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Keywords:

  • senile osteoporosis;
  • murine;
  • bone formation;
  • bone marrow;
  • aging

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We used the SAMP6 osteoporotic mouse to examine the link between marrow osteogenic potential and in vivo cortical bone formation. SAMP6 marrow supported less in vitro osteogenesis than marrow from SAMR1 controls; SAMP6 mice had a corresponding deficit in endocortical mineralizing surface. This marrow/endocortical defect did not affect the periosteum, where SAMP6 mice had normal to enhanced bone formation.

Introduction: With aging, there may be a reduction in the number or proliferative capacity of bone marrow osteoprogenitors that may contribute to age-related decreases in bone formation. To examine the link between the ability of the marrow to support osteogenesis and age-related changes in bone formation, we measured in vitro and in vivo indices of osteogenesis in a model of osteoporosis, the senescence-accelerated mouse SAMP6.

Materials and Methods: Femora and tibias from SAMP6 and SAMR1 (control) mice were harvested at 2, 4, 6, and 12 months of age (168 bones total). Bone marrow cells were cultured under osteogenic conditions and stained for alkaline phosphatase (ALP) and alizarin red. Dynamic indices of bone formation were assessed histologically from calcein labels.

Results: ALP+ and alizarin red-positive areas were significantly less in cultures from SAMP6 bones versus SAMR1 (p < 0.05), indicating less osteogenic potential. For example, SAMP6 tibial cultures had 21% less ALP+ area and 36% less alizarin red-positive area than SAMR1. Marrow from tibias had 2-fold greater osteogenesis than femoral marrow (p < 0.001). SAMP6 mice had a deficit in endocortical mineralizing surface across all age groups (p < 0.05), but no deficit in mineral apposition rate. Last, despite the marrow and endocortical deficits, SAMP6 mice had normal or slightly increased periosteal bone formation, consistent with their larger bone size.

Conclusion: SAMP6 bone marrow supports less in vitro osteogenesis than SAMR1, consistent with a lower concentration of marrow osteoprogenitors in SAMP6. SAMP6 mice have less endocortical mineralizing surface than SAMR1 at all ages but no detectable deficit in mineral apposition rate, which suggests a reduction in osteoblast number but normal function. Periosteal bone formation is unimpaired in SAMP6 mice, indicating that the marrow/endocortical defect does not affect the periosteal surface.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

AFTER THE AGE of peak bone mass, there is a steady progressive loss of bone in both women and men, with a superimposed, transient period of rapid bone loss in postmenopausal women.(1,2) The progressive loss of bone with aging in women and men represents a deficit in bone formation relative to bone resorption(3) and leads to what has been termed senile or type II osteoporosis.(2,4)

Histological and cell culture data support the notion that reduced osteoblast number rather than reduced osteoblast function is the basis for senile osteoporosis.(5) Trabecular wall thickness is reduced with aging(6,7) and is reduced further in osteoporosis,(8) a change attributed to a reduction in osteoblast recruitment.(8) Moreover, there are reports of age- or osteoporosis-related reductions in dynamic indices of trabecular bone formation, including reduced mineralizing surface and/or mineral apposition rate,(8–10) consistent with decreased osteoblast numbers. Osteoblasts originate from pre-osteoblasts that derive from pluripotent mesenchymal stem cells (MSCs) in the bone marrow stroma.(11–15) Although several recent studies have reported no age-related decrease in the osteogenic potential of adult marrow,(16–18) many studies support the concept that, with aging, the osteogenic potential of bone marrow is reduced by a decrease in the number of MSCs in the bone marrow,(11,19) a decrease in the fraction of MSCs that can differentiate into osteoblast progenitors,(20–23) or a decrease in the proliferative capacity of MSCs.(24) Thus, fewer osteoblast progenitors in the marrow or a reduced ability of progenitors to differentiate may contribute to reduced trabecular bone formation in senile osteoporosis.

Whereas loss of trabecular bone with aging is critical to the increased fracture risk observed in the spine, hip, and forearm,(25) there is greater loss of cortical bone mass with aging than trabecular bone mass.(26) Cortical bone loss occurs at endocortical surfaces as a result of increased resorption with insufficient formation; it does not occur at periosteal surfaces. In fact, with aging in humans, there is an increase in both periosteal and medullary widths,(27–30) indicating that continued periosteal bone formation must occur with aging. However, there is little data on endocortical and periosteal rates of bone formation with aging, and the relationship, if any, between bone formation at these two surfaces is not well understood.

The senescence accelerated mouse, strain P6 (SAMP6) has been described as a model of senile osteoporosis because it exhibits (relative to the SAMR1 control strain(31)) reduced areal BMD,(32,33) reduced rates of trabecular bone formation,(33,34) reduced marrow osteogenesis,(33,34) increased periosteal and medullary widths,(35) and decreased bone strength.(35) An advantage of the SAMP6 model compared with a true aging model is that osteoporotic-like traits are clearly developed by 3–4 months. Jilka et al.(33) showed a direct correlation between marrow osteogenic potential and trabecular bone formation in SAM mice, but there has been no examination of the relationship of marrow osteogenesis to endocortical bone formation. Moreover, previous reports have not focused on differences between SAMP6 and SAMR1 as each strain ages. Our overall goal was to use the SAMP6 model to better characterize the link between age-related changes in bone formation and the ability of the marrow to support osteogenesis. Specifically, we asked the following questions. (1) What is the relationship between marrow osteogenesis and endocortical bone formation in the long bones of SAM mice? (2) Do differences between SAMP6 and SAMR1 persist with aging? (3) Does the defect in endosteal bone formation in SAMP6 affect periosteal bone formation?

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animals and tissue processing

A total of 84 male mice were obtained from our breeding colony after approval by our institutional animal studies committee. SAMR1 and SAMP6 mice were maintained as separate inbred strains from breeders originally provided by the Council for SAM Research (Kyoto University, Kyoto, Japan). Mice were housed up to five per cage, with 12:12-h light:dark cycles and access to standard mouse chow and water ad libitum. Mice were injected with calcein (7 mg/kg, IP; Sigma) 9 and 2 days before death. Mice were killed by CO2 asphyxiation, and their right femurs and tibias were removed immediately for marrow harvest followed by bone fixation in 70% ethanol. We examined a total of 168 bones: femurs and tibias from SAMP6 and SAMR1 (control) mice at 2, 4, 6, and 12 months of age (n = 10–11/group).

Cell culture

We cultured adherent bone marrow stromal cells to assess the relative osteogenic potential of the marrow based on a classical approach.(11) The epiphyses of the bones were removed, and whole marrow was flushed from the diaphyses using a syringe and standard culture medium, which consisted of α-MEM (Mediatech cellgro, MT10022CV; Fisher Scientific) supplemented with 10% heat-inactivated FBS (SH30071.03; Hyclone Laboratories) and penicillin/streptomycin (Washington University Tissue Culture Support Center). All FBS used in this study was from a single manufacturer's lot. The marrow was passed through a 70-μm filter before being centrifuged at 1150 rpm for 10 minutes at 4°C. The supernatant was removed, and the cell pellet was resuspended in fresh standard medium. The resuspended cells were counted using a hemacytometer, and then cells were diluted to a concentration of 1.8 × 106 cells/ml. Typical yields were 80 × 106 cells per femur and 20 × 106 cells per tibia.

Cells from individual bones were plated on 12-well plastic culture plates at a concentration of 3.6 × 106 cells/well. We did not pool cells from different bones or mice. We plated 5–6 replicate wells per femur and 2–4 wells per tibia. Each plate contained samples from one SAMR1 and one SAMP6 mouse. Plates were placed in a humidified incubator with a 5% CO2 atmosphere at 37°C. On day 3, nonadherent cells were discarded, and the medium was changed to one that supports osteoblast differentiation, that is, standard media plus 13 mM β glycerophosphate (G-9891; Sigma) and 50 mg/liter ascorbic acid (A-4544; Sigma). This osteogenic medium was used throughout the rest of the culture period, with complete changes twice per week.

Cell staining and quantitation of in vitro osteogenesis

On day 14, one-half of the plates from each experimental group were stained for the alkaline phosphatase (ALP) enzyme. In this assay, positive stained fibroblastic colonies (sometimes termed CFU-F/ALP+) represent osteoprogenitor colonies,(22,36) and the percent positive area is taken as an in vitro index of the osteogenic potential of the bone marrow in vivo. Staining was done using a commercial kit (85-L; Sigma). Each well was first rinsed with 1× PBS. The wells were fixed for 5 minutes in a solution of 60% acetone (A4206; Sigma), 39.2% DI water, and 0.8% citrate concentrate solution (85-4C; Sigma). The wells were then rinsed with water and stained using an alkaline-dye mixture composed of 96% water, 4% naphthol AS-MX phosphate alkaline solution (85-5; Sigma), and one fast blue RR salt capsule (FBS-25; Sigma). Cells were exposed to the stain for 30 minutes at room temperature in the absence of light. The wells were rinsed with water and counterstained with Mayer's hematoxylin solution (MHS-1; Sigma) for 10 minutes. Finally, the wells were rinsed with water and dried in air.

On day 28, mineral deposits on the remaining plates were stained with alizarin red (A5533; Sigma). In this assay, positive stained colonies (sometimes termed CFU-OB) represent osteoblast colonies that have deposited matrix that has become mineralized(33,37); again, the percent positive area is taken as an in vitro index of the osteogenic potential of the bone marrow in vivo. First, the wells were rinsed with 1× PBS, and the cells were fixed with 10% neutral buffered formalin (245-684; Fisher Scientific) for 5 minutes at room temperature. After fixation, the formalin was removed by rinsing with water before exposing the wells to a 2% alizarin red solution (pH 4.1-4.3). After 10 minutes, the wells were rinsed with water and dried in air.

Images of each well were obtained using a digital camera (DC 300; Leica) mounted to a dissecting microscope (Photomakroskop M400; Wild Heerbrugg). Camera settings, magnification, and lighting conditions were standardized. The images were loaded into an analysis program (ImageJ; NIH) as grayscale images (0-255 arbitrary density). Threshold values separating positive cells from background were chosen based on a sensitivity analysis of a subset of wells and applied uniformly to all images. Threshold values (150 for ALP, 140 for alizarin red) were set relatively high to minimize false positives, and consequently, some regions of light staining (e.g., the periphery of colonies) were excluded. Three parameters were determined: percent positive area (100 × positive area/total well area), mean positive density (average density of positive stained pixels), and total stain index (percent positive area × mean positive density). Values from replicate wells were averaged to get a single value of each parameter for each bone.

Histomorphometric analysis of in vivo bone formation

Femora and tibias were dehydrated in ascending concentrations of ethanol (70-100%) and embedded in methylmethacrylate (Osteo-Bed; Polysciences) using standard procedures for undecalcified bone. Transverse sections at the midshaft were cut at 100 μm thickness using a saw microtome (SP 1600; Leica) and mounted on glass slides. Slides were viewed under UV excitation on a fluorescence equipped microscope (Leitz Orthoplan). Dynamic measures of cortical bone formation were determined based on calcein labels using commercial software (Osteomeasure; Osteometrics). We determined percent single- (sLS/BS) and double-labeled (dLS/BS) bone surface, mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) based on histomorphometry standards.(38) Endocortical (Ec) and periosteal (Ps) surfaces were analyzed separately.

Data analysis

Differences between groups were assessed by ANOVA (Statview 5.0; SAS Institute), with strain (SAMP6, SAMR1), age (2, 4, 6, 12 months), and bone (femur, tibia) as factors. Fisher's protected least squares differences test was used for posthoc multiple comparisons. Significance was defined as p ≤ 0.05. Initial assessment was done using a three-factor ANOVA, with bone as a repeated (paired) factor, followed by a two-factor ANOVA for femur and tibia separately. Marrow cultures were compared using a paired (repeated measures) analysis comparing SAMP6 versus SAMR1 samples from the same culture plates. Correlations between in vitro and in vivo outcomes and between periosteal and endocortical formation indices were examined using linear regression analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

In vitro marrow osteogenesis

Marrow cultures from SAMP6 mice had significantly less ALP and alizarin red staining compared with SAMR1 controls, indicating less osteogenic potential (Table 1). Differences between strains were consistently greatest at 4 months of age (Fig. 1) and were similar for both femoral and tibial cultures. Specifically, ALP+ area in SAMP6 cultures was less than in SAMR1 cultures by an average of 28% for the femur (p = 0.003) and 21% for the tibia (p = 0.007), with peak differences at 4 months of 47% and 54%, respectively (p < 0.05). Alizarin red-positive area in SAMP6 cultures was less then in SAMR1 by an average of 36% for the tibia (p = 0.005), with a peak difference of 70% at 4 months (p < 0.05). Alizarin red-positive area in femoral cultures did not differ between strains (p = 0.31), which we attribute to the few numbers of positive colonies for both groups (1-3% positive area).

Table Table 1. Measures of In Vitro Osteogenesis in Bone Marrow Cell Cultures From SAMR1 (Control) and SAMP6 mice at 2, 4, 6, and 12 Months of Age (Mean ± SD; n = 10-11/group)
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Figure Fig. 1.. Results from in vitro cell cultures from SAMR1 and SAMP6 mice at 4 months of age showing reduced osteogenic potential in SAMP6 bone marrow. Cells were stained for ALP and alizarin red as indicators of osteoprogenitor and osteoblast colonies, respectively. (A) Representative images of individual wells. Each SAMP6 mouse was paired with a SAMR1 control; marrow cells from the pair were cultured on the same plate. Images are shown from two pairs of mice whose values were near the group means. (B) Mean ± SD values for positive stained area. Note the significantly greater positive areas for tibial cultures. *Significant difference between SAMR1 and SAMP6 (p < 0.05).

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Decreases in stained area and slight decreases in staining density together contributed to significantly lower total stain index in SAMP6 cultures. ALP (p < 0.001) and alizarin red (p = 0.034) staining density was reduced in SAMP6 versus SAMR1 by 3% for femoral cultures, whereas alizarin red staining density was reduced by 3% in tibial cultures (p = 0.032); ALP staining density in tibial cultures did not differ between strains (p = 0.42). ALP total staining index was decreased by 30% in femoral (p = 0.002) and 26% in tibial (p = 0.003) cultures from SAMP6 versus SAMR1, with peak differences at 4 months of 50% and 61%, respectively (p < 0.05). Alizarin red total stain index was decreased by an average of 39% in tibial cultures from SAMP6 versus SAMR1 (p = 0.005), with a peak difference of 70% at 4 months. Alizarin red total stain index in femoral cultures did not differ between strains (p = 0.80), reflecting the lack of difference in stained area.

We consistently observed less staining in femoral cultures than in tibial cultures from the same mice. Compared with tibial cultures, femoral cultures had 59% less ALP+ and 45% less alizarin red-positive area (p < 0.001), 3% less ALP and 5% less alizarin red staining density (p < 0.001), and 57% less ALP and 49% less alizarin red total stain index (p < 0.001).

There were no consistent changes in in vitro staining with aging, and differences between strains were generally observed at all ages. In femoral cultures, ALP+ area (p = 0.096) and alizarin red-positive area (p = 0.023) showed evidence of decreasing from 2 to 4 months, but then increased from 4 to 6 or 4 to 12 months. Tibial cultures did not vary significantly with age (ALP, p = 0.44; alizarin red, p = 0.89).

In vivo bone formation

Histomorphometric analysis indicated that, on the endocortical surface, SAMP6 mice had a deficit in mineralizing surface but no deficit in mineral apposition rate (Table 2; Fig. 2). These findings were noted for both femur and tibia at the earliest age (Fig. 3) and persisted up to 12 months. (Because formation indices did not differ between femur and tibia, statistical analysis of pooled data are presented.) Endocortical mineralizing surface (Ec.MS/BS) was reduced by an average of 43% in SAMP6 bones compared with SAMR1 (p < 0.001) because of a significant reduction in single-labeled surface (sLS/BS, p < 0.001) but not double-labeled surface (dLS/BS, p = 0.24). In contrast, mineral apposition rate (Ec.MAR) was not different between strains (p = 0.47). Bone formation rate (Ec.BFR/BS) was significantly less in SAMP6 versus SAMR1 (p = 0.048) because of the reduced mineralizing surface.

Table Table 2. Endocortical Bone Formation Parameters in Long Bones From SAMR1 (Control) and SAMP6 Mice at 2, 4, 6, and 12 Months of Age (Mean ± SD; n = 7-11/group)
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Figure Fig. 2.. Fluorescent photomicrographs of representative femoral cross-sections from SAM mice showing impaired endocortical but normal periosteal calcein labeling in SAMP6 mice compared with SAMR1 controls. (Double-labeled surfaces seen at 10× objective magnification are noted by arrowheads.) At 2 months, SAMP6 had markedly reduced single- and double-labeled endocortical surface compared with SAMP6, whereas more modest differences were evident at 4 months. Tibial cross-sections revealed similar findings (data not shown). Periosteally, femora from SAMP6 had increased single-labeling and equivalent double-labeling compared with SAMR1 at 2 months, and there were no significant differences at 4 months. In both strains, endocortical and periosteal labeling decreased markedly from 2 to 4 months.

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Figure Fig. 3.. Mineralizing surface (mean ± SD) at 2 months of age showing that SAMP6 bones had less mineralizing surface endocortically but normal mineralizing surface periosteally. *Significant difference between SAMR1 and SAMP6 (p < 0.05).

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In contrast to the deficit in endocortical mineralizing surface, periosteal formation indices were not impaired in SAMP6 mice (Table 3; Fig. 2). In the femur, there was evidence for increased periosteal formation in SAMP6 mice compared with SAMR1, although findings were not consistent at all ages. Femoral single-labeled bone surface (Ps.sLS/BS) was significantly greater in SAMP6 compared with SAMR1 (p = 0.040), whereas periosteal mineralizing surface (Ps.MS/BS, p = 0.49), mineral apposition rate (Ps.MAR, p = 0.061), bone formation rate (Ps.BFR/BS, p = 0.26), and double-labeled bone surface (Ps.dLS/BS, p = 0.40) were not different between strains. There was a trend for greater femoral mineralizing surface in SAMP6 mice at 2, 6, and 12 months, but not at 4 months. In the tibia, there were no significant differences between SAMP6 and SAMR1 in any periosteal formation index (p > 0.05). There was a trend for higher periosteal formation indices in SAMP6 tibias versus SAMR1 at 2 months of age (Fig. 3), with a reversal of this trend at 4 months.

Table Table 3. Periosteal Bone Formation Parameters in Long Bones From SAMR1 (control) and SAMP6 Mice at 2, 4, 6, and 12 Months of Age (Mean ± SD; n = 7-11/group)
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For both SAMP6 and SAMR1 strains, formation indices were highest in the youngest mice (Figs. 2 and 4). Endocortical mineralizing surface, mineral apposition rate, and bone formation rate were significantly higher at 2 months than at 4, 6, and 12 months (p < 0.001). There were no significant changes in endocortical formation parameters after 4 months (p > 0.05). Similarly, periosteal mineralizing surface, mineral apposition rate, and bone formation rate were significantly higher at 2 months than at 4, 6, and 12 months (p < 0.05). Periosteal indices continued to decrease up to 12 months; mineralizing surface and mineral apposition rate were significantly less at 12 months than at 4 months (p < 0.05).

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Figure Fig. 4.. Mineralizing surface (mean ± SD) from tibias of SAMR1 and SAMP6 mice at 2, 4, 6, and 12 months showing decreases with age. Formation indices on the endocortical and periosteal surfaces were 2- to 3-fold greater at 2 months of age compared with other ages (except for the periosteal surface of SAMR1, where values at 2 and 4 months did not differ). Endocortical surface did not decrease after 4 months, whereas periosteal mineralizing surface decreased from 4 to 12 months.aSignificantly different from 2-month-old group (p < 0.05).bSignificantly different from 4-month-old group (p < 0.05).

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Periosteal measures of bone formation were positively correlated (p < 0.05) with endocortical measures. However, correlations were weak: mineralizing surface, r2 = 0.07; mineral apposition rate, r2 = 0.20; bone formation rate, r2 = 0.13. Thus, there was little evidence for tight coupling of bone formation on endocortical and periosteal surfaces.

Correlations between in vitro osteogenesis and in vivo bone formation

Measures of in vivo endocortical bone formation correlated poorly with measures of in vitro osteogenesis when data from individual bones were used in the regression, whereas stronger correlations were observed when group means were analyzed. For all specimens pooled (n = 145), endocortical mineralizing surface (Ec.MS/BS) correlated significantly but extremely weakly with ALP+ area (p = 0.05; r2 = 0.03) and ALP total stain index (p = 0.037; r2 = 0.03). These correlations were improved slightly when femoral data were analyzed separately, but the coefficient of determination (r2) was never >0.18; correlations for tibial data separately were not significant. There were no other significant correlations between endocortical mineralizing surface or mineral apposition rate versus in vitro indices of osteogenesis. To reduce the effects of assay variability for individual samples, we also analyzed correlations between group means. For femoral samples, mean mineralizing surface correlated strongly with mean ALP+ area (r2 = 0.71), whereas for tibial samples, the correlation was less strong (r2 = 0.30).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Our study was designed to address three questions. First, what is the relationship between marrow osteogenesis and endocortical bone formation in the long bones of SAMP6 mice? We observed an overall reduction in the ability of the marrow to support in vitro osteogenesis in femurs and tibias of SAMP6 mice compared with SAMR1 controls. This deficit corresponds to an in vivo decrease in endocortical mineralizing surface but not mineral apposition rate in these same bones. Thus, our overall findings support a link between the osteogenic potential of the marrow assayed in vitro and the extent of the adjacent mineralizing surface in vivo. However, this relationship was weak when values from individual bones were correlated and was moderately strong only when mean values were correlated. We attribute this in part to the large variability in both the in vitro and in vivo measures of osteogenesis. In particular, we believe that use of an in vitro assay that requires 14–28 days of incubation has limitations as an index of the osteogenic capacity of the marrow in vivo.

Our findings are consistent with previous reports of reduced in vitro osteoblastogenesis and reduced in vivo trabecular bone formation in SAMP6 mice.(33,34) Notably, Jilka et al.(33) reported 3-fold fewer osteoblastic colonies (CFU-OB) in femoral bone marrow cultures from SAMP6 mice at 3–4 months age, similar to the relative differences in ALP and alizarin staining we observed at 4 months (Fig. 1). They further showed that the in vitro marrow deficit correlated significantly to reduced in vivo bone formation in vertebral trabecular bone. We have extended these findings by analyzing femora and tibias separately and by relating the osteogenic potential of the bone marrow to bone formation on the adjacent endocortical surface. Jilka et al.(33) also noted a significant reduction in osteoblast numbers in trabecular bone. Whereas we did not quantify osteoblast numbers on the endocortical surface, the previous findings with respect to trabecular bone and the reduced mineralizing surface we observed lead us to hypothesize that there are fewer endocortical osteoblasts in SAMP6 mice.

Second, we asked the following: do differences between SAMP6 and SAMR1 persist with aging? Our data indicate that differences between strains are evident at 2 months and largely are maintained through 12 months of age. Both strains had a sharp reduction in in vivo bone formation indices from 2 to 4 months age, followed by no further declines in endocortical formation but modest declines in periosteal formation indices from 4 to 12 months. This is consistent with the end of rapid growth in mice near 3 months.(39) The in vitro data showed evidence of declines from 2 to 4 months in marrow osteogenesis in the femur, but we observed no evidence of age-related declines in the tibia. Notably, we found no consistent declines in in vitro osteogenesis or endocortical bone formation in either strain from 4 to 12 months, a period representing young adulthood to middle age. We did not design our experiment to include more advanced ages because of concerns about longevity in SAMP6 mice. (Previous reports indicated an average lifespan of 10 months in SAMP mice,(40) although we have noted no increase in mortality in a small number of mice aged 12 to 24 months.)

Third, we asked the following: does the defect in endocortical bone formation affect periosteal bone formation? Our data indicate that, in contrast to the deficit in endocortical mineralizing surface, periosteal mineralizing surface was normal or slightly enhanced in SAMP6 mice compared with SAMR1. In the femur, periosteal mineralizing surface was increased in SAMP6 mice compared with SAMR1 at 2, 6, and 12 months, but not at 4 months. In the tibia, SAMP6 had a trend for increased formation at 2 months, but not at other ages. These results clearly indicate that a deficit in the ability of the marrow to support osteogenesis does not impair periosteal bone formation in SAMP6 mice. Moreover, the poor correlations between endocortical and periosteal formation indices that we observed suggests that formation on these two surfaces is not tightly coupled. Thus, lower endocortical formation secondary to a marrow defect neither stimulated nor suppressed periosteal formation.

Taken together with our previous morphological findings,(35) the bone formation data indicate that long bones from SAMP6 mice have increased periosteal apposition compared with SAMR1 early in life, leading to increased periosteal width by 4 months of age. Subsequently, SAMP6 and SAMR1 mice have comparable rates of periosteal apposition, thus maintaining, but not significantly increasing, their relative, external size differences. Endocortically, a reduced rate of bone formation in SAMP6 mice early in life is consistent with the larger medullary area observed at 4 months.(35) Interestingly, the relative difference in the medullary area between strains is maintained from 4 to 12 months, despite the diminished endocortical formation in SAMP6, which suggests that SAMP6 bones also have reduced endocortical resorption. Although we did not assess endocortical resorption, reduced trabecular osteoclast surface has been reported in SAMP6 mice,(33) supporting the notion that the endosteal surface of SAMP6 skeleton is in a state of low turnover.

One unexpected finding was that tibial cultures produced approximately twice the number of osteoprogenitor and osteoblast colonies as femoral cultures from the same mice (both plated at 3.6 × 106 cells/well). We do not know the basis for this bone-specific difference in marrow osteogenic potential, but we hypothesize that it is related to the total number of marrow cells. We typically obtained three to four times more total cells from the femur because of its larger medullary cavity. Thus, the relative femoral deficit in in vitro osteogenesis per million cells plated is offset by an increase in the total number of marrow cells. Possibly, each marrow cavity has an appropriate number of osteoprogenitor cells for the endosteal bone surface that it supports, but the concentration of these cells in the marrow may be diluted in bones with larger marrow cavities. This unproven concept suggests that the deficit in marrow osteogenesis in the SAMP6 mouse (per million cells plated) may reflect, in part, the larger marrow cavity in the SAMP6 bones compared with SAMR1.(35) Nevertheless, even if the total osteogenic potential in SAMP6 bone marrow is normal, there remains a deficit in endocortical and trabecular(33) bone formation. This in turn suggests that the deficit in bone formation in SAMP6 relates more to a defect in differentiation in vivo (perhaps related to interleukin-11(41)) rather than a deficit in osteoprogenitor number. Further work is needed to resolve this issue.

Our data support the following conclusions regarding the femur and tibia of SAMP6 mice compared with SAMR1. First, SAMP6 marrow supports less in vitro osteogenesis (per million cells plated), consistent with a defect in the concentration of osteoprogenitors cells in the bone marrow of SAMP6 mice. Second, SAMP6 mice have a persistent reduction in endocortical mineralizing surface from 2 to 12 months, but no detectable deficit in mineral apposition rate. Finally, periosteal bone formation is normal or marginally enhanced in SAMP6 mice, which indicates that the defect on the endocortical surface does not impair formation on the periosteal surface.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study was supported by National Institute of Arthritis, Musculoskeletal and Skin Diseases Grant AR47867 (MJS).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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