Our results show that continuous Gs activation in mouse osteoblasts leads to deposition of large quantities of immature trabecular bone with reduced mineralization. FTIR spectroscopy revealed a 25–29% deficit in mineral-to-matrix ratio in the bones of mice expressing Rs1, and synchrotron imaging showed a reduced mean TMD. These findings are consistent with low levels of bone mineralization. Measures of mineral composition, mineral maturity, and collagen maturity also indicate significant abnormalities in bone formation induced by Gs activation in maturing osteoblasts. These alterations in tissue quality accompany dramatic structural changes, including greatly increased bone mass, increased heterogeneity of mineralization, disorganization of trabecular morphology, effacement of the cortical shell, and elimination of the medullary canal.
Our model is particularly relevant for GPCR diseases found in humans, including primary hyperparathyroidism and fibrous dysplasia of the bone such as that occurring in McCune-Albright syndrome. Patients with primary hyperparathyroidism present with marked cortical thinning and decreased cortical BMD [30
], possibly related to increased cortical porosity [32
]. The elimination of the cortical compartment in DT mice is an extreme version of the cortical loss seen in primary hyperparathyroidism. However, the massive increase in trabecular volume observed in DT mice is not generally seen in patients with hyperparathyroidism. Bone from McCune-Albright syndrome patients shows a fibrous infiltrate, significant increases in trabecular bone formation, ablation of the marrow cavity, and an increased propensity to deformation and fracture [35
]. In addition, these patients accumulate unmineralized osteoid with a nonlamellar structure as well as mineralized tissue with markedly reduced mineral content [1
]. The mineralization abnormalities found in our mouse model of fibrous dysplasia may reflect those in patients with fibrous dysplasia of the bone.
FTIR spectroscopic measures provide bone tissue characterization at the molecular level and, importantly, are correlated with mechanical integrity and remodeling properties of the tissue. Mineral-to-matrix ratio increases as both primary and secondary mineralization progress and, therefore, is positively associated with tissue age [12
]. Studies of human tissue and animal models demonstrated positive correlations between mineral-to-matrix ratio and tissue stiffness and hardness [12
]. Mineral-to-matrix ratio explains 50–60% of the variation in both tissue modulus and hardness [37
]. In our study, decreased mineral-to-matrix ratio in DT bone suggests the presence of immature bone with reduced resistance to deformation. Since FTIR crystallinity values are positively associated with tissue age, tissue yield strength, and stiffness [12
], the decreased crystallinity we observed in DT bone substantiates the presence of immature tissue with reduced mechanical properties. We previously showed that DT animals have markedly elevated bone-turnover markers; display irregular, punctate bone formation by von Kossa staining and double fluorescent labeling; and have increased numbers of TRAP-positive osteoclasts [6
]. These characteristics support the conclusion that DT bone lesions contain regions with extremely high rates of bone formation and turnover, consistent with our findings of accumulated immature tissue.
Increased carbonate substitution has been associated with increased tissue age [12
], increased tissue indentation modulus and hardness [40
], and—by a mechanism of reduced ductility—increased incidence of fracture [43
] and inferior mechanical properties at the whole-bone level [20
]. Carbonate substitution in the hydroxyapatite lattice leads to a change in lattice dimensions and increased disorder of the crystalline structure [44
]. Further, mineral solubility is affected by carbonate content and increased carbonate content is thought to enhance bone resorption [45
]. Carbonate-to-phosphate ratio was significantly elevated in DT bone, indicating an alteration in crystal synthesis and perhaps playing a role in the high turnover observed in DT animals [6
Collagen matrix biochemistry is also related to tissue age, mineralization, and mechanics. Intermolecular cross-linking provides the matrix tensile strength and influences whole-bone strength [46
]. Cross-link formation also alters the rate of mineralization and microdamage accumulation [50
], thereby providing a second mechanism for regulating the mechanical properties of bone [51
]. The ratio of mature to immature, reducible cross-links—quantified as cross-link ratio—increases with tissue maturity [52
] and correlates positively with indentation modulus [12
]. In DT bone, low cross-link ratio indicates immature tissue with reduced stiffness.
FTIR analyses showed that DT bone does not exhibit maturation between 6 and 15 weeks. In contrast, specimens from 6- and 15-week-old WT mice showed significant differences in carbonate-to-matrix and carbonate-to-phosphate ratios, indicative of increasingly mature tissue. Cross-link ratio decreased in WT animals, in contrast to the established association between cross-link ratio and matrix maturation.
SRμCT imaging was used to quantify mean TMD and distribution of TMD values, measures complementary to those determined through FTIR analysis. Consistent with the deficit in mineral-to-matrix ratio identified by FTIR, DT bone had mean TMD values 39% and 33% lower than WT bone at 3 and 9 weeks, respectively. SRμCT also revealed increased heterogeneity in mineral content in DT mice as reflected by the large standard deviation of TMD in DT bones at 3 and 9 weeks (151% and 88% higher, respectively). The alteration in distribution of mineralization values might significantly affect the overall material properties of DT bone; heterogeneous regions of tissue mineralization may hinder crack propagation and toughen tissue. It is intriguing to consider that the increased variation in TMD may represent a compensatory mechanism by which DT mice counteract the deficit in tissue mineralization. Increased tissue volume in DT animals may represent an additional adaptive response to the formation of hypomineralized tissue and loss of cortical structure. These changes may partially or fully compensate for any loss of whole-bone strength or stiffness. No spontaneous fractures have been identified in DT animals, perhaps supporting this hypothesis.
The presence of immature bone in our mouse model of fibrous dysplasia suggests that strategies for modulating bone formation, such as antiresorptive medications, may have therapeutic value for these patients. Since the fibrous dysplastic bone lesions appear to be reversible by inhibiting Gs
], the immature bone formation seen in our DT mice might also be reversed. This would be an important metric as potential therapies for fibrous dysplasia are developed.
Despite identifying significant effects of Rs1 signaling on bone mineralization, our study has several limitations. First, we were unable to perform direct measurement of mechanical competence in DT mice. Mechanical testing is challenging in DT specimens due to morphological abnormalities and heterogeneous mineralization. However, the compositional measures derived from FTIR and SRμCT data are highly associated with mechanical properties of bone tissue and give us insight into the mechanisms of increased fragility in fibrous dysplastic bone. Second, the ages of animals characterized by FTIR and SRμCT were different; this should be taken into consideration when making direct comparisons between analysis results. However, for each analysis technique, the younger animals (3 and 6 weeks old) were in the rapid skeletal growth phase, while the older animals (9 and 15 weeks old) had reached a plateau of bone accumulation. This is supported by BMD measurements and consistent with the observed sexual maturity window of 7–8 weeks in both WT and DT mice. Finally, our findings are based on samples from a relatively small number of animals. The significant abnormalities in tissue quality and skeletal structure we observed in our TD mice were surprisingly conserved between the two different anatomic sites sampled (femora and parietal bones of calvariae). This finding allowed us to compile results from the two skeletal sites and perform a conservative statistical analysis. Despite these limitations, we believe that our results provide new insight into the roles of Gs signaling in regulating the matrix formation process.
In conclusion, our results illustrate that activation of the Gs signaling pathway in maturing osteoblasts leads to a significant degradation of bone tissue quality in a mouse model of fibrous dysplasia. The striking influence of the fibrous dysplasia model on tissue quality metrics reinforces the paradigm that tissue quality, and not just quantity and structure, must be considered in the evaluation of any disease processes or potential therapies.