While prior studies have examined the effects of cannabis exposure on bone, few have investigated chronic inhaled THC specifically in the context of estrogen deficiency for a clinically relevant at-risk population, particularly with respect to trabecular microarchitecture and bone remodeling outcomes. Therefore, we developed an inhalation-based rat model to determine whether chronic THC exposure mitigates or heightens the effects of ovariectomy on osteoporosis. The trabecular deterioration observed in the OVX + Air rats, including reduced bone volume fraction, increased trabecular spacing, decreased tissue mineral density, and loss of trabecular number and connectivity density, is consistent with previous research patterns of a well-characterized estrogen deficiency-induced bone loss due to accelerated osteoclastic resorption leading to perforation and disappearance of trabeculae [59,60,61]. Interestingly, THC exposure compounded these osteoporotic changes, as shown by OVX + THC animals exhibiting further reductions in trabecular thickness and bone mineral density beyond those attributed to ovariectomy alone. This additive’s deleterious effect aligns with previous in vitro research evidence that THC exerts dose-dependent stimulatory effects on osteoclastic bone resorption at lower concentrations, as well as with clinical data that associates heavy cannabis use with a lower bone mineral density and elevated bone turnover markers [36,48,62]. It is likely the mechanism involves THC activation of cannabinoid receptors expressed on osteoblasts and osteoclasts, CB1 and CB2. Previous research has found CB1 receptor signaling to regulate ovariectomy-induced bone loss, and pharmacological antagonism of both receptors has been osteoprotective in ovariectomy models [63,64]. In contrast to the widespread trabecular changes observed, cortical bone was largely spared, with the only significant finding being increased endosteal volume in OVX + THC rats compared to OVX + Air controls. This compartment-specific pattern is consistent with previous literature and the established understanding that estrogen deficiency preferentially targets trabecular microarchitecture, while the cortical changes manifest primarily as endocortical resorption and bone marrow cavity expansion, rather than gross cortical thinning [65,66]. The selective increase in endosteal volume observed with THC exposure to ovariectomized animals suggests that THC may potentiate endocortical resorption when in an estrogen-deficient state, a finding that warrants further investigation given the importance of bone integrity for fracture resistance.

Building on these findings, the present study suggests that chronic THC inhalation following the onset of the estrogen deficiency does not attenuate osteoporotic bone loss and may instead further impair select aspects of trabecular bone microarchitecture. While OVX alone produced the expected reductions in trabecular integrity, chronic THC exposure was associated with additional deficits, particularly in trabecular thickness and bone mineral density when compared to OVX controls. These changes were also accompanied by reductions in trabecular number and connectivity density, further contributing to deterioration of trabecular network structure. The observed increase in structural model index indicates a shift toward a more rod-like trabecular structure, which is generally considered mechanically weaker and more susceptible to fracture [67]. Together, these results suggest that THC may contribute to a more fragile trabecular microarchitectural phenotype in the context of osteoporosis.

The mechanisms underlying these effects may involve dysregulation of the ECS, which plays a critical role in maintaining the balance between bone formation and bone resorption [68]. Osteoblasts and osteoclasts express cannabinoid receptors, with CB1 signaling associated with the bone remodeling processes and CB2 generally linked to anti-resorptive and protective effects [63,69]. Importantly, estrogen was shown to regulate ECS activity, as estradiol increases CB1 and CB2 receptor expression and levels of the endocannabinoid AEA [70], while also inhibiting osteoclast activity through CB2-mediated mechanisms [71]. In the context of estrogen deficiency, this regulatory balance is disrupted, promoting increased osteoclast activity and bone resorption. This is supported by the ELISA analysis, which demonstrated a significant reduction in serum estradiol levels in both OVX groups compared to sham controls, with OVX + THC animals showing a 61% decrease and OVX + Air animals showing a 71% decrease. No significant differences were observed between OVX + THC and OVX + Air animals, indicating that the THC exposure did not alter circulating estradiol levels.

Under these conditions, chronic THC exposure may further dysregulate ECS signaling. Although THC acts as a partial agonist of both CB1 and CB2 receptors, it exhibits greater affinity for CB1 receptors [20]. Prolonged THC exposure has also been shown to induce receptor desensitization and impair downstream signaling pathways [72,73]. Both CB1 and CB2 receptors have been implicated in bone remodeling by influencing osteoblast differentiation and osteoclast activity [20,63,69]. Within bone tissue, CB1 receptors expressed on osteoblasts and sympathetic nerve terminals have been associated with regulation of bone formation and osteoclast activity, with excessive CB1 signaling linked to increased bone resorption and bone loss [63,74]. In contrast, CB2 receptors are generally associated with anti-resorptive effects through suppression of osteoclast differentiation and activity (Idris 2008 [69] and Ofek 2006 [75]).

Consistent with this, inhibition of CB1 signaling was shown to attenuate osteoporosis-related bone loss [63,76,77], whereas CB2 agonism has been associated with increased osteoblast activity and reduced osteoclast activity [34,75,78,79]. Disruption of the balance of osteoblast and osteoclast activity in favor of osteoclast-mediated resorption would therefore be expected to promote bone loss and reduced bone formation [63,72]. When combined with estrogen deficiency, which independently promotes osteoclast-driven bone loss [80], these effects may act synergistically to exacerbate deterioration in trabecular bone microarchitecture. Given THC’s greater affinity for CB1 receptors, the trabecular deterioration observed in the present study may therefore reflect predominantly CB1-mediated effects on bone remodeling. However, because the downstream mechanisms of CB1 and CB2 signaling in osteoporosis remain incompletely understood, these interpretations should be considered cautiously. These hypothesized mechanisms of action should be interrogated by future studies.

The selective impact of THC on trabecular bone observed in this study is consistent with the known biological properties. Trabecular bone has a higher surface area, greater metabolic activity, and a more rapid turnover compared to cortical bone, making it particularly sensitive to hormonal and physiological changes [81]. As such, early osteoporotic changes are typically most pronounced in trabecular regions, with cortical alterations and biomechanical deficits emerging later in disease progression [82]. This likely explains why significant differences were observed in trabecular parameters, while cortical structure and biomechanical strength remained largely unchanged. This may also reflect the relatively short interval between OVX induction and tissue collection, as well as the age at which osteoporosis was induced, both of which can influence the extent to which structural changes translate into measurable biomechanical deficits.

Despite clear microarchitectural deterioration, no significant differences were detected in biomechanical properties. This may reflect the relatively short duration of THC exposure or the possibility that the bone’s internal structure worsens before changes in overall strength can be detected, which is consistent with the greater sensitivity of trabecular bone to early osteoporotic changes. Similarly, the increase in endosteal volume observed in OVX + THC animals suggests enhanced resorption along the inner cortical surface, which may represent an early indicator of cortical remodeling that has not yet translated into functional deficits.

Previous clinical and preclinical studies examining cannabis and bone health have yielded mixed findings, and other drugs of abuse have routinely been shown to alter bone health and development [53,54,55,83,84], supporting a broader role for substance exposure in disrupting skeletal homeostasis. One clinical study linked chronic cannabis use to reduced BMD and increased fracture risk [48], while a preclinical study suggested potential therapeutic effects for specific cannabinoids [85]. Differences in cannabinoid composition, dosing, route of administration, and timing relative to disease onset likely contribute to the inconsistency of these studies. The present findings highlight that in the context of established estrogen deficiency, chronic THC exposure alone does not provide skeletal benefit and may instead worsen bone microarchitecture.

Translationally, these results suggest that THC use in populations at risk for osteoporosis, particularly post-menopausal individuals, may negatively impact bone quality by altering remodeling processes rather than improving skeletal integrity. These findings have important implications given the increasing prevalence of cannabis use, particularly among populations at risk for osteoporosis, and underscore the need for further research into the skeletal effects of cannabinoids.