Mechanical and material properties of cortical and trabecular bone from cannabinoid receptor-1-null (Cnr1−/−) mice

https://doi.org/10.1016/j.medengphy.2016.06.024Get rights and content

Highlights

  • Cannabinoid receptor 1 in C57BL/6 mice regulates trabecular and cortical bone.

  • Deleting Cnr1 improves trabecular bone quality in females.

  • Cnr1−/− adversely affects cortical bone strength and stiffness in both sexes.

  • Cnr1−/− results in slower bone growth and smaller bones.

Abstract

The endocannabinoid system is known for its regulatory effects on bone metabolism through the cannabinoid receptors, Cnr1 and Cnr2. In this study we analysed the mechanical and material properties of long bones from Cnr1−/− mice on a C57BL/6 background. Tibiae and femora from 5- and 12-week-old mice were subjected to three-point bending to measure bending stiffness and yield strength. Elastic modulus, density and mineral content were measured in the diaphysis. Second moment of area (MOA2), inner and outer perimeters of the cortical shaft and trabecular fractional bone volume (BV/TV) were measured using micro-CT. In Cnr1−/− males and females at both ages the bending stiffness was reduced due to a smaller MOA2. Bone from Cnr1−/− females had a greater modulus than wild-type controls, although no differences were observed in males. BV/TV of 12-week-old Cnr1−/− females was greater than controls, although no difference was seen at 5-weeks. On the contrary, Cnr1−/− males had the same BV/TV as controls at 12-weeks while they had significantly lower values at 5-weeks. This study shows that deleting Cnr1 decreases the amount of cortical bone in both males and females at 12-weeks, but increases the amount of trabecular bone only in females.

Introduction

The strength of a bone is determined by the material properties of the matrix and the shape of the bone. These, in turn, are determined by a cellular process of modelling and remodelling involving matrix resorption and formation; osteoclasts resorb bone while osteoblasts lay down new bone [1]. The balance between formation and resorption determines the overall amount of bone, with osteoporosis resulting from resorption outweighing formation during remodelling [2] and high bone mass disorders when formation exceeds resorption, either because of increased anabolic activity [3] or defective osteoclastic resorption as in osteopetrosis [4], [5] due to genetic abnormalities. These processes are governed centrally, e.g. via leptin and the hypothalamus [6], via the autonomic nervous system [7], [8], as well as by local signals such as mechanical loads. Central and local regulation are coordinated to ensure that gross imbalances do not occur in bone deposition or resorption at different sites in the body.

Among many factors now identified, recent studies have uncovered a role for cannabinoid signalling in the regulation of bone [9], [10], [11], [12], [13], [14]. The endogenous cannabinoid (endocannabinoid) system is widely studied for its regulatory effects on numerous physiological functions, including appetite, pain sensitivity and immune function [15], [16], [17], [18]. In addition, it is being increasingly recognised as having a complex regulatory role in bone metabolism [10], [19], [20], [21]. There are two classical cannabinoid receptors, Cnr1 and Cnr2 and these belong to the family of G-protein coupled receptors that, when activated, inhibit adenyl cyclase activity, and activate the MAPK signalling cascade [22]. Cnr1 is expressed ubiquitously throughout the brain [23] and also on immune cells, in vascular tissue and adipocytes [16], [24]. Cnr2, on the other hand, is predominately located in peripheral immune tissue such as macrophages [24], [25]. Both Cnr1 and Cnr2 have been reported in osteoblasts and osteoclasts [11], [13].

Several studies have indicated a role for the cannabinoid receptor Cnr1 in bone metabolism [9], [10], [11], [14], [19], [21], [26] but, in trabecular bone, the phenotype resulting from deleting Cnr1 in mice has been found to depend on mouse strain and sex. On a CD1 background, Cnr1−/− male mice exhibited a high trabecular bone mass, while the females had normal trabecular bone with slight cortical expansion [11]. Another group suggested that females also had a high bone mass and loss of Cnr1 protected against ovariectomy-induced bone loss [10]. They later extended this to show that trabecular bone volume fraction, BV/TV, was significantly greater at 3 months of age in both male and female Cnr1−/− mice compared with wild-type animals, although it had fallen to become significantly lower by 12 months [26]. Different results were reported from mice on a C57BL/6 background; both male and female Cnr1−/− mice at 9–12 weeks of age exhibited a low bone mass phenotype, accompanied by an increase in osteoclast number and a reduction in bone formation rate [11].

The effects of Cnr1 deletion on bone properties have mostly been investigated within the trabecular compartment. This is commonly done because the larger surface area of trabeculae results in a higher turnover rate and greater sensitivity to manipulation. It does not, however, reflect fully the range of bone properties. Bones can balance the quantity and quality of bone matrix and factors regulating bone properties could feasibly affect either or both of these; for instance, a weaker matrix may be compensated by increased geometrical properties. The purpose of this paper is to characterize cortical and trabecular bone from the tibia and femur of Cnr1−/− mice on a C57BL/6 background to address the discrepancies found in previous studies and extend our knowledge of bone regulation by Cnr1.

Section snippets

Animals

C57BL/6 Cnr1−/− mice were available from a previous study in which they were generated by homologous recombination, as described previously [27]. For studying the effect of knocking out Cnr1 on the mechanical, material and geometrical properties of bone, 5 and 12-week-old male (5 Wild-type (WT), 7 knockouts (KO) and 5 WT, 4 KO) and female mice (5 WT, 6 KO and 5 WT, 9 KO) were euthanized and the hind limbs cleaned and stored in phosphate-buffered saline (PBS) at −20 °C until measurements were

Results

Both tibiae and femora from Cnr1−/− mice were significantly shorter than WT in young (5-week-old) mice (Fig. 2). By 12 weeks of age, however, the lengths of the KO femora were not significantly different from those of the WT animals.

Discussion

Studies using animal models to investigate the regulation of bone commonly make measurements only on trabecular bone at only one site, frequently the proximal tibia. Reports, however, have indicated that not only can trabecular bone behave differently to cortical bone, but that even femur and tibia may show different responses [35], [36], [37]. Here, using an array of mechanical and material testing techniques on both tibia and femur, we have demonstrated that deleting the gene for the Cnr1

Competing interests

None declared.

Funding

ABK was funded by a studentship from the University of Aberdeen, Institute of Medical Sciences, and the Overseas Research Students Awards Scheme.

Ethical approval

Not required.

Disclosure statement

The authors have no conflicting interests to declare with respect to the work published in this paper.

Acknowledgments

We are grateful to Dr J.S. Gregory for assistance with Image J and Mr K. Mackenzie for assistance with Micro-CT analysis.

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