leucine-rich repeat containing G protein–coupled receptor 6
- OCN
osteocalcin
- PBS
phosphate-buffered saline
- PTH
parathyroid hormone
- RANKL
receptor activator of nuclear factor κ β ligand
- Runx2
RUNX family transcription factor 2
- TPTD
teriparatide
Contributor Information
Margaret M Kobelski, Endocrine Unit, Massachusetts General Hospital, Boston, MA 02114, USA.
Sabashini K Ramchand, Endocrine Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA.
Joy N Tsai, Endocrine Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA.
Benjamin Z Leder, Endocrine Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA.
Marie B Demay, Endocrine Unit, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA.
Funding
This work was supported by the National Institutes of Health (NIH grant Nos. R01 AR073191 and P30 AR075042) and NIH National Center for Advancing Translational Sciences (grant No. 1UL1TR002541-01).
Disclosures
J.N.T. has a financial interest in Amgen. J.N.T.'s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. All other authors have no conflicts of interest to declare.
Data Availability
Data sharing is not applicable to this article as no data sets were generated or analyzed during this study.
References
- 1. Shoback D, Rosen CJ, Black DM, Cheung AM, Murad MH, Eastell R. Pharmacological management of osteoporosis in postmenopausal women: an endocrine society guideline update. J Clin Endocrinol Metab. 2020;105(3):dgaa048. [DOI] [PubMed] [Google Scholar]
- 2. Dempster DW, Zhou H, Recker RR, et al. Differential effects of teriparatide and denosumab on intact PTH and bone formation indices: AVA osteoporosis study. J Clin Endocrinol Metab. 2016;101(4):1353‐1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Recker RR, Delmas PD, Halse J, et al. Effects of intravenous zoledronic acid once yearly on bone remodeling and bone structure. J Bone Miner Res. 2008;23(1):6‐16. [DOI] [PubMed] [Google Scholar]
- 4. Frolik CA, Black EC, Cain RL, et al. Anabolic and catabolic bone effects of human parathyroid hormone (1-34) are predicted by duration of hormone exposure. Bone. 2003;33(3):372‐379. [DOI] [PubMed] [Google Scholar]
- 5. Cosman F, Eriksen EF, Recknor C, et al. Effects of intravenous zoledronic acid plus subcutaneous teriparatide [rhPTH(1-34)] in postmenopausal osteoporosis. J Bone Miner Res. 2011;26(3):503‐511. [DOI] [PubMed] [Google Scholar]
- 6. Finkelstein JS, Hayes A, Hunzelman JL, Wyland JJ, Lee H, Neer RM. The effects of parathyroid hormone, alendronate, or both in men with osteoporosis. N Engl J Med. 2003;349(13):1216‐1226. [DOI] [PubMed] [Google Scholar]
- 7. Leder BZ, Tsai JN, Uihlein AV, et al. Two years of denosumab and teriparatide administration in postmenopausal women with osteoporosis (the DATA extension study): a randomized controlled trial. J Clin Endocrinol Metab. 2014;99(5):1694‐1700. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Sfeir JG, Drake MT, Khosla S, Farr JN. Skeletal aging. Mayo Clin Proc. 2022;97(6):1194‐1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Modder UI, Roforth MM, Nicks KM, et al. Characterization of mesenchymal progenitor cells isolated from human bone marrow by negative selection. Bone. 2012;50(3):804‐810. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402‐408. [DOI] [PubMed] [Google Scholar]
- 11. Li C, Huang Q, Yang R, et al. Targeted next generation sequencing of nine osteoporosis-related genes in the Wnt signaling pathway among Chinese postmenopausal women. Endocrine. 2020;68(3):669‐678. [DOI] [PubMed] [Google Scholar]
- 12. Khedgikar V, Charles JF, Lehoczky JA. Mouse LGR6 regulates osteogenesis in vitro and in vivo through differential ligand use. Bone. 2022;155:116267. [DOI] [PubMed] [Google Scholar]
- 13. Doherty L, Wan M, Peterson A, et al. Wnt-associated adult stem cell marker Lgr6 is required for osteogenesis and fracture healing. Bone. 2023;169:116681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Seike M, Omatsu Y, Watanabe H, Kondoh G, Nagasawa T. Stem cell niche-specific Ebf3 maintains the bone marrow cavity. Genes Dev. 2018;32(5-6):359‐372. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Zhang J, Cohen A, Shen B, et al. The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin. Proc Natl Acad Sci U S A. 2021;118(25):e2026176118. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Data sharing is not applicable to this article as no data sets were generated or analyzed during this study.
