Metaphyseal and diaphyseal bone loss in the tibia following transient muscle paralysis are spatiotemporally distinct resorption events

Abstract

When the skeleton is catabolically challenged, there is great variability in the timing and extent of bone resorption observed at cancellous and cortical bone sites. It remains unclear whether this resorptive heterogeneity, which is often evident within a single bone, arises from increased permissiveness of specific sites to bone resorption or localized resorptive events of varied robustness. To explore this question, we used the mouse model of calf paralysis induced bone loss, which results in metaphyseal and diaphyseal bone resorption of different timing and magnitude. Given this phenotypic pattern of resorption, we hypothesized that bone loss in the proximal tibia metaphysis and diaphysis occurs through resorption events that are spatially and temporally distinct. To test this hypothesis, we undertook three complimentary in vivo/μCT imaging studies. Specifically, we defined spatiotemporal variations in endocortical bone resorption during the 3weeks following calf paralysis, applied a novel image registration approach to determine the location where bone resorption initiates within the proximal tibia metaphysis, and explored the role of varied basal osteoclast activity on the magnitude of bone loss initiation in the metaphysis using μCT based bone resorption parameters. A differential response of metaphyseal and diaphyseal bone resorption was observed throughout each study. Acute endocortical bone loss following muscle paralysis occurred almost exclusively within the metaphyseal compartment (96.5% of total endocortical bone loss within 6days). Using our trabecular image registration approach, we further resolved the initiation of metaphyseal bone loss to a focused region of significant basal osteoclast function (0.03mm(3)) adjacent to the growth plate. This correlative observation of paralysis induced bone loss mediated by basal growth plate cell dynamics was supported by the acute metaphyseal osteoclastic response of 5-week vs. 13-month-old mice. Specifically, μCT based bone resorption rates normalized to initial trabecular surface (BRRBS) were 3.7-fold greater in young vs. aged mice (2.27±0.27μm(3)/μm(2)/day vs. 0.60±0.44μm(3)/μm(2)/day). In contrast to the focused bone loss initiation in the metaphysis, diaphyseal bone loss initiated homogeneously throughout the long axis of the tibia predominantly in the second week following paralysis (81.3% of diaphyseal endocortical expansion between days 6 and 13). The timing and homogenous nature are consistent with de novo osteoclastogenesis mediating the diaphyseal resorption. Taken together, our data suggests that tibial metaphyseal and diaphyseal bone loss induced by transient calf paralysis are spatially and temporally discrete events. In a broader context, these findings are an essential first step toward clarifying the timing and origins of multiple resorptive events that would require targeting to fully inhibit bone loss following neuromuscular trauma.

Keywords: Bone; Bone resorption; Image registration; MicroCT; Muscle paralysis; Osteoclast.

Fig. 1

Trabecular image registration within the metabolically active metaphysis was achieved through coupling with image registration in the unchanged diaphysis. After obtaining two non-contiguous scan volumes within a single scanning session (1A), rotational and translational parameters were defined by spatially registering the volumetrically identical IRVs and then applied to the ROI volumes. This approach enabled unbiased superimposing of the trabecular compartment despite the potential for morphologic alteration on any bone surfaces (1B).

Fig. 2

Endocortical bone resorption in the metaphysis and diaphysis following transient muscle paralysis had distinct spatial and temporal patterns. Resorptive activity within the tibia following transient muscle paralysis was quantified in each of six discrete bins (2A) over a near three week time period. The initiation of endocortical expansion (mean ± s.e.) occurred exclusively within the metaphysis (2B). Within the first 6 days following muscle paralysis, endocortical expansion was focused nearest the growth plate (2B, * significantly different from Bins B-F at same time point, all p<0.001). The resorptive initiation in the metaphysis did not migrate to the diaphysis. (2C). Instead, diaphyseal expansion occurred homogeneously in the day 6 to 13 time period (2C, ^ significantly greater than the day 0 to 6 and day 13 to 20 time periods at each bin location, all p<0.01) and lacked a significant main effect of location or significant location:timepoint interaction.

Fig. 3

Separating the metaphysis into equal-volume longitudinal stacks does not provide sufficient resolution to identify focal bone loss initiation in the proximal metaphysis. Bone volume alterations in registered and discretized trabecular bone volumes were first analyzed in layers of Elements (i.e., Stacks) perpendicular to the growth plate (3A, Stack¹ highlighted in blue). There was no significant difference in bone alterations at any location or across treatment groups within 2 (3B) or 3 days (3C), though in both cases the greatest bone loss was adjacent to the growth plate in BTxA treated animals. However, a representative coronal composite cross-section 3 days following BTxA treatment suggests that bone loss occurred most predominately nearest the growth plate (3D, Red=Bone Loss, Orange=Bone Formation).

Fig. 4

Metaphyseal trabecular bone resorption following transient muscle paralysis initiated in locations of high basal osteoclast activity nearest the growth plate. In both Saline and BTxA treated mice, significant bone loss occurred in Element^3,4,1 by day 2 (4A-B, light red). Unlike the Saline group where the bone loss pattern was consistent across day 2 and day 3 (4C), significant bone loss expanded to adjacent bins in BTxA treated mice by day 3 (4D, dark red).

Fig. 5

The rate of acute metaphyseal bone resorption following transient muscle paralysis varied between Young and Aged mice independent of trabecular morphology. Serial μCT imaging of the proximal tibia metaphysis (5A) enabled quantification of dynamic bone morphology measures normalized to either bone volume or bone surface. Within the first 3 days following muscle paralysis, the Bone Resorption Rate per initial bone volume (BRR_BV) was significantly greater in Young versus Aged mice (5B, * P<0.001). When normalized to initial bone surface, Bone Resorption Rate (BRR_BS) was also significantly elevated in Young versus Aged mice (5C, * P<0.005).