Old age causes de novo intracortical bone remodeling and porosity in mice

Abstract

Decreased cortical thickness and increased cortical porosity are the key anatomic changes responsible for osteoporotic fractures in elderly women and men. The cellular basis of these changes is unbalanced endosteal and intracortical osteonal remodeling by the osteoclasts and osteoblasts that comprise the basic multicellular units (BMUs). Like humans, mice lose cortical bone with age, but unlike humans, this loss occurs in the face of sex steroid sufficiency. Mice are therefore an ideal model to dissect age-specific osteoporotic mechanisms. Nevertheless, lack of evidence for endosteal or intracortical remodeling in mice has raised questions about their translational relevance. We show herein that administration of the antiosteoclastogenic cytokine osteoprotegerin to Swiss Webster mice ablated not only osteoclasts, but also endosteal bone formation, demonstrating the occurrence of BMU-based endosteal remodeling. Femoral cortical thickness decreased in aged male and female C57BL/6J mice, as well as F1 hybrids of C57BL/6J and BALB/cBy mice. This decrease was greater in C57BL/6J mice, indicating a genetic influence. Moreover, endosteal remodeling became unbalanced because of increased osteoclast and decreased osteoblast numbers. The porosity of the femoral cortex increased with age but was much higher in females of both strains. Notably, the increased cortical porosity resulted from de novo intracortical remodeling by osteon-like structures. Age-dependent cortical bone loss was associated with increased osteocyte DNA damage, cellular senescence, the senescence-associated secretory phenotype, and increased levels of RANKL. The demonstration of unbalanced endosteal and intracortical remodeling in old mice validates the relevance of this animal model to involutional osteoporosis in humans.

Keywords: Aging; Bone Biology; Cellular senescence; Osteoporosis.

Figure 1. Age-related bone loss is greater in B6 than in CB6F1 mice.

Bone mineral density was determined in the (A) femur, (B) lumbar vertebrae, and (C) total body excluding the head. Number of mice analyzed, ordered by increasing age: female B6 (20, 20, 19), male B6 (10, 10), female CB6F1 (10, 10, 10), male CB6F1 (9, 9, 8). *P < 0.05 vs. 6- to 7-month-old sex-matched animals of the same strain, **P < 0.05 vs. 6- to 7-month and 18- to 19-month sex-matched animals of the same strain, as determined by two-tailed t test (B6 males) or one-way ANOVA (B6 females and CB6F1 females and males).

Figure 2. Age-related decrease in cortical thickness is greater in B6 than in CB6F1 mice.

Cortical thickness was determined at the (A) mid-diaphysis and (B) distal metaphysis of the femur, as well as at the (C) anterior vertebra (L4), as indicated by the red arrows and pink fill in the microCT images. Number of mice analyzed, ordered by increasing age: female B6 (8, 8, 7), male B6 (10, 10), female CB6F1 (8, 10, 10), male CB6F1 (9, 9, 8); n = 5 for metaphyseal cortical thickness of 26-month female B6 mice because of trabecularization of the endosteal boundary. *P < 0.05 vs. 6- to 7-month-old sex-matched animals of the same strain, **P < 0.05 vs. 6- to 7-month and 18- to 19-month sex-matched animals of the same strain, †P < 0.05 vs. 19-month sex-matched animals of the same strain, as determined by two-tailed t test (B6 males)or one-way ANOVA (B6 females and CB6F1 females and males).

Figure 3. Osteoprotogerin eliminates endosteal bone formation in adult mice.

Female 7-month-old Swiss-Webster mice were injected with vehicle (Veh) or OPG. (A) Longitudinal sections of the femur stained for TRAP to visualize osteoclasts (red arrowheads). Scale bar: 20 μm. (B) Unstained cross-sections made at the distal metaphysis (met) and middiaphysis (dia) to visualize alizarin (red) and calcein (yellow) fluorochromes. White arrowheads, sites of endosteal bone formation; yellow arrowhead, site of bone formation within a cortical pore. Original magnification: 40×.

Figure 4. Multiple fluorochrome labeling of femoral bone during growth and adulthood.

Female B6 mice were given calcein, alizarin, and demeclocycline at the indicated ages (A, left hand panel). Nondecalcified cross sections were prepared at 11 months of age from the (B) proximal, (C) diaphyseal, and (D) distal metaphyseal parts of the femur as indicated (A, middle panel). Photomicrographs from two mice, shown side-by-side (B-D), are oriented as illustrated (A, right panel); M, medial; P, posterior; L, lateral; A, anterior. Scale bar: 200 μm. (E) Representative high-power images from proximal (left), diaphyseal (middle), and metaphyseal (right) cross sections. White arrowheads, sites of endosteal bone formation. Scale bar: 50 μm.

Figure 5. Endocortical remodeling becomes unbalanced in favor of resorption with advancing age.

(A) Unstained cross-sections of the distal metaphysis (met) and the diaphysis (dia) with endosteal surface labeled by tetracycline (yellow arrowheads) given at 7 and 3 days before euthanasia of 6- and 26-month-old female B6 mice. Original magnification: 40×. Right hand panels depict high power brightfield (upper) and fluorescent images of the area denoted by the box. (B) Toluidine blue–stained longitudinal sections with irregular scalloped cement lines (yellow arrowheads) in 7- and 21-month-old female B6 mice. Scale bar: 20 μm. (C–H) Histomorphometric determination of endosteal osteoclast number (N.Oc./B.Pm), osteoblast number (N.Ob/B.Pm), mineralizing surface (MS/BS), wall width (W.Wi), mineral apposition rate (MAR), and bone formation rate (BFR) in 7-month-old mice (n = 9) and 21-month-old (n = 10) mice. *P < 0.05 vs. 7-month-old animals by two-tailed t test.

Figure 6. Femoral cortical porosity increases with age in females, but not males, in B6 and CB6F1 mice.

(A) Inverse microCT images of femora (proximal at top) from female B6 mice, excluding epiphyses. Cortical voids are depicted in gray within a blue bone matrix. Lines show location of single microCT slices (before binarization). Orientation of slices: P, posterior; A, anterior; M, medial; L, lateral. (B) Quantification of porosity of the distal half of the femur, as indicated by the red box. Number of mice analyzed (ordered by increasing age): female B6 (8, 8, 7); male B6 (10,10); female CB6F1 (8, 10, 10); male CB6F1 (8, 10, 10). *P < 0.05 vs. 6-month B6 females; †P < 0.05 vs. 7-month and 19-month CB6F1 females, using a permutation approach to Student’s two-tailed t test.

Figure 7. The femoral cortex of young-adult B6 female mice contains capillaries but does not exhibit intracortical remodeling.

(A–D) Representative photomicrographs of pores in the femoral cortex of 7-month-old female B6 mice viewed in nondecalcified toluidine blue–stained (A–C) or TRAP-stained (D) longitudinal sections. Cement lines are marked by black arrowheads. (E) Yellow demeclocycline fluorescence labeling of small pores (yellow arrowheads) in a longitudinal section from an 11-month-old female B6 mouse given this fluorochrome at 2.6 months of age, as described in Figure 4. Scale bars: 20 μm (C–E).

Figure 8. De novo osteonal remodeling in 21-month-old female B6 mice.

(A–G) Representative photomicrographs of pores in the femoral cortex of 21-month-old female B6 mice viewed in nondecalcified toluidine blue–stained sections. The right portion of D and E show an unstained adjacent section under fluorescence illumination to visualize tetracycline (white arrowheads) given at 7 and 3 days before euthanasia. B is a high-power image of the area denoted by the box in A. Arrowheads mark red blood cells (yellow), cement lines (green), canalliculi (turquoise), and TRAP-positive osteoclasts (red); ad, adipocytes. Scale bars: 20 μm. (H and I) Histomorphometric determination of pore number and pore area per cortical bone area (B.Ar) in 7-month-old (n = 8) and 21-month-old (n = 10) female B6 mice.*P < 0.01 vs. 7-month-old mice using a permutation approach to two-tailed Student’s t test.

Figure 9. Osteocyte-enriched bone from aged mice exhibits markers of cellular senescence.

Western blot analysis and quantification of markers of DNA damage (γ-H2AX) and cellular senescence (GATA4 and p62) in femoral extracts from female (A and B) or male (D and E) B6 mice. Transcript levels of the senescence marker p16^ink4a (p16), and the SASP proteins Sdf1 and Mmp-13 were determined by qPCR of extracts from 7-month-old (n = 9) and 21-month-old (n = 10) female B6 mice (C), or from 6-month-old (n = 9) and 20-month-old (n = 9) male B6 mice (F). *P < 0.05 vs. 7-month-old sex-matched animals by two-tailed Student’s t test, or by Rank Sum test, after adjustment of P values for repeated measurements by the Benjaminji-Hochsberg method.

Figure 10. Osteocyte-enriched bone from aged mice exhibits changes in the expression of factors involved in the regulation of osteoclast and osteoblast differentiation.

(A–L) Transcript levels and femoral marrow plasma (mp) protein levels of indicated genes were determined by qPCR or ELISA, respectively, using extracts from the 7-month-old (n = 9) and 21-month-old (n = 10) female B6 mice of described in Figure 9. *P < 0.05 vs. 7-month-old mice by two-tailed t test or Rank Sum test. P values for the effect of age on transcript levels were adjusted for repeated measurements by the Benjaminji-Hochsberg method.