(D-133) Sex, Not Osteocyte-specific Endocannabinoid Receptor 1 (CB1) Deficiency, Influences Osteoclast Activity In Response To Hindlimb Unloading

Introduction: The absence of mechanical loading, or disuse, leaves individuals at risk of experiencing disuse-induced bone loss, which can increase fracture risk. Endocannabinoid Receptor 1 (CB1) has been found in all bone cell types, including osteocytes, osteoblasts, and osteoclasts. CB1 and its ability to affect bone mass loss is dependent on the age, sex, and strain of mice from which the bones are harvested. Previous work in our lab found that global CB1 deficiency in disuse conditions results in sex- and site-specific effects on bone loss1. However, the role of CB1 deficiency in osteocytes, the primary mechanoresponsive cell in bone, in disuse induced bone loss remains unknown. We hypothesized that mice with osteocyte-specific CB1 deficiency that undergo unloading would experience greater disuse-induced bone loss, partially through increased osteoclast activity, than wildtype mice.

Materials and Methods: We generated novel homozygous DMP1-Cre(+); CB1flox/flox mice (cKO) and DMP1-Cre(-); CB1flox/flox mice (WT) from a C57BL/6J for this experiment. 20-week-old male and female mice were assigned to either hind-limb suspension (HLS) or control group (n=7-9/group) for three weeks. After 21 days, all groups were sacrificed and femurs were harvested. Femurs were fixed in 10%Neutral Buffered Formalin before being decalcified by 14%EDTA for 10-14 days. Samples were then embedded in paraffin and sectioned for further histological analysis in longitudinal segments of 5 um thickness. Sections were stained for Tartrate-resistant acid phosphatase (TRAP) to determine osteoclast number (OcN) and fast green to determine bone surface (BS). Osteoclast number and surface (OcS) were normalized to BS per ASBMR standards². Slides were imaged by PhenoImager (Akoya Biosciences) and ROIs consisted of 20x regions. Statistics were done using 3-way ANOVA with Sidak post-hoc test (p< 0.05).

Results, Conclusions, and Discussions: In the femoral epiphyseal region, only the male WT mice had a significant increase in Oc.N/BS and Oc.S/BS due to disuse (Figure 1). In the metaphyseal region of the bone, the male WT mice had significantly increased Oc.N/BS and Oc.S/BS ratios due to disuse compared to the ratios of female WT mice (data not shown). Furthermore, male mice, regardless of genotype, in the femoral metaphyseal region had greater Oc.N/BS and Oc.S/BS values due to unloading than female mice. These results indicate that sex, rather than CB1 deficiency, plays a greater role in osteoclast activity during disuse conditions. In addition, these results suggest that CB1 deficiency in DMP1 cells in male mice may attenuate osteoclast numbers.

These results found that, different from the original hypothesis, females from both strains in the HLS group had significantly decreased Oc.N/BS and Oc.S/BS ratios in comparison to males from both strains, also in the HLS group. This implies that disuse-induced bone loss due to increased osteoclast activity has a higher dependency on sex than it does on CB1 deficiency. However, the above results could also be due to female mice tending to have lower bone mass to begin with when compared to male mice bones (data not shown).

Acknowledgements (Optional): A big thank you to the B.E.S.T. Lab at VCU for its support throughout my research!


References (Optional): [1] DeNapoli, R.C. et al. Journal of biomechanics (2023). [2] Dempster, David W et al. Journal of bone and mineral research (2013).