Long-term cannabidiol treatment did not restore bone microstructural defects in skeletally mature ovariectomized Sprague-Dawley rats
1 Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2 Food Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand
3 Center of Excellence for Preclinical Toxicity and Efficacy Assessment of Medicines and Chemicals, Chulalongkorn University, Bangkok 10330, Thailand
4 Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok 10400, Thailand
5 Department of Nutritional Sciences, University of Texas, Austin, TX 78712, United States
6 Central Animal Facility, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
7 Food Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand
8 Food Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand
9 Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok 10400, Thailand
10 Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
11 Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok 10400, Thailand
12 Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
13 Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand
14 The Academy of Science, The Royal Society of Thailand, Bangkok 10210, Thailand
Abstract
Cannabidiol (CBD) effects on bone metabolism in postmenopausal osteoporosis remain unclear. While endocannabinoids and phytocannabinoids bind to receptors in bone cells, direct evidence of CBD’s bone-protective effects is lacking. We evaluated the effects of CBD on bone metabolism in ovariectomized (OVX) rat model of estrogen deficiency. Twelve-week study with treatment initiated 2 wk after the surgery was conducted. Five experimental groups were established: sham-operated with vehicle (SHM/VEH), sham with CBD (SHM/CBD5), OVX with vehicle (OVX/VEH), OVX with 17β-estradiol (OVX/E2), and OVX with CBD (OVX/CBD5). Cannabidiol was administered at 5 mg/kg/d via osmotic pumps. Micro-CT of the distal femur revealed that trabecular bone mass in OVX/CBD5 decreased similarly to OVX/VEH, indicating no protective effect. Serum bone turnover markers showed increased bone resorption in OVX/CBD5 compared to OVX/VEH. Gene expression analysis revealed that estrogen significantly reduced Ctsk gene expression compared to OVX/VEH, while CBD showed no significant differences. No significant changes were observed in cannabinoid receptor expression or bone metabolism in sham-operated rats receiving CBD. While CBD (5 mg/kg/d) was well-tolerated, it did not mitigate OVX-induced bone loss in skeletally mature rats. Consequently, CBD should not be considered a monotherapy for postmenopausal osteoporosis, though it appears safe for other potential medical applications.
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Keywords: aging, bone micro-CT, preclinical studies, osteoporosis, therapeutics
Graphical Abstract
Article notes
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Received 2025 May 10; Revised 2025 Dec 5; Accepted 2025 Dec 19; Collection date 2026 Feb.
Introduction
Osteoporosis is a debilitating skeletal problem that is characterized by low bone mass, disruption of bone microarchitecture, and compromised bone strength.1 In 2050, a global share of the population aged 65 yr or over is estimated to increase from 9% in 2019 to 16%.2 Bone fragility leads to fractures, disability, an increased rate of mortality and morbidity, as well as economic burden.3 Women are more susceptible to osteoporosis, because they lose bone mass more rapidly after menopause.1 Although current osteoporosis treatments (eg, bisphosphonates, selective estrogen receptor modulators, and antiresorptive and anabolic drugs) are generally effective in reducing fracture risk, some (such as denosumab) may result in increased risk of vertebral fractures if stopped abruptly due to rapid bone loss. Other treatments are linked to adverse effects, such as atypical femoral fractures, osteonecrosis of the jaw, or thromboembolic events.4 This results in poor compliance and subsequent ineffectiveness of the treatment. Although new effective and safe treatments that attenuate and delay bone loss in the elderly are of paramount importance, estrogen-deficient osteoporosis remains intractable to many treatments, and it is not surprising to observe some interventions lacking efficacy.
Recent studies have indicated the relationship between the endocannabinoid (EC) system and bone homeostasis.5 The EC system involves several receptors, endogenous ligands (also known as ECs), and their metabolizing enzymes. Endogenous ligands are N-arachidonoylethaolamide (AEA or anandamide) and 2-arachidonoylglycerol (2-AG) that have been shown to regulate bone homeostasis through canonical cannabinoid receptors, CB1 and CB2.6 Furthermore, AEA and 2-AG also activate non-cannabinoid receptors like an orphan G protein coupled receptor GRP557 and transient receptor potential vanilloid type 1 (TRPV1) channels.8 These receptors have been reported to play a role in the regulation of bone phenotypes. The foregoing receptors and their ligands are expressed in human and mouse osteoblasts and osteoclasts.5 While the brain showed a 10-fold higher AEA level compared to the calvarial tissue,9 comparative studies by Tam et al.10 and Eger et al.9 revealed consistent 2-AG levels across bone (femoral and tibial metaphysis and the calvarial bone, respectively) and brain tissues in 12-wk-old mice. Therefore, the EC system has been increasingly shown to modulate skeletal development and maintenance.
Phytocannabinoids have attracted tremendous interest as the use of cannabis extracts is legalized in several countries around the world. Cannabidiol (CBD) is one of the principal non-addictive constituents found in Cannabis sativa. The U.S. FDA approved Epidiolex®, an oral CBD solution, for the treatment of 2 rare forms of epilepsy, Lennox-Gastaut syndrome and Dravet syndrome. In addition, various in vitro and preclinical studies have reported its anti-inflammatory,11 neuroprotective,12 anti-psychotic,13 anxiolytic,14 and anti-cancer effects.15 For example, Bielawiec et al.11 reported that CBD (10 mg/kg BW for 2 wk) suppressed high fat diet-induced proinflammatory markers in the muscle in vivo. However, the molecular mechanisms through which it produces these actions are largely elusive, and whether its long-term use is safe remains debatable.
Cannabidiol exhibits a dose-dependent relationship in humans, with higher plasma levels generally linked to greater clinical effects in epilepsy but with significant variability.16,17 For example, in epilepsy patients receiving CBD ranged from 5 to 50 mg/kg/d), higher plasma CBD levels (7.1-1200 ng/mL) are associated with greater seizure reduction, and both children and adults show similar dose-response trends.16,18 In vitro, CBD acts on multiple targets (eg, CB1, CB2, TRPV1, 5-HT1A, GPR55, and PPAR-γ), but many effects require higher concentrations (~1 to 10 μM or 314 to 3140 ng/mL)19 than typically achieved in vivo, underscoring the importance of dose selection and target validation for therapeutic use.
While there are some preclinical studies examining the effects of CBD in ovariectomized (OVX) mice,20,21 these studies used mice that were relatively young (8-12 wk old). Ovariectomy performed in young animals represents premature ovarian failure,22 thus not appropriately representing the scenario of postmenopausal women. WHO recommends using adult rats, not young growing rats, as an animal model for osteoporosis.23 At approximately 6 mo or older, bone formation in rats dramatically decreases as compared to at the age of 3 mo indicating the maturation of the bone and the expansion of cortical bone as well as longitudinal bone growth was stabilized.24 Therefore, rats aged 6 mo or older are preferred to ensure that the bone is mostly undergone remodeling instead of the bone modeling process. Interventions administered during the growth phase would yield different outcomes when compared to those administered to animals with mature skeletons.
Several in vitro studies pointed toward effective doses of CBD between 3 and 5 μM in promoting osteoblast25–27 and suppressing osteoclast differentiation.28 In this study, we tried to achieve approximately 4 μM of CBD concentration in the plasma to investigate whether the positive effects of CBD observed in cell cultures are translatable to the in vivo experiments. In addition, the effects of CBD on bone metabolism in skeletally mature OVX rats have not been conducted to date, we herein aimed to investigate the effect of CBD in preventing bone deterioration resulting from estrogen deprivation in adult OVX rats.
Materials and methods
Animals
Female Sprague-Dawley (SD) rats were purchased from Nomura Siam Co., Ltd. at 8 wk of age. The animals were acclimatized to the new environment for 1 wk and maintained in polycarbonate shoe box cages at a temperature of 22-24 °C, with 50%-60% relative humidity, and a 12:12-h light/dark cycle. After acclimatization, the animals were group-housed (2-3 rats/cage) in a strict hygienic conventional housing system, and body weights (BWs) were recorded weekly. The animals were fed standard chow (Product no. 082G15, Perfect Companion Group Co., Ltd.) and reverse osmosis water ad libitum until the age of 28-wk-old when the intervention started. All animal procedures have been approved by the Institutional Animal Care and Use Committee, Faculty of Science, Mahidol University (Protocol number: MUSC65-009-602). All studies related to animals were performed in accordance with relevant guidelines and regulations, particularly the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://arriveguidelines.org/).
Chemicals
Natural CBD pure active pharmaceutical ingredient (2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol, M.W.: 314.47 g/mol) was purchased from Lipomed AG (Cat. No. CBD-303-005G, Lot no.: 303b.1B2.1-A) and was kept and controlled according to the institute guideline. Purity was 99.5% as determined by high-performance liquid chromatography according to the manufacturer’s protocol; 17β-estradiol (Cat. No. E1024, Sigma-Aldrich); Isoflurane (Attane, Minrad INC.); RNAlater stabilization solution (Cat. No. AM7020, Thermo Fisher Scientific); Direct-zol RNA MiniPrep (Zymo Research); iScript cDNA Synthesis kit (BioRad); qPCRBIO SyGreen Lo-ROX (PCR Biosystems).
Experimental design
Because ovariectomy generally leads to increased BW,29 and BW can affect bone parameters,30 a controlled feeding providing ~15 g of chow/day was chosen in order to minimize the BW increase that could confound the effect of treatments tested.
Twenty-six-week-old, nulliparous SD rats weighing approximately 300 g underwent either a sham operation (SHM) or bilateral ovariectomy (OVX). Two weeks after surgery (week 0), the rats were randomly divided into 5 experimental groups of n = 7-10 (Figure 1A and B). Treatments include a vehicle (VEH), 17β-estradiol (30 μg/kg BW) (E2), or CBD (5 mg/kg BW) (CBD5). Simple random allocation was used to assign rats to the SHM and OVX operational groups. An investigator generated a unique, continuous random number for each rat using the RAND() function in Microsoft Excel (Microsoft 365). The rats were then sorted in ascending order based on the generated random number. The first 20 sorted rats were assigned to SHM group, and the remaining 30 rats were assigned to OVX group. Using the same random allocation procedure, the SHM rats were randomized to receive either VEH or CBD5, whereas OVX rats were randomized to receive either VEH, E2, or CBD5. The SHM/VEH group is considered normal control, the OVX/SHM group is negative control, and the OVX/E2 group serves as positive control. The treatment was subcutaneously infused into each rat for 12 wk using Alzet osmotic pumps (model: 2ML4, DURECT Corp) (from the age of 28-40 wk or week 0 to week 12 of treatment, respectively). The veterinarian who performed the implantation was blinded. Sterile technique was used to prepare the vehicle before dissolving the treatment in the solvent. Drug loading was performed in a Class II biosafety cabinet. Each osmotic pump provided a continuous release of each tested compound for 4 wk, so a new pump was installed every 4 wk (a total of 3 replacements during the intervention period). Older animals were more prone to skin inflammation and necrosis. In that case, skin inflammation was treated by topical steroid and antibiotics, and a new pump was implanted in a new location to avoid the occurrence of these skin conditions.
Bilateral ovariectomy (OVX)
Rats were subjected to general anesthesia using isoflurane (flow rate of 5% for induction and 2% for maintenance) and the oxygen flow rate of 1 L/min. After confirming the absence of movement, such as eyelid flicking, tail reflex, and paw retraction, they were subjected to bilateral ovariectomy. Briefly, two incisions were made, and the dorsal subcutaneous adipose tissue was separated from the skin. The muscle layer below was opened, and the ovaries were isolated and removed. The muscles and skin were closed with 4/0 silk sutures. Sham-operated rats underwent the same surgical procedure, but without ovary removal. To ensure the success of the ovariectomy, both ovaries were confirmed absent by inspection at the end of the surgery. Euthanasia was humanely performed via an overdose of an injectable mixture containing 40 mg/kg Zoletil (tiletamine HCl and zolazepam HCl) and 5 mg/kg xylazine. Finally, the uterus and uterine horns were dissected and weighed.
Collection of blood and bone samples
After 4 wk of intervention, blood samples (500 μL) from the tail vein were collected into heparinized tubes and centrifuged at 12 000 × g for 15 min at 4 °C to prepare plasma for 17β-estradiol measurement. At euthanasia, blood samples were collected by cardiac puncture and processed to prepare plasma and serum for measuring CBD concentration and bone turnover markers, respectively. The right tibiae, left femurs, and L5 vertebrae were cleaned and wrapped in saline-soaked gauze for storage at −20 °C. The right tibia and L5 vertebra were subjected to micro-CT analysis, while the left femur was used for a three-point bending test. The right femur was further cleaned to remove bone marrow and was immediately frozen in liquid nitrogen for total RNA extraction. Femoral and tibial lengths were recorded using a vernier caliper.
Measurement of bone turnover markers
Levels of bone turnover markers, including NH2-terminal propeptide of type 1 procollagen (P1NP; cat. no. AC-33F1, Immunodiagnostic Systems) and COOH-terminal telopeptide of type 1 collagen (CTX-1; cat. no. AC-06F1, Immunodiagnostic Systems), were determined using commercial ELISA kits according to the manufacturers’ protocols.
Plasma levels of cannabidiol
Plasma CBD levels were determined using a validated gas chromatography–mass spectrometry (GC–MS) method.31 A 100 μL plasma sample was extracted with 95% methanol (400 μL) via vortexing and centrifuged at 10 000 × g for 10 min at 4 °C. A 100 μL of supernatant was transferred to a GC vial, and a 20 μL myristic acid-D27 internal standard (1 μmol/mL in hexane) was added. The mixture was dried twice to remove the residual water, followed by trimethylsilylation using 50 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide containing 1% of trimethylchlorosilane, incubated at 37 °C for 30 min. The derived samples were cooled and capped immediately for analysis.
A 2 μL sample was injected into the gas chromatography triple quadrupole tandem mass spectrometry (GC-TQ/MS; GC 7890B/MSD 7000D; Agilent Technologies) coupled to a PAL3 autosampler system (CTC Analytics AG) with a DB-5MS UI column (30 m, 0.25 mm i.d.; Agilent Technologies). The injector temperature of 250 °C with a split ratio of 10:1, and Helium was the carrier gas (1.0 mL/min). The oven temperature was programmed to increase from 60 to 325 °C at a rate of 10 °C/min, followed by a 10-min hold time. The mass spectrometer operated in dynamic multiple reaction monitoring mode. The transition at m/z 389.9 > 301.2 m/z was detected for CBD. To maintain stability and accuracy, derivatized samples were limited to 30 injections per day and stored at <10 °C. The detector tuning and calibration curve (1–800 ng/mL, R2 = 0.9999) were performed daily. Data were acquired and analyzed using the MassHunter software (version 10.0; Agilent Technologies).
Micro-CT
Ultra-high resolution micro-CT (model: VECTor6/CT, MILabs) was used for both in vivo and ex vivo examination of trabecular and cortical bone in the tibiae. In addition, L5 vertebra was analyzed ex vivo. For in vivo assessment, rats were anesthetized in an induction chamber with 4% isoflurane vaporized in a flow of 700 mL/s oxygen (Univentor 410 Anaesthesia Unit). Once anesthetized, they were transferred to a secure nose cone with a continuous flow of 1.9% isoflurane in 400 mL/s O2, maintaining anesthesia during the scan. The left tibia was analyzed for all rats. They were placed in a prone position on a warm pad, with the hindlimb secured using dental wax and a foam roll fitted inside the animal bed, held firmly with medical adhesive tape. Bone assessments were conducted monthly as previously described.32 Briefly, the radiation dose was 0.83 Gy with an isotropic resolution of 18 μm. Total radiation exposure was approximately 10 min. The in vivo scan was performed on all rats at each time point (n = 7–10). Blurry scans were excluded to ensure accuracy in image analysis. Table S1 shows the number of in vivo micro-CT scans that were analyzed at each timepoint.
Regarding ex vivo micro-CT, the left tibia and L5 vertebra were cleaned, wrapped with saline-soaked gauze, and stored in a 5-mL polypropylene tube with a screw cap at −20 °C. Before scanning, the tibia and L5 vertebra were thawed in the fridge overnight, and the tibia length was measured using a vernier caliper. The sample tube was secured on a mouse bed of VECTor6/CT MILabs system and exposed to X-ray at 65 kV, 615 μA with the isotropic resolution of 10 μm. Scanned images were reconstructed using MILabs Rec. Serial cross-sectional images of tibia and L5 vertebra were rendered as bone stacks, and a 3D analysis of the trabecular bone was performed using CTan (version 1.23.0.2).
For trabecular bone analysis of the tibia, the start slice of the region of interest (ROI) was defined as 100 slices (1 mm) from the end of proximal growth plate, and the ROI was 300 slices (a stack of 3 mm in height) from the start slice. For trabecular bone analysis of the L5 vertebra, the ROI was the entire body of the vertebra beginning from the end of proximal growth to the slice before the beginning of the distal growth plate. We manually contoured trabecular bone every 20 slices, and the intermediate slices were automatically interpolated by the software to create a volume of interest. The investigator rechecked all the contoured slices before proceeding with the 3D analysis. We measured bone volume fraction [bone volume/tissue volume (BV/TV)], trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), trabecular number (Tb.N, 1/mm), structure model index (SMI), and connectivity density (Conn.D, mm−3) as recommended.33 Only the tibia was then subjected to the measurement of cortical bone geometry. Cortical bone geometry was evaluated from a cross-sectional stack of 100 slices (a total of 1 mm thick) from the midshaft. Cortical bone parameters include total area (Tt.Ar, mm2), cortical area (Ct.Ar, mm2), defined the cortical area fraction (Ct.Ar/Tt.Ar, %) cortical thickness (Ct.Th, mm) and also the polar moment of inertia (J, mm4), maximum moment of inertia around the shorter axis (Imax, mm3), and minimum moment of inertia around the longer axis (Imin, mm3).
Bone mechanical properties
The left femur was dissected and cleaned to remove connective tissue. Hydration was maintained by keeping it in moist gauze. Bone mechanical properties were determined using a three-point bending test (model: 5943, Instron). Femur length was recorded before performing the test. Each femur was positioned with the posterior part facing down on 2 lower spans, with an 18 mm distance between them. A preload of 2 N was applied at the midshaft, followed by a displacement rate of 2 mm/min until structural failure. The biomechanical parameters were automatically calculated from the force-displacement curve using Bluehill 3 software. The measured parameters included stiffness (N/mm), maximum load (N), yield load (N), yield displacement (μm), ultimate displacement (μm), and energy absorption (N·mm).
Total RNA extraction and quantitative real-time PCR (RT-qPCR) analysis
The right femur was harvested for RNA extraction. Briefly, bone ends were cut off, and bone marrow was flushed out from the bone cavity using a 25-gauge needle attached to a syringe filled with sterile PBS. The distal femur end was chopped into small fragments and washed thoroughly with ice-cold PBS to remove all bone marrow. These bone fragments were stored in RNAlater according to the manufacturer’s protocol. Total RNA was extracted from the distal femur end using TRIzol Reagent (Invitrogen). Direct-zol RNA MiniPrep was used to isolate RNA from the TRIzol homogenate. Total RNA purity was assessed by measuring an OD at 260 and 280 nm using NanoDrop-2000c spectrophotometer (the 260/280 ratio ranged between 1.8 and 2.0). Thereafter, cDNA synthesis was performed by reverse transcription of 0.5 μg of total RNA with an iScript cDNA synthesis kit. Rpl13a was used as a housekeeping gene and its expression was used to normalize the obtained expression levels of genes of interest. qPCRBIO SyGreen Lo-ROX was used to carry out the RT-qPCR to quantify specific gene transcripts on the CFX384 Touch Real-Time PCR Detection System (BioRad). Oligonucleotide sequences of the primers used for gene expression analysis are shown in Table S2. Each primer pair was confirmed to give specific amplification of a single product using melting curve analysis. Changes in gene expression were calculated from the threshold cycles (Ct) based on the standard 2−∆∆Ct method and expressed as relative expression to the housekeeping gene.
Statistical analysis
The sample size was calculated using G*Power (version 3.1.9.7). This study was powered to capture the effect of BV/TV = 0.68, α = 0.05, and power = 0.8. All data were analyzed using GraphPad Prism 10 (GraphPad Software). The primary outcome is BV/TV and secondary outcomes are other trabecular parameters and bone turnover markers. Data are expressed as means ± SEM (means, SD, and SEM are provided in the Table S3). Gene expression data were plotted as dot plots superimposed with violin plots, with the central dashed line indicating the median and the dotted line spanning the 25th to 75th percentiles. An unpaired Student’s t-test to compare differences between the means of SHM and OVX groups at 26-wk-old (before the operation). To compare the effects among treatment groups, differences between pairs of means were assessed by one-way analysis of variance (ANOVA) with Dunnett’s post-test, comparing each group to the normal control (SHM/VEH), or Tukey’s multiple comparisons test. Brown–Forsythe test and Bartlett’s test was used to assess the ANOVA assumptions of the equality of group variances and homoscedasticity, respectively. To analyze changes in in vivo micro-CT measurements over time within each group, repeated measures one-way ANOVA was used. A p-value of less than .05 was considered significant.
Results
Body weight, bone length, and blood levels of 17β-estradiol and cannabidiol
Since the start of the intervention period (2 wk post-operation, 28 wk of age), food intake was controlled (~15 g/d) and monitored daily. Twelve weeks after treatment, the BW of sham-operated rats was significantly lower than that of OVX rats (370.46 ± 17.63 vs 415.11 ± 19.35 g, p < .05). Within the same operation type, the BW of rats receiving CBD was comparable to those receiving the vehicle (eg, 370.46 ± 17.63 vs 360.06 ± 18.29 g for SHM/VEH and SHM/CBD5, respectively). Only rats in the OVX/E2 group maintained their BW (352.04 ± 26.56 g) comparable to the SHM/VEH group. The longitudinal measurements and the final BW are presented in Figures 1C and 5C, respectively. Tibial lengths were comparable among all experimental groups (overall value: 40.69 ± 0.14 mm, Figure 1D).
Serum concentrations of 17β-estradiol, the major estrogenic hormone, were measured after 4 wk of treatment. The OVX/VEH and OVX/CBD5 groups had significantly lower estradiol levels compared to their sham-operated counterparts (−195.91% and −146.60%, respectively, Table S4). Only the OVX/E2 group showed a dramatic increase in estradiol compared to the SHM/VEH group (+143.81%, p < .001). For plasma CBD levels, the concentrations in SHM/CBD5 and OVX/CBD5 were 1144 ± 65.67 and 1280 ± 89.51 ng/mL, respectively.
OVX-induced bone loss was rescued by 17β-estradiol but not CBD
At the 26-wk-old baseline (before surgery), in vivo micro-CT scans of the left tibia showed rats, that were later assigned to SHM and OVX groups, had similar BV/TV, Tb.Th, Tb.Sp, Tb.N, SMI, and Conn.D (Figures 1E-J). In vivo micro-CT was also performed to monitor longitudinal changes in the trabecular bone microstructure in the left tibia over the course of the intervention (Figure 2 and Figures S1 and S2). At week 0 (aged 28-wk-old, before the start of the intervention), a significant decrease in BV/TV and Tb.N, and an increase in SMI were observed between SHM and OVX rats, whereas Tb.Th, Th.Sp, and Conn.D were not different between the groups at this point. From 4 wk of treatment onward, no differences in trabecular bone parameters were observed between the SHM/VEH and SHM/CBD5 groups. The BV/TV of the OVX/E2 group was similar to the healthy controls, while BV/TV in the OVX/VEH and OVX/CBD5 groups dramatically plummeted by approximately 35% (p < .0001) compared to the 2 SHM groups. Tb.Sp, Tb.N, SMI, and Conn.D differed significantly between the SHM and OVX groups, with the exception of the OVX/E2 group whose trabecular bone did not deteriorate as observed in other OVX groups. From 8 to 12 wk after treatment, all trabecular bone parameters in the OVX/VEH and OVX/CBD5 groups continued to deteriorate at a marginally reduced rate. Trabecular bone parameters of the OVX/E2 group remained comparable to those of the SHM/VEH (Figure 2).
Ex vivo micro-CT was also used to examine trabecular bone of the left tibia and L5 vertebra. In the tibia, BV/TV of the SHM/VEH, SHM/CBD5, and OVX/E2 groups were not different from each other, whereas BV/TV of the OVX/VEH and OVX/CBD5 were significantly decreased when compared to the SHM/VEH group (−95.36% and −109.73%, respectively; p < .0001). The BV/TV of the OVX/CBD5 group was not different from the OVX/VEH group either. Similar patterns of changes were observed in other parameters, including Tb.Th, Tb.Sp, Tb.N, SMI, and Conn.D (Figure 3).
The L5 vertebra also showed the same trend in trabecular changes, where BV/TV of the SHM/VEH, SHM/CBD, and OVX/E2 were not significantly different. However, the BV/TV of the OVX/VEH and OVX/CBD were apparently reduced compared to the SHM/VEH group (−32.08% and −32.35%, respectively; p < .0001). Unlike the tibia, there were no significant differences in Conn.D of the L5 vertebra among the 5 groups (Figure 4).
Cortical bone parameters and bone mechanical properties
The OVX rats, excluding the OVX/E2 group, weighed more than the SHM counterparts (p < .001, Figure 5C), we examined the correlation between the BW and each cortical parameter of bone geometry, as shown in Figure 5A and B. Body weight was positively correlated with all the cortical parameters (p < .05) except for Ct.Ar/Tt.Ar (p = 0.27, Figure 5A and B). Multiple linear regression analysis examines the relationship between treatment group and each cortical parameter when corrected for the effect of BW. We found that only the means of Tt.Ar among treatment groups are significantly different. Specifically, Tt.Ar of OVX/E2 were smaller than those of SHM/CBD5 and OVX/CBD5 (−11.93% and +15.44%, respectively; p < .05). For Ct.Ar/Tt.Ar, we used one-way ANOVA to compare means of each treatment group and found that the OVX/E2 group showed a significant increase in Ct.Ar/Tt.Ar compared to other groups (+11.85% and +8.86% vs SHM/CBD5 and OVX/VEH, respectively; p < .05). However, Tt.Ar and Ct.Ar/Tt.Ar of OVX/CBD5 were not different from those of OVX/VEH (Figure 5D and E).
In addition to micro-CT evaluation of cortical bone, mechanical properties were measured by three-point bending test; stiffness of the OVX/VEH group did not differ from the SHM/VEH. However, the OVX/CBD5 group had significantly lower stiffness than the OVX/E2 (−16.19%; p < .05) and a trend toward a significant decrease as compared to the SHM/VEH (p < .10) (Figure S3).
Effect of CBD on gene expression of osteoblast-related markers
The expression profile of genes involved in osteoblast differentiation and maturation including Alpl, Col1a1, Ocn, and Runx2, was examined (Figure 6A-D). No significant differences were observed in the gene expression of Alpl, Col1a1, and Runx2 even when comparing the OVX/VEH against the SHM/VEH, where severe bone loss was detected from micro-CT imaging. For the expression of Ocn, we detected a trend toward a significant increase in the OVX/VEH compared to the SHM/VEH (+83.28%, p = .082). The OVX/E2 group had a lowered expression of Ocn which was significantly different from the OVX/VEH group (−154.79, p < .01). The OVX/CBD5 group did not show significant changes from the SHM/VEH and the OVX/VEH groups.
Changes in serum levels of bone turnover markers
Serum concentrations of CTX-1 of the OVX/VEH group were significantly increased when compared to the normal controls (+54.7%, p < .049). No differences were detected among the control SHM/VEH, SHM/CBD5, and OVX/E2 groups. While the CTX-1 level of the OVX/CBD5 did not differ from the OVX/VEH group, the CTX-1 level of the OVX/CBD5 significantly increased when compared to both SHM/VEH (+67.69, p < .0079) and OVX/E2 rats (+53.24, p < .02). Similar observations were found in the level of P1NP (Figure 6G and H).
Unaltered expression of cannabinoid receptors in bone
In addition to examining the expression of osteoblast- and osteoclast-related markers, we also assessed changes in the gene expression of potential receptors in the EC system, including Cnr1, Cnr2, Gpr55, and Trpv1. However, no significant differences in the expression of these genes were observed among the experimental groups (Figure S4).
Discussion
This study investigated the potential protective effect of CBD against OVX-induced bone loss in skeletally mature rats. Over a 12-wk period, CBD was continuously administered via subcutaneous infusion using osmotic pumps at 5 mg/kg/d. Despite the intervention, CBD could not prevent bone loss associated with estrogen deficiency. Serum biomarkers revealed increased bone resorption (CTX-1) and bone formation (P1NP) in OVX rats. While 17β-estradiol effectively reduced these elevated bone turnover markers, CBD did not suppress them. Gene expression analysis showed that the OVX/E2 group exhibited significantly decreased expression of Ctsk (osteoclast marker) and Ocn (late osteoblast marker) compared to the OVX/VEH group. In contrast, the OVX/CBD5 group’s gene expression remained similar to the OVX/VEH group. Additionally, no changes in cannabinoid receptor expression were detected across any experimental groups.
In this study, we used a mature rodent model to simulate postmenopausal conditions seen in women. The rats were 6-mo-old at the time of ovariectomy. This is the minimum recommended age for evaluation of OVX-induced bone loss, and it ensured that longitudinal bone growth and radial expansion were stabilized.23,24 We also chose to start the treatment 2 wk after the surgery, because others have reported that this is when endogenously produced estradiol levels reached their nadir in the plasma22 and when some trabecular bone loss is detected but not to the degree that trabecular bone connectivity has severely deteriorated.34
While no clinical studies on the effect of CBD in bone have been reported, CBD displays a dose-dependent relationship in humans, with higher plasma concentrations generally linked to greater clinical effects, such as seizure reduction in epilepsy.16,17,35 In clinical epilepsy studies, effective CBD doses typically range from 5 to 50 mg/kg/d, and higher plasma levels (7.1-1200 ng/mL) are associated with greater seizure reduction in both children and adults.16,18 In anxiety and other neuropsychiatric conditions, the relationship is less linear, with some evidence for an inverted U-shaped or bell-shaped dose-response curve, where moderate doses (eg, 300-600 mg) are more effective than lower or higher doses.35,36
In vitro, CBD acts on multiple receptors but many effects require higher concentrations (~1–10 μM or 314–3140 ng/mL)19 than typically achieved in preclinical settings. In vivo, CBD is considered a weak antagonist or negative allosteric modulator at CB1 and CB2, and may also act as an inverse agonist at CB2, but its pharmacological effects are mediated through a broader polypharmacological profile, including indirect modulation of the EC system and other receptor pathways.37
Several studies have investigated CBD’s mechanisms in bone cells. Whyte et al.38 found that CBD (500 nM) suppressed osteoclast activity in GPR55−/− mice, reducing resorption pit area and active osteoclast numbers. Nielsen et al.28 similarly demonstrated the inhibitory effect of CBD (3-10 μM) on bone resorption in human monocytes. Research suggests CBD can promote mesenchymal stem cell migration and osteoblastic differentiation via CB2 receptor activation39 and GPR55 inhibition.40 In cell culture studies, CBD showed mixed effects: it enhanced pre-osteoblast proliferation but decreased alkaline phosphatase activity and collagen production at later stages.41 Kang et al.25 observed that CBD-induced osteoblast differentiation in cell lines, potentially through angiopoietin 1 upregulation and p38/MAPK signaling. Collectively, these findings support CBD’s potential to suppress osteoclast activity while promoting osteoblast differentiation. While in vitro evidence supports the use of CBD in osteoporosis, no direct investigations of CBD in preventing postmenopausal osteoporosis are available.
Despite several existing preclinical studies examining the impact of CBD on bone-related endpoints, no studies have directly investigated the effect of CBD on skeletally mature animals. For example, Napimoga et al.42 reported that a daily dose of 5 mg/kg of CBD decreased alveolar bone loss in male Wistar rats, whose age was not reported, that were induced to have periodontitis. Another study by Li et al.43 studied the effect of CBD (5 mg/kg/d) in 12-wk-old male SD rats given a severe spinal cord injury (SCI) and observed that CBD reduced the severity of SCI-induced bone loss. However, the pathology and physiological responses to SCI-induced skeletal deterioration do not model postmenopausal osteoporosis. Third, CBD (5 mg/kg/d given 12-h post-surgery and over 14 d) markedly improved the biomechanical properties of healing mid-femoral fractures in 3-mo-old male SD rats.44 Additionally, CBD has been shown to exert a pain-relieving effect and promote healing in a mouse model for tibial fracture.45 Lastly, Whyte et al.38 observed that the treatment of 3-mo-old male C57BL/6 mice with CBD (10 mg/kg, 3 times per week for 8 wk) decreased serum type 1 collagen C-terminal telopeptide fragments (a biomarker of bone resorption), but did not significantly improve any trabecular parameters.
A number of earlier studies have seen beneficial effects of CBD treatment in rodents OVX at younger ages. Sui et al.20 reported that CBD could reduce trabecular bone loss in 12-wk-old OVX mice. In their study, mice were fed CBD at a dose of 25 mg/kg/d for 18 wk starting 2 wk after OVX. This was a higher per BW dose compared to other previous studies38,42,43; we believe they used this dose to compensate for higher rates of drug metabolism and degradation that happen when drugs were orally administered, and it allowed them to ensure that an adequate blood concentration of CBD was achieved (2 ng/μL). Ihejirika-Lomedico et al.21 also showed that CBD administered (5 mg/kg/d by an implanted osmotic pump) at the time of ovariectomy (OVXpre) in growing 8-wk-old mice could prevent OVX-induced bone loss and also increase recovery from an induced bone fracture performed at 6-wk post-OVX. In contrast, an OVX group that received CBD only after the bone fracture surgery was not protected. More recently, de Oliveira et al.46 reported improvements in trabecular bone parameters and mechanical properties of 9-wk-old OVX SD rats given CBD (5 mg/kg/d) for 3 wk (5 d on/2 d off) starting 12 wk after OVX.
Previous research shows CB2 receptors are highly expressed in bone cells and their activation by cannabinoids, including CBD, promotes osteoblast activity, and bone formation, while inhibiting osteoclast differentiation and function, supporting bone regeneration and maintenance.5 CB1 receptors, though less abundant, also modulate osteoclast activity and bone turnover.5 GPR55, present in osteoclasts and osteoblasts, is antagonized by CBD, leading to reduced osteoclast function and bone resorption, which helps preserve bone mass.7,38 TRPV1, another target of CBD, is involved in osteoclast activity,47 pain and inflammation pathways within bone tissue.48
Since CBD is a highly pleiotropic molecule and these receptors are all part of the EC system or are closely related, we determined the effect of CBD on the expression of these targets. We did not detect any significant differences in the expression of Cnr1, Cnr2, Gpr55, or Trpv1 between the OVX/ CBD5 group compared to the normal, negative, and positive controls. This suggests that CBD may not act directly on CB1 and CB2. In addition, the low expression of GPR55 and TRPV1 may contribute to the null results.
While the findings of our study contrast with previous investigations, several factors could account for the discrepancy. Earlier studies varied in the timing of CBD treatment post-ovariectomy, animal age (using growing vs our growth-stable adult rats), dose regimens, and experimental models. The use of growing animals in previous work suggests an existing anabolic hormonal environment that could make them more responsive to CBD effects than our adult cohort. The dosing regimen also matters; for example, Ihejirika-Lomedico et al.21 administered CBD concurrent with OVX so it is possible to observe bone-protective effect while residual estrogen is present. Ihejirika-Lomedico et al.21 also saw a benefit of CBD following on healing of a fracture induced 6 wk after OVX. Fracture stimulates endochondral bone formation while OVX is uncoupling osteoblast-osteoclast activities that normally maintain adult bone mass. Thus, the impact of CBD on activated chondrocytes and osteoblasts may be more robust than their inhibitory impact on osteoclasts. However, additional studies will be needed to test these hypotheses. Lastly, we restricted the diet to 15 g/d of the standard chow to prevent confounding positive effects of inadvertently increased BW of OVX rats, contrasting with the ad libitum feeding in other studies. While this represents modest caloric restriction, previous studies affirms the overall health of Sprague-Dawley rats was maintained.49
The limitation of the present study is that CBD does not selectively target the canonical cannabinoid receptors CB1 and CB2, which remain important regulators of bone metabolism. Although CBD exhibits weak or indirect actions on these receptors, its pharmacological profile is more strongly aligned with TRPV1 modulation. As a result, the extent to which CB1/CB2 pathways contribute to the observed cellular responses cannot be conclusively determined in this model. Future studies should therefore incorporate selective CB1/CB2 agonists or antagonists to determine the receptor-specific mechanisms and to clarify whether the bone-related effects of CBD are mediated independently of these classical cannabinoid signaling pathways.
In conclusion, this research demonstrates that CBD—with blood levels exceeding 1200 ng/mL (~4 μM)—did not prevent OVX-induced bone loss in adult rats. While CBD elicits beneficial effects on bone cells as reported previously, these effects may not be translatable to in vivo and are likely specific to bone during growth or certain bone diseases that are not caused by estrogen deficiency. Cannabidiol is apparently a safe compound for use in adult rats and does not worsen bone health in a preclinical model of postmenopausal women. Although the present CBD regimen lacks the efficacy to protect bones against osteoporosis induced by estrogen deprivation, it did not aggravate bone loss or impair bone mechanical properties.
Supplementary Material
Acknowledgments
The authors thank Dr. Sisi Cao and Pamela Lachcik for their guidance on experimental design and methods and would like to thank Sirichai Suksai and Thanaporn Thanikakornkul for assisting in animal work and micro-CT scanning. The authors also thank Thitapha Kiattisirichai for artwork. The ultra-high-resolution micro-CT platform was supported by Mahidol University-Frontier Research Facility (MU-FRF) and MUSC-Central Animal Facility.
Contributor Information
Krittikan Chanpaisaeng, Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; Food Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand; Center of Excellence for Preclinical Toxicity and Efficacy Assessment of Medicines and Chemicals, Chulalongkorn University, Bangkok 10330, Thailand; Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
James C Fleet, Department of Nutritional Sciences, University of Texas, Austin, TX 78712, United States.
Visut Rawiwet, Central Animal Facility, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Natthaporn Phonsatta, Food Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand.
Atikorn Panya, Food Biotechnology Research Team, National Center for Genetic Engineering and Biotechnology, Pathum Thani 12120, Thailand.
Nattapon Panupinthu, Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok 10400, Thailand; Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Narattaphol Charoenphandhu, Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok 10400, Thailand; Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand; The Academy of Science, The Royal Society of Thailand, Bangkok 10210, Thailand.
Funding
This research is supported by National Research Council of Thailand (NRCT, N42A650188) and National Science and Technology Development Agency (to K.C.). The authors also express their gratitude for the research funding from the Faculty of Pharmaceutical Sciences, Chulalongkorn University (Grant number Phar2567_RG006; to K.C.), the National Research Council of Thailand (NRCT)-Mahidol University (Distinguished Research Professor Grant; to NC), Thailand Science Research and Innovation (TSRI)-Mahidol University [Fundamental Fund/Basic Research Fund by National Science Research and Innovation Fund (NSRF): fiscal year 2023-2025; to N.C.], Program Management Unit for Human Resources & Institutional Development, Research and Innovation (PMU-B; to N.C.), and Mahidol University under the Research Cluster Development Fund (to N.C.).
Conflicts of interest
All authors state that they have no conflicts of interest.
Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.
References
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Data Availability Statement
The data underlying this article will be shared on reasonable request to the corresponding author.