## 3 Estrogen Receptors
### Role of Estrogen and Its Receptors in Bone Regeneration  
Fracture healing is a series of postnatal events that resemble biological processes taking place during the embryonic development of the skeleton (for review refer to [71]). This complex process occurs in three marked stages: inflammation, callus formation, and bone remodeling. Each step is characterized by a series of cellular and biochemical events. At the site of injury, there is disruption of vascular vessels and formation of a hematoma [72]. This leads to an almost immediate increase in the expression of inflammation markers such as tumor necrosis factor (TNF\(\alpha\)) [73], interleukins (IL) 1\(\beta\), 6, 7, 11, 17A & 18 [74]. This environment promotes the movement and infiltration of inflammatory cells allowing for the recruitment of mesenchymal stem cells (MSCs) from the bone marrow [75], periosteum [76,77], muscle, and peripheral blood supply (see [78] for review). The second step is callus formation. At the inner core of the injury site, there is a remarkable depletion of oxygen due to the disrupted vasculature. It is at this location that bone regeneration occurs via endochondral bone formation. The newly recruited MSCs undergo chondrogenesis: proliferation, hypertrophy, and terminal differentiation. This step is marked by the increased expression of chondrocyte markers including Sox-9, Col II, Col X, MMP-9, MMP-13, and Ihh. The newly differentiated chondrocytes start producing vascular endothelial growth factor (VEGF) [79] to promote the formation of new blood vessels in the injured area, a process called angiogenesis. Then, chondrocytes undergo apoptosis attracting osteoclasts to the area to start the bone remodeling process. This stage is marked by an increase in osteoclasts-related genes such as M-CSF (macrophage colony-stimulating factor), RANK, RANK-L, and osteoproterozoic (OPG). At the periphery of the fracture where vasculature is still intact, bone regeneration occurs via intramembranous ossification which is the process when the bone is formed from a fibrous membrane. The late phase of fracture healing is characterized by the formation of the neocortex and its remodeling to mature lamellar bone. During this late stage, the healing process is characterized by the remodeling of the periosteal callus which is enhanced in the presence of estrogen. Estrogen plays an important role in all aspects of bone healing. In callus formation by enhancing cartilage homeostasis, growth, and differentiation [80]. In 2010, [PERSON] and colleagues showed that ovariectomized (OVX) mice subjected to bone fracture have less newly formed cartilage at the fracture gap (at 7 and 14-days post-fracture) as compared to sham and estrogen-treated groups. In the same paper, they reported that the estrogen-treated group had increased chondrocyte formation compared to Sham and OVX mice. Authors have noticed that there were no differences in terms of periosteal callus size, femur diameter, or BMD in the fracture area up to this point of the fracture healing process [81]. Estrogen also plays an important role in the last step of bone remodeling. The ability to remove cartilage and allow deposition of new mineralizing cells into the area is critical in the fracture healing process, and lack of estrogen impairs this process. In OVX mice, the amount of cartilage remaining in the fracture site is higher when compared to control groups of mice receiving estrogen treatment, the latest group having the highest mineralized area [81]. As described previously in this review, estrogen signals through its receptors, ER\(\alpha\), ER\(\beta\), and GPER1/GPR30, and it should not be surprising to expect that these receptors play an important role in the bone healing process. Recently, [PERSON] et al. (2020) studied the levels of ERs in the bone healing process using a metaphyseal bone defect. Mice were subjected to a monocortical bone circular perforation (1.0 mm diameter) of the femur.