Mechanical and material properties of cortical and trabecular bone from cannabinoid receptor-1-null (Cnr1−/−) mice
Graphical abstract
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.
References (42)
- et al.
A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait
Am J Hum Genet
(2002) - et al.
Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations
Am J Hum Genet
(2008) - et al.
Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass
Cell
(2000) - et al.
Leptin regulates bone formation via the sympathetic nervous system
Cell
(2002) - et al.
Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase
J Biol Chem
(2005) - et al.
Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system
Neuroscience
(1998) Presence and functional regulation of cannabinoid receptors in immune cells
Life Sci
(1999)- et al.
Cannabinoid receptor type 1 protects against age-related osteoporosis by regulating osteoblast and adipocyte differentiation in marrow stromal cells
Cell Metab.
(2009) - et al.
PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures
Bone
(2011) - et al.
A comparison of cortical and trabecular bone from C57 Black 6 mice using Raman spectroscopy
Bone
(2009)
A comparison of mechanical properties derived from multiple skeletal sites in mice
J Biomech
Bone development and age-related bone loss in male C57BL/6 J mice
Bone
Bone remodelling at a glance
J Cell Sci
Pathogenesis of osteoporosis: concepts, conflicts, and prospects
J Clin Invest
Localization of the gene causing autosomal dominant osteopetrosis type I to chromosome 11q12-13
J Bone Miner Res
Skeletal parasympathetic innervation communicates central IL-1 signals regulating bone mass accrual
Proc Natl Aacd Sci USA
Cannabinoids and bone: friend or foe?
Calcif Tiss Int
Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors
Nature Med
Involvement of neuronal cannabinoid receptor CB1 in regulation of bone mass and bone remodeling
Mol Pharmacol
Regulation of bone mass, osteoclast function, and ovariectomy-induced bone loss by the type 2 cannabinoid receptor
Endocrinology
Peripheral cannabinoid receptor, CB2, regulates bone mass
Proc Natl Aacd Sci USA
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- 1
Present address: Department of Orthopaedic Surgery, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
- 2
Present address: Department of Pharmacology and Toxicology, University of Toronto, Toronto, Canada.