Sclerostin Immunoreactivity Increases in Cortical Bone Osteocytes and Decreases in Articular Cartilage Chondrocytes in Aging Mice

Abstract

Sclerostin is a 24-kDa secreted glycoprotein that has been identified as a negative modulator of new bone formation and may play a major role in age-related decline in skeletal function. Although serum levels of sclerostin markedly increase with age, relatively little is known about whether cells in the skeleton change their expression of sclerostin with aging. Using immunohistochemistry and confocal microscopy, we explored sclerostin immunoreactivity (sclerostin-IR) in the femurs of 4-, 9-, and 24-month-old adult C3H/HeJ male mice. In the femur, the only two cell types that expressed detectable levels of sclerostin-IR were bone osteocytes and articular cartilage chondrocytes. At three different sites along the diaphysis of the femur, only a subset of osteocytes expressed sclerostin-IR and the percentage of osteocytes that expressed sclerostin-IR increased from approximately 36% to 48% in 4- vs. 24-month-old mice. In marked contrast, in the same femurs, there were ~40% fewer hypertrophic chondrocytes of articular cartilage that expressed sclerostin-IR when comparing 24- vs. 4-month-old mice. Understanding the mechanism(s) that drive these divergent changes in sclerostin-IR may provide insight into understanding and treating the age-related decline of the skeleton.

Keywords: aged; articular cartilage; cortical bone; hypertrophic chondrocyte; joint; osteocyte subtype.

Figure 1.

Detectable levels of sclerostin immunoreactivity (sclerostin-IR) are present in osteocyte cell bodies, osteocyte dendrites/canaliculi, and the periosteum in the cortical bone of the mouse femur. (A) Low power magnification, medial-lateral view of an H&E section of a young (4-month-old) male mouse femur. Boxed region indicates the mid-diaphyseal region where images in Figs. 1B, 1C, 2 and 3 were obtained. (B) Representative mid-power confocal image showing sclerostin-IR (fire red) in the osteocyte cell bodies and periosteum. (C) High-power confocal image showing sclerostin-IR within an osteocyte cell body (arrowhead) and the dendrite/canaliculi process (arrow). Scale (B) 25 µm; (C) 10 µm.

Figure 2.

Sclerostin-IR in mineralized bone and adjacent periosteum (marked with black double-ended line; right), along with serially adjacent sections processed as absorption controls. (A) High-power confocal images of sclerostin-IR in mouse femoral cortical bone of young (4-month-old) and (B) old (24-month-old) adult animals. Note that sclerostin-IR is present in the osteocyte cell body, dendrite/canaliculi network, and the periosteum of young and old animals. The periosteum layers—cambium and fibrous—are demarcated by a white dashed line. (C) and (D) are serial sections of (A) and (B), respectively, and were processed the same as (A & B) except that the sclerostin antibody was replaced by a recombinant mouse SOST. Abbreviations: C, cambium layer; F, fibrous layer. Scale, 10 µm.

Figure 3.

A subset of osteocytes that bind phalloidin (which binds to F-actin and that is expressed by osteocytes) also express detectable levels of sclerostin-IR. (A) Mid-power confocal image of cortical bone from a 4 month old animal showing co-localization of phalloidin (green) and sclerostin-IR (red). The periosteal surface is on the right and the endosteal surface is on the left. (B) High power confocal image of DAPI (blue) / phalloidin (green) overlay. Since >95% of cells in cortical bone are osteocytes there is nearly a 1:1 overlap of phalloidin and DAPI in cortical bone. (C) High power confocal image shows that the co-localization of phalloidin (green) and sclerostin (red) is only present in a subset of osteocytes (closed arrowhead). Sclerostin-negative osteocytes are demarcated by open arrowhead. (D-F) High power confocal image of phalloidin (green), DAPI (blue), and sclerostin (fire red) showing that when sclerostin-IR signal is maximized, there is clear distinction between sclerostin-IR positive and sclerostin-IR negative osteocytes. Scale bar (A) 30 mm; (B) 20 mm; (C) 10 mm; (D-F) 8 mm.

Figure 4.

The percentage of osteocytes that express detectable levels of sclerostin-IR increases with age. (A) Low-power magnification, medial-lateral view H&E section of a young mouse femur showing the regions where osteocyte sclerostin-IR was analyzed in young (4-month-old), middle-aged (9-month-old), and old (24-month-old) adult C3H male mice. Three sites were selected along the diaphysis: the proximal (4 mm from the proximal head), mid-diaphysis (8 mm from the proximal head), and distal (3 mm from the distal head). (B–D) High-power confocal images of sclerostin-IR (upper panel), DAPI (middle panel) and sclerostin/DAPI overlay (lower panel) in mouse femoral cortical bone of young (B) middle-aged (C) and old (D) adult animals. (E) Histogram illustrating the percentage of osteocytes expressing detectable levels of sclerostin-IR in cortical bone in young, middle-aged, and old adult diaphyseal cortical bone. All ages were statistically significantly different from each other at each site, except middle-aged vs. old at the proximal diaphyseal region (p>0.06). The numbers in parentheses above each bar indicate the number of animals analyzed for each age group. Bars represent the mean ± SEM; p<0.05 after a one-way ANOVA, Tukey’s post-hoc test. Scale (A) 1 mm; (B–D) 5 µm.

Figure 5.

Sclerostin-IR in hypertrophic chondrocytes present in articular cartilage decreases in the old as compared with that in the young adult mice. (A) Representative low-power H&E section of the proximal end of the mouse femur in medial-lateral view. Boxed areas correspond to where images were obtained and analysis was performed. (B, D) Mid-power confocal image showing sclerostin-IR (white) expressed by hypertrophic chondrocytes in the articular cartilage (designated by AC) in young (4-monthold, B) and old (24-month-old, D) adult animals. (C, E) High-magnification confocal overlay of sclerostin-IR (red) and DAPI (blue) show that lower levels of sclerostin-IR and lower number of sclerostin-expressing hypertrophic chondrocytes are present in the old vs. young adult animals. (F) Histograms showing the decline in the number of sclerostin-IR hypertrophic chondrocytes in the old vs. young animals. (G) Histogram showing that the relative fluorescence of sclerostin-IR in chondrocytes declines by approximately 40% in the old vs. young animals. Bars represent mean ± SEM; p<0.05 after a one-way ANOVA, Tukey’s post-hoc test. Abbreviations: AC, articular cartilage; MB, mineralized bone. Scale (A) 1 mm; (B, D) 100 µm; (C, E) 10 µm.