Abstract
Sexually dimorphic tissues are formed by cells that are regulated by sex hormones. While a number of systemic hormones and transcription factors are known to regulate proliferation and differentiation of osteoblasts and osteoclasts, the mechanisms that determine sexually dimorphic differences in bone regeneration are unclear. To explore how sex hormones regulate bone regeneration, we compared bone fracture repair between adult male and female mice. We found that skeletal stem cell (SSC) mediated regeneration in female mice is dependent on estrogen signaling but SSCs from male mice do not exhibit similar estrogen responsiveness. Mechanistically, we found that estrogen acts directly on the SSC lineage in mice and humans by up-regulating multiple skeletogenic pathways and is necessary for the stem cell’s ability to self- renew and differentiate. Our results also suggest a clinically applicable strategy to accelerate bone healing using localized estrogen hormone therapy.
Introduction
Sexually dimorphic tissues are formed by cells that are regulated by sex hormones1,2,3,4. While a number of systemic hormones and transcription factors are known to regulate proliferation and differentiation of osteoblasts and osteoclasts, the mechanisms that determine sexually dimorphic differences in bone regeneration have been elusive5,6,7. Aging and age-related disease also show a marked sexual bias8. Previously, complex changes associated with aging were seen in many endocrine glandular tissues, resulting in dysregulation of steroid9,10 and amino acid-derived hormones11,12. Increasing evidence suggests that sex hormones, beyond their role in promoting sexual dimorphism, regulate stem cell self-renewal, differentiation, and proliferation13,14,15,16,17,18. Skeletal tissue exhibits sex differences in morphology and physiological function19. Clinically this is also manifested in differential epidemiology of osteoporosis and fracture risk20,21. In women, the risk of developing osteoporosis accelerates after menopause while incidence of the disease in men is progressive with age22. Current therapies of osteoporosis target fracture prophylaxis. However, due to non-union or disability up to a third of hip fracture patients can become totally dependent23, and the risk of institutionalization is great24. In a prospective study of 1133 patients with intracapsular fractures of the femoral neck, investigators found a significant increase in the incidence of non-union in females25. This raised the question of whether sex hormones, such as estrogen regulate skeletal stem cells in fracture regeneration.
In this study, to explore how sex hormones regulate bone regeneration, we analyzed bone fracture repair in young adult male, female, and female ovariectomized mice. We find the repair response to be significantly stronger in male mice is corresponding to a higher level of activated skeletal stem cell activity (SSC). We also find that the healing response in females to be dependent on ovarian function as ovariectomy nearly abolished activated SSC activity, leading to impaired skeletal regeneration and corresponding to a drop in systemic estrogen levels. These effects are mirrored clinically in postmenopausal women where lower rates of fracture healing are reported20. Mechanistically, we found that estrogen acts directly on the SSC lineage in mice and humans by up-regulating multiple skeletogenic pathways, and is necessary for the stem cell’s ability to self-renew and differentiate. Moreover, the response to estrogen also appears to be sexually dimorphic as SSCs from male mice do not exhibit similar estrogen responsiveness. Strikingly, SSC deficiency and bone loss following ovariectomy and aging can be reversed with a single, highly localized delivery of estrogen to the fracture site. Our results indicate that the osteoprotective actions of estrogen are mediated by direct SSC activation and suggest a clinically applicable strategy to accelerate bone healing using localized estrogen hormone therapy.
Results
Skeletal regeneration demonstrates sexual dimorphism
In this study, to determine whether sex hormones can affect fracture healing in mice we created pin-stabilized transverse femoral fractures in a well-established ovariectomized mouse model. Fracture healing was compared in adult ovariectomized mice (denoted as OVX), 14-week-old sham-operated female mice (denoted as sham), and male mice (denoted male), (Fig. 1A). OVX mice share multiple features and endocrine changes that are associated with human menopause26,27,28. We measured bone mass/volume in OVX mice and found that by 6 weeks after ovariectomy, there was a statistically significant decrease in bone mass compared to sham. In subsequent experiments, all OVX mice refer to mice 6 weeks after ovariectomy. It is well accepted that bone loss in OVX mice is the result of an imbalance of bone deposition and resorption leading to reduced skeletal mass and structural deterioration of skeletal tissue5,29. Enhanced osteoclast activity has previously been defined as the hallmark of osteoporosis in quiescent bone27,30,31. However, elimination of senescent osteoclast progenitors has no effect on age-related bone loss32. Despite this very little is known about the anabolic processes in bone integrity and fracture healing. At post-fracture week 2, macroscopic inspection of the dissected femora suggests a larger fracture callus in sham and male mice relative to OVX mice (Fig. 1B). Comparing male and sham female mice, we also observed a significant reduction in bone mass and mechanical strength in sham female calluses (Fig. 1C, D). Additionally, there is a significant difference in bone mass and mechanical strength in fracture calluses between male and OVX mice (Fig. 1C, D). These findings suggest that bone healing could be sexually dimorphic, and that skeletal regeneration is dependent on ovarian function in female mice. This is supported by the work of Manolagas’ group who have demonstrated that estrogens influence mammalian skeletal growth and maintain resulting in sexual dimorphism33….
