The authors have no conflict of interest.
Aging Increases Stromal/Osteoblastic Cell-Induced Osteoclastogenesis and Alters the Osteoclast Precursor Pool in the Mouse†
Article first published online: 2 MAY 2005
Copyright © 2005 ASBMR
Journal of Bone and Mineral Research
Volume 20, Issue 9, pages 1659–1668, September 2005
How to Cite
Cao, J. J., Wronski, T. J., Iwaniec, U., Phleger, L., Kurimoto, P., Boudignon, B. and Halloran, B. P. (2005), Aging Increases Stromal/Osteoblastic Cell-Induced Osteoclastogenesis and Alters the Osteoclast Precursor Pool in the Mouse. J Bone Miner Res, 20: 1659–1668. doi: 10.1359/JBMR.050503
- Issue published online: 4 DEC 2009
- Article first published online: 2 MAY 2005
- Manuscript Accepted: 27 APR 2005
- Manuscript Revised: 1 APR 2005
- Manuscript Received: 4 JAN 2005
- stromal/osteoblastic cell;
Stromal/osteoblastic cell expression of RANKL and M-CSF regulates osteoclastogenesis. We show that aging is accompanied by increased RANKL and M-CSF expression, increased stromal/osteoblastic cell-induced osteoclastogenesis, and expansion of the osteoclast precursor pool. These changes correlate with age-related alterations in the relationship between osteoblasts and osteoclasts in cancellous bone.
Introduction: Bone mass is maintained through a balance between osteoblast and osteoclast activity. Osteoblasts regulate the number and activity of osteoclasts through expression of RANKL, osteoprotegerin (OPG), and macrophage-colony stimulation factor (M-CSF). To determine whether age-related changes in stromal/osteoblastic cell expression of RANKL, OPG, and M-CSF are associated with stimulation of osteoclastogenesis and whether the osteoclast precursor pool changes with age, we studied cultures of stromal/osteoblastic cells and osteoclast precursor cells from animals of different ages and examined how aging influences bone cell populations in vivo.
Materials and Methods: Osteoclast precursors from male C57BL/6 mice of 6 weeks (young), 6 months (adult), and 24 months (old) of age were either co-cultured with stromal/osteoblastic cells from young, adult, or old mice or treated with M-CSF, RANKL, and/or OPG. Osteoclast precursor pool size was determined by fluorescence-activated cell sorting (FACS), and osteoclast formation was assessed by measuring the number of multinucleated TRACP+ cells and pit formation. The levels of mRNA for RANKL, M-CSF, and OPG were determined by quantitative RT-PCR, and transcription was measured by PCR-based run-on assays. Osteoblast and osteoclast numbers in bone were measured by histomorphometry.
Results: Osteoclast formation increased dramatically when stromal/osteoblastic cells from old compared with young donors were used to induce osteoclastogenesis. Regardless of the origin of the stromal/osteoblastic cells, the number of osteoclasts formed from the nonadherent population of cells increased with increasing age. Stromal/osteoblastic cell expression of RANKL and M-CSF increased, whereas OPG decreased with aging. Exogenously administered RANKL and M-CSF increased, dose-dependently, osteoclast formation from all donors, but the response was greater in cells from old donors. Osteoclast formation in vitro positively, and the ratio of osteoblasts to osteoclasts in vivo negatively, correlated with the ratio of RANKL to OPG expression in stromal/osteoblastic cells for all ages. The effects of RANKL-induced osteoclastogenesis in vitro were blocked by OPG, suggesting a causal relationship between RANKL expression and osteoclast-inducing potential. The osteoclast precursor pool and expression of RANK and c-fms increased with age.
Conclusions: Our results show that aging significantly increases stromal/osteoblastic cell-induced osteoclastogenesis, promotes expansion of the osteoclast precursor pool and alters the relationship between osteoblasts and osteoclasts in cancellous bone.
BONE MASS IS maintained through a delicate balance between bone formation and resorption. During growth, bone formation exceeds resorption, and total bone mass increases. As adulthood is approached, the balance shifts and resorption exceeds formation. Bone is lost beginning early in adulthood and continues unabated into old age. In the female mouse, vertebral mass, cortical thickness in long bones and BMD in the femoral neck decrease with age. (1–3) In the male mouse, fat-free weight of the tibia decreases by roughly 20%, and cancellous bone volume in the proximal tibia falls by >60% with advancing age. (4)
On a cellular level, the rates of bone formation and resorption reflect the number and activity of stromal/osteoblastic cells and osteoclasts. Stromal/osteoblastic cells regulate the number and activity of osteoclasts through expression of RANKL, macrophage-colony stimulation factor (M-CSF), and osteoprotegerin (OPG). (5–15) RANKL, a member of the TNF family, is expressed on the stromal/osteoblastic cell surface, binds to its receptor RANK on the surface of osteoclast progenitors, and stimulates osteoclast differentiation and activity. M-CSF stimulates proliferation and differentiation of osteoclast progenitors through its receptor (c-fms), enhances mononuclear cell fusion, and promotes osteoclast survival. The differentiating effects of RANKL on osteoclast progenitors require M-CSF, whereas the stimulating effects of RANKL on mature osteoclast activity do not. (9) OPG acts as a secreted, nonsignaling decoy receptor, which binds RANKL and prevents the activation of RANK. Thus, the RANK/RANKL/OPG and M-CSF/c-fms axes provide a means of coupling stromal/osteoblastic cell to osteoclast activity and controlling the balance between bone formation and resorption.
With advancing age, expression of RANKL in whole bone and in cultured marrow cells from both humans and animals gradually increases, and expression of OPG either decreases or remains unchanged. (16–19) In humans, Eghbali-Fatourechi et al. (20) reported that estrogen deficiency associated with menopause increases RANKL expression in marrow cells. In mice, RANKL expression in whole bone and early stromal/osteoblastic cell cultures of marrow stromal cells is increased in cells from older animals, whereas OPG expression is decreased with age. (19) Furthermore, the osteoclast progenitor pool is reported to increase with advancing age in mice. (21)
To determine (1) whether age-related changes in stromal/osteoblastic cell expression of RANKL, OPG, and M-CSF are associated with stimulation of osteoclastogenesis; (2) whether the osteoclast precursor pool increases with age; and (3) whether these putative changes are associated with alterations in the relative numbers of osteoblasts and osteoclasts in vivo, we studied cultures of stromal/osteoblastic cells and osteoclast precursor cells from animals of different ages and examined how aging influences bone cell populations in vivo. Our results show that aging significantly increases stromal/osteoblastic cell-induced osteoclastogenesis, promotes expansion of the osteoclast precursor pool, and alters the relationship between osteoblasts and osteoclasts in cancellous bone.
MATERIALS AND METHODS
Chemicals and reagents
Ketamine, xylazine HCl, and acepromazine were purchased from Abbott Laboratories (North Chicago, IL, USA), Boehringer Ingelheim (St Joseph, MO, USA), and Animal Health Co. (Kansas City, MO, USA), respectively. α-MEM was from Mediatech (Herndon, VA, USA), FBS was from Atlanta Biologicals (Nocross, GA, USA), and the TRACP staining kit, RANKL, OPG, and M-CSF were purchased from Sigma (St Louis, MO, USA). Pit formation cell culture system (BD Biocoat Osteologic bone cell culture system) was from BD Biosciences (Bedford, MA, USA), and dentin disks were purchased from IDS (Fountain Hill, AZ, USA). RNA-STAT 60 was purchased from Tel-Test (Friendwood, TX, USA). The oligonucleotide primers and TaqMan probes for PCR amplification were designed by the Primer Express software (Version 1.0) from Applied Biosystems and synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA) with HPLC purification.
Male C57BL/6 mice, 6 weeks (young), 6 months (adult), and 24 months (old) of age, were obtained from the National Institute of Aging (NIA) colony of aging rodents (Harlan Sprague Dawley, Bethesda, MD, USA). The animal protocol for these studies was approved by the Animal Care and Use Committee at the Veterans Affairs Medical Center, San Francisco. Animals were maintained and processed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Preparation of bone marrow stromal cells and stromal/osteoblastic cell-osteoclast co-cultures
Bone marrow stromal cells were prepared and cultured as previously described. (19) Sequential expression of alkaline phosphatase, collagen, and osteocalcin and mineralized nodule formation confirmed the stromal/osteoblastic cell nature of our cells (data not shown). (19, 22, 23)
For co-culture experiments, marrow stromal cells from animals of different ages were cultured in primary media in T-75 flasks at a density of 100 × 106 cells/flask. On day 5 after seeding, the cells were harvested with 1× trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA, Cellgro), and the cells were replated at 20,000 cells/well in 24-well plates or onto either Biocoat substrates or dentin disks (n = 3–6 wells/experimental point). The cells, referred to as stromal/osteoblastic cell cultures, were incubated in secondary media (osteogenic inducing) for 4 or more days, with media changes every 2 days. On day 4 or as indicated, the nonadherent fraction (which includes osteoclast precursors) of fresh marrow stromal cells prepared from a separate group of animals and previously cultured for 2 days was counted and added (1 × 106 cells/well) to the stromal/osteoblastic cell cultures to create stromal/osteoblastic cell-nonadherent cell co-cultures prepared from animals of the same or different ages. The co-cultures were carried for 6 days in secondary media, with changes every 2 days. On the sixth day of co-culture, the cells were rinsed with PBS and prepared for TRACP staining or pit analysis. In other experiments, the effects of addition of exogenous RANKL (0, 1, 3, 10, 15, 20, 30, 60, 100 ng/ml), OPG (100 ng/ml), and M-CSF (0, 1, 10, 30, 100 ng/ml) on osteoclast formation were studied.
To determine whether RANKL expression is induced in response to endogenous soluble stimuli in our cultures, we determined whether conditioned media from cells harvested from animals of different ages contain soluble factors that can induce RANKL expression. Bone marrow cells from different ages of mice were cultured, and on day 5, the culture medium was changed to secondary differentiating medium. Conditioned media were collected at day 7 and added to separate plates of young stromal/osteoblastic cells at 10%, 20%, and 40% of the total medium. The expression of RANKL and OPG was measured after 2 days of culture in conditioned media.
TRACP staining was performed using a commercial kit from Sigma. The TRACP+ cells were observed with an inverted microscope at ×200 magnification. Multinucleated cells (more than three nuclei) that were a dark, reddish purple were counted as TRACP+ cells. Those osteoclasts having 3–15 nuclei were considered small, 15–30 nuclei were medium, and >50 nuclei osteoclasts were considered large.
Equal numbers of stromal/osteoblastic cells from young, adult, and old donors were added in each co-culture experiment, but to insure that differences in cell growth during co-culture did not affect the outcome, total cell number at the end of the experiment was determined using crystal violet staining as described earlier. (23)
Resorption pit assay
Co-cultures were prepared as described above using the BD Biocoat Osteologic bone cell culture system. For the Biocoat system, stromal/osteoblastic cells were plated onto 12-mm-diameter round coverslips (20,000 cells/disc) coated with a submicron synthetic calcium-phosphate film in a 24-well culture plate. Nonadherent cells (1 × 106 cells/well) were added on day 4 of stromal/osteoblastic cell culture, and the co-cultures continued for up to 10 days. To measure pit number and size on the Biocoat discs, a bleach solution (1 ml, 6% NaOCl, 5.2% NaCl) was added to each well, washed, and air dried. Dark-field microscopy was used to visualize the pits on the calcium-phosphate film. Images of three random fields from each disc were captured and analyzed using OpenLab 3.0 (Improvision, Lexington, MA, USA). Pits >75 μm2 in area were counted, and total pit number and total area were calculated using Bioquant software (Nashville, TN, USA).
Measurement of mRNA levels and PCR-based nuclear run-on assay
The extraction of mRNA and measurement of mRNA levels were as previously described. (19, 23, 24) Primers and probes for RANKL, OPG, alkaline phosphatase, collagen, and osteocalcin have been described. (19) The primers for RANK, c-fms, and M-CSF are shown in Table 1.
To assess whether changes in mRNA levels are a function of transcription or of subsequent RNA degradation, we used a PCR-based nuclear run-on assay. (25) On day 7, stromal/osteoblastic cells were harvested with 1× trypsin-EDTA for 10 minutes at 37°C. Cells were washed three times with ice-cold PBS and pelleted at 1000g for 10 minutes. Cells were resuspended in 2 ml ice-cold lysis buffer (10 mM NaCl, 5 mM MgCl2, and 10 mM Tris, pH 7.5) containing 0.3% NP-40 (Sigma) and incubated on ice for 10 minutes. After centrifugation at 2000g for 10 minutes at 4°C, the nuclear pellet was resuspended in 150 μl 2× Transcription Mix (180 mM KCl, 50 mM DTT, 20 mM Tris, pH 8.3, and 10 mM MgCl2) and 40 U ribonuclease inhibitor (PE Biosystems) with or without 30 μl Nucleotide Mix (10 mM dATP, 10 mM dCTP, 10 mM dGTP, and 10 mM dUTP) and incubated for 30 minutes at 37°C. RNA was extracted and subjected to RT-PCR.
Analysis of macrophage population with flow cytometry and magnetic sorting
To assess whether the population of osteoclast precursors changes with age, a single cell suspension of marrow stromal cells was prepared, washed two times with PBS calcium magnesium free (CMF), and incubated with 2.4G2 (Courtesy of Dr M Nakamura) on ice for 30 minutes to block the Fc receptor. After centrifuging at 1400 rpm for 3 minutes, cells were stained for 30 minutes on ice in either 100 μl fluorescein isothyocyanate (FITC)-conjugated CD11b antibody or isotype control antibody, rat IgG2b. Cells were washed three times with PBS and resuspended in 500 μl PBS. Macrophage-positive cells were measured by a Becton Dickinson FACScan and analyzed by CellQuest software. To isolate cells of the monocyte/macrophage cell linage, 107 fresh marrow stromal cells in a single cell suspension were incubated in 80 μl labeling buffer (PBS supplemented with 2 mM EDTA and 0.5% BSA) and 20 μl of MACS CD11b antibody (Miltenyi Biotec) for 15 minutes. Cells were washed once with 1.5 ml of labeling buffer and applied to a MACS LS sorting column, and the CD11b+ cells were collected.
Animals were treated with calcein (10 mg/kg) and demeclocycline (10 mg/kg) by subcutaneous injection 12 and 5 days, respectively, before death. The distal end of the right femur and lumbar vertebrae 2 and 3 were prepared for quantitative histomorphometry as described. (26) Cancellous bone volume (BV/TV), osteoblast and osteoclast surface, mineral apposition rate, and bone formation were quantified.
All in vitro experiments were performed at least twice with similar results. Results are reported as mean ± SD and analyzed where appropriate using Student's t-test or ANOVA and the Student Newman-Keuls methods (SigmaStat; SPSS, Chicago, IL, USA).
The effects of co-culturing stromal/osteoblastic cells with nonadherent marrow stromal cells from young, adult, and old donors are shown in Fig. 1. In the first group of bars on the left, nonadherent cells from young donors were cultured without any stromal/osteoblastic cells or with the same number of stromal/osteoblastic cells from young, adult, or old donors. Without stromal/osteoblastic cells to support osteoclastogenesis, either no or only a few osteoclast (multinucleated, TRACP+) cells were generated. There were 56 ± 16, 63 ± 12, and 123 ± 17 osteoclasts counted in the presence of stromal/osteoblastic cells from young, adult, and old mice, respectively. In each case, the same number of stromal/osteoblastic cells, regardless of age, and the same number of nonadherent cells, regardless of age, were combined and co-cultured. Separate dishes of stromal/osteoblastic cells and nonadherent cells were stained with crystal violet to confirm that all dishes had the same number of cells at the end of the co-culture. The number of stromal/osteoblastic cells in the dishes was the same, but the ability of the stromal/osteoblastic cells from the aged animals to increase the number of osteoclasts was increased 2.2-fold (p < 0.01) over that of cells from young animals.
In the middle and right groups of bars of Fig. 1, nonadherent cells from adult and old mice were cultured with stromal/osteoblastic cells from young, adult, and old donors. The pattern was the same. Stromal/osteoblastic cells from old donors had a greater propensity to recruit and stimulate the growth of multinucleated TRACP+ cells. The change in both the capacity to recruit by the stromal/osteoblastic cell from old donors and in the apparent number of osteoclast precursors in the nonadherent population resulted in a synergism in overall osteoclast recruitment. This was manifest by a 4.4-fold increase in osteoclast number when culture of stromal/osteoblastic and nonadherent cells from old donors were compared with cells from young donors. The size of the osteoclasts formed from co-culture ranged from relatively small (3-6 nuclei) to giant (>50 nuclei) cells, but a difference in the size distributions could not be detected (Table 2).
To confirm our results from the TRACP measurements, we examined the ability of different combinations of cells from young and old donors to form resorption pits (Fig. 2A). The left top panel of Fig. 2A shows the response of co-cultured stromal/osteoblastic cells and nonadherent cells from young donors. A few small resorption pits were formed. In the left bottom panel, stromal/osteoblastic cells from an old donor were cultured with nonadherent cells from a young donor. Consistent with our TRACP findings, cells from old donors tended to increase the formation of resorption pits. In the right panels, stromal/osteoblastic cells from young and old donors were cultured with nonadherent cells from old donors. The pattern is virtually identical with the TRACP findings. Despite the culture of identical numbers of cells, the old-old combination produced a dramatic increase in the number of osteoclasts. The pit area was also consistently greater (Fig. 2B). To directly compare osteoclast formation as judged by TRACP activity and resorption pit formation, the same cells were co-cultured in either standard culture dishes or on calcium-phosphate coated coverslips, and TRACP expression and pit formation, respectively, were quantified. In all cases, stromal/osteoblastic cells from old donors increased the number of pits formed (Fig. 3), and for a given co-culture cell combination, the number of TRACP+ cells correlated with the number of pits.
We next measured mRNA levels for RANKL, OPG, and M-CSF at day 7 of stromal/osteoblastic cell culture (i.e., the midpoint of the co-culture). Message levels for RANKL, OPG, and M-CSF in cells from young, adult, and old donors, relative to young animals, are shown in Fig. 4A. Between 6 weeks and 24 months, RANKL and M-CSF expression increased by >5- and 2-fold, respectively. OPG expression gradually fell by 40% with age. Transcriptional activity as measured by PCR-based nuclear run-on assay correlated with mRNA levels (Fig. 4A). Addition of conditioned media from young, adult, and old stromal/osteoblastic cells had no effect on expression of RANKL and OPG (data not shown). Levels of expression reflected age only, suggesting that endogenous soluble factors are not likely responsible for the observed differences in RANKL and OPG expression across age.
The ratio of RANKL:OPG increases with age (p < 0.05, one-way ANOVA) and positively correlates (p < 0.05) with the increase in the ability of stromal/osteoblastic cells to induce osteoclastogenesis (Fig. 4B). Expression of alkaline phosphatase and osteocalcin at day 7 of culture was not affected by age (data not shown).
To determine whether the increase in the ability to induce osteoclastogenesis exhibited by stromal/osteoblastic cells from old donors was mediated, in part, through the RANK/RANKL pathway, we attempted to block recruitment with exogenous administration of OPG. Osteoprotegerin decreased the number of multinucleated TRACP+ cells in all combinations of co-cultured cells by roughly 90%. In the presence of OPG, pit number and pit area decreased by 76% and 88%, respectively.
Throughout our studies, culture of nonadherent cells from old donors with stromal/osteoblastic cells from young, adult, and old donors produced more TRACP+ cells than culture of cells from young or adult donors. To determine whether this is related to an increase in the monocyte/macrophage population (osteoclast precursors), we quantified the number of monocyte/macrophages in the marrow of young, adult, and old animals using fluorescence-activated cell sorting (FACS). With advancing age, the proportion of CD11b+ cells over total nucleated cells increased from 43% in young animals to 57% in aged animals (Fig. 5).
Changes in responsiveness of the nonadherent cell population were examined by studying the effects of increasing doses of RANKL and M-CSF and by measuring the levels of RANK and c-fms expression in cells from young, adult, and old animals. Increasing RANKL and M-CSF increased osteoclast formation in cells from all ages, but the increase was greatest in cells from old donors (Figs. 6 and 7). Expression of RANK and c-fms in CD11b+ marrow cells increased by roughly 2-fold between 6 weeks and 24 months (Fig. 8).
To determine whether the changes in stromal/osteoblastic cells and apparent increase in the osteoclast precursor pool was reflected in vivo, we examined the effect of age on cancellous bone. In the femur, osteoblast surface decreased with age, whereas osteoclast surface remained unchanged (Table 3). The ratio of osteoblasts to osteoclasts decreased. In the lumbar vertebrae, osteoblast surface did not change, but the osteoclast surface increased. Mineralizing surface, mineral apposition, and bone formation rates in the distal femur decreased by 40–70% with advancing age. Although the same trends existed in the vertebrae, none were significant.
Our results indicate that stromal/osteoblastic cells from aged animals have a much greater ability than stromal/osteoblastic cells from young or adult animals to induce osteoclastogenesis as judged by both TRACP expression and resorption pit formation. In the absence of stromal/osteoblastic cells, osteoclast formation is rare. In the presence of stromal/osteoblastic cells from young (6 weeks) and adult (6 months) animals, the number of osteoclasts generated was similar. Thus, the ability of stromal/osteoblastic cells to induce osteoclastogenesis does not change significantly as animals transition from growth to adulthood but does change during the transition from adulthood to old age.
The increased tendency of stromal/osteoblastic cells from old animals to induce osteoclastogenesis is associated with increased expression of RANKL and M-CSF, both of which have been shown to contribute to the recruitment and formation of osteoclasts. Thus, our data suggest that the age-related increase in stromal/osteoblastic cell expression of RANKL and M-CSF is responsible, in part, for the increase in osteoclast formation induced by stromal/osteoblastic cells from old donors. This is supported by the ability of exogenously administered OPG to block osteoclastogenesis equally in young and old animals. Although alterations in OPG expression with age are modest in the mouse, OPG has been reported to decrease significantly with aging in the human. (17) Aging induces a shift in expression of RANKL and OPG that favors osteoclast formation. The age-related increase in M-CSF expression would be expected to further augment osteoclastogenesis. Although the changes in stromal/osteoblastic cell expression of RANKL and OPG associated with aging have not been proven to contribute to increased osteoclast formation and bone loss in vivo, overexpression of RANKL or underexpression of OPG have been shown to lead to severe osteopenia. (12, 13)
Although our results suggest that the stromal/osteoblastic cell is responsible for the age-related increase in RANKL and M-CSF expression, as well as the increase in osteoclast number, it is possible that other cells in the marrow stromal cell population contribute directly or indirectly to the increase in osteoclastogenesis. It is well established that the bone marrow stromal cell population is heterogeneous. Thus, changes in the cell make-up of this population related to aging could also influence osteoclast formation.
In addition to the age-related changes in stromal/osteoblastic cell function, our data suggest that the number of preosteoclasts may also increase with age. Co-culture of stromal/osteoblastic cells from a given aged animal with equal numbers of nonadherent cells from young, adult, and old animals results in incremental increases in the number of osteoclasts generated. This occurs regardless of stromal/osteoblastic cell donor age as long as the same aged stromal/osteoblastic cell is used to activate osteoclast formation. FACS analysis suggests that the monocyte/macrophage population, as judged by expression of CD11b, increases with age by about 30%. The osteoclast precursor population, however, may not necessarily change proportionately. From our dose-response studies, the maximum number of osteoclasts formed from the nonadherent population is higher in cells from old than from young donors by about 2-fold. Taken together, these data suggest that the precursor pool from which osteoclasts are derived increases with aging, findings consistent with those of Perkins et al. (21) in mice and with those of Koshihara et al. (27) in humans. Both of these investigators report an increase in pre-osteoclast numbers in the bone marrow compartment with advancing age. Sudo et al. (28) conclude that the hematopoietic stem cell population gradually increases with age. Despite substantial evidence to suggest that the precursor pool of osteoclasts increases with age, these changes may not necessarily translate into changes in osteoclast number in the bone. For example, the osteoclast surface in the femur, unlike in the vertebrae, does not change with age. This may be a consequence of age-related changes in osteoclast turnover or cell death or in other factors that regulate osteoclast recruitment.
The elevation of RANK and c-fms expression in CD11b+ cells could reflect either an increase in the relative number of precursor cells expressing RANK and c-fms or an increase in the number of receptors/cell. An elevation in the number of receptors per cell would be expected to increase the sensitivity to RANKL and M-CSF and shift the half-maximal response in the dose curves to a lower concentration of ligand. Not being the case, our data are more consistent with the notion that there are more osteoclast precursor cells in aging animals.
The increasing ability of stromal/osteoblastic cells from aging animals to induce osteoclastogenesis and the apparent age-related increase in the osteoclast precursor pool suggest that the balance between osteoblasts and osteoclasts in vivo is likely to be disrupted. This seems to be the case. Our histomorphometry data show that the cancellous bone surface occupied by osteoblasts is lower in the long bones of the aged animal. This is consistent with the findings that the number of marrow-derived osteoprogenitors is lower in the long bones of aged mice. (29) The decrease in ratio of osteoblasts to osteoclasts with aging may well be a consequence of the combined age-related effects of a dysfunctional stromal/osteoblastic cell population that is overexpressing RANKL and M-CSF and an increase in the osteoclast precursor pool.
In the vertebrae, the osteoblast to osteoclast ratio also decreases, but as a consequence principally of an increase in osteoclast number. Site specificity is well known in the skeleton, and the observation that the decrease in osteoblast to osteoclast ratio in the femur is a consequence of a decrease in osteoblast number, whereas in the vertebrae it is a consequence of an increase in osteoclast number, is not surprising. Functionally, the effect is the same. Our findings are similar to those of Erben et al. (30) These investigators reported that, during postmaturational aging in the rat, osteoclast number decreases by 40%, whereas osteoblast number decreases by 76%. The progressive decrease in mineralizing surface in the femur correlates predictably with the fall in osteoblast surface, and the decrease in mineral apposition rate suggests that individual osteoblast bone-forming activity is blunted in the aging animal. Combined, the decrease in mineralizing surface and mineral apposition rate produce a 73% decrease in bone formation rate in the femur, changes that may contribute to the 75% decrease in BV/TV.
In summary, our results suggest that aging increases stromal/osteoblastic cell expression of RANKL and M-CSF and that these changes increase the propensity of the aged stromal/osteoblastic cell to recruit and stimulate the formation of osteoclasts. At the same time the pre-osteoclast pool is increasing, thus further aggravating the imbalance in osteoblasts and osteoclasts in vivo. Coincident with these changes, the efficacy of osteoblasts to form bone is impaired. There are dozens of factors including systemic, local, and inherent changes in the bone cell population that likely contribute to age-related bone loss. Our data suggest that among these are an age-related increase in stromal/osteoblastic cell-induced osteoclastogenesis and an increase in the osteoclast precursor pool size. Whether the changes in stromal/osteoblastic cell function and osteoclast precursor number reflect inherent cell senescence or whether the extracellular milieu in the aged animal alters the metabolic activity of the maturing pre-osteoblast-osteoclast remains to be determined.
This work was supported by the Veterans Affairs Merit Review program.
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