Bone tissue consists of three types of proper bone cells, such as osteoclast, osteoblast, and osteocyte. Osteoclast is a bone-resorbing cell, which is a multi-nucleated giant cell differentiated from hematopoietic stem cells by M-CSF [5] and RANKL [6] stimulation. Osteoblast is a bone-forming cell, which is differentiated from mesenchymal stem cell by function of master regulators, such as Runx2 [7] and Osterix [8]. Osteocyte is differentiated from osteoblast and is embedded in extracellular bone matrix formed by osteoblast itself. Osteocyte has a lot of processes to contact with osteoblasts and osteoclasts as well as other osteocytes. Recently, it has been reported that osteocytes can control bone metabolism by indirectly regulating osteoblastic bone formation and osteoclastic bone resorption. Bone mass is precisely maintained by balances between osteoblastic bone formation and osteoclastic bone resorption. If this balance shifts into either one, which means abnormal remodeling, metabolic bone disorder can be developed. Osteoporosis can be caused by more increased osteoclastic bone resorption than increased osteoblastic bone formation, which called high turnover bone metabolism. This bone turnover can be determined by bone metabolic biomarkers, which are derived from degraded extracellular bone matrix or active osteoclasts or osteoblasts. Bone resorption markers include deoxypyridinoline (DPD), type 1 collagen cross-linked N-telopeptide (NTX), type 1 collagen cross-linked C-telopeptide (CTX), and tartrate-resistant acid phosphatase 5b (TRACP-5b). On the other hand, bone formation markers include osteocalcin, bone alkaline phosphatase (BAP), and type 1 procollagen-N-propeptide (P1NP). These bone metabolic markers are useful for diagnosis and evaluation of effects of therapies for osteoporosis [9].

Bone microstructure is one of the factors, which consists of bone quality, and can be assessed by high-resolution microcomputed tomography (μCT). μCT measurements can visualize and quantitatively evaluate 2D and 3D bone microstructures. μCT measurements should be performed according to the guideline [10], which determined technical terms and recording methods. Trabecular microstructure analyses were analyzed based on 3D voxel data. These data can provide not only basic parameters, such as bone volume (BV), total tissue volume (TV), relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), but also 3D structural parameters, such as connectivity of trabecular per unit volume (Connectivity density) and structure model index (SMI), which is a quantitative parameter of trabecular morphology, rod or plate shape.

The trabecular bone microstructure in osteoporotic bone is characterized by decreased BV/TV, decreased connectivity, increased SMI, increased Tb.Sp, and decreased Tb.N. In addition to trabecular bone, cortical bone in osteoporosis exhibits reduction of cortical thickness and increased cortical porosity. These osteoporotic changes in bone microstructure impair bone quality, followed by increased fragility and fracture risks.

These changes in microstructure are varied among a kind and region of the bone. Distal femur and vertebral bone are usually used for evaluation in an experimental model.

Postmenopausal osteoporosis is caused by deficiency of female sex steroid hormone, estrogen, and is the main reason of primary osteoporosis. Postmenopausal osteoporosis patients and ovariectomized rodents, an experimental model of postmenopausal osteoporosis, exhibit bone loss with high turnover bone metabolism due to increased osteoclastic bone resorption, which is more significant than increased osteoblastic bone formation at same time [11].

Estrogen exerts its function by binding to its own receptor, estrogen receptor (ER), which is a member of nuclear receptor superfamily. ER has subtype ERα and ERβ. Ligand (estrogen) bound ERs can form dimer and translocate into nucleus, and work as a transcription factor to regulate gene expression of target gene [12]. Estrogen/ER signaling is essential for the development, maturation, and maintenance of sex differences and characters in various organs/tissues, especially in reproductive organs. Regarding bone metabolism, the patients harboring loss-of-function mutation at gene locus of ESR1, which encodes ERα, exhibit remarkable osteoporosis that is irresponsible to estrogen treatment [13]. From this point of view, it has been considered that ERα is dominant subtype in regulation of estrogen/ER signaling and estrogen’s osteoprotective effects have studied focusing on ERα.

To clarify the effects of estrogen in bone metabolism, the mice lacking ERα (ERαKO) were generated and analyzed. As a result, ERαKO mice exhibited estrogen-deficient phenotypes like postmenopausal women in various tissues/organs [14]. However, bone mass of ERαKO was increased with low bone turnover. This inconsistent bone phenotype can be explained by abnormally high levels of serum estrogen/androgen due to disruption of negative feedback loop of estrogen biosynthesis.

Because ERαKO did not exhibit bone loss phenotype like postmenopausal osteoporosis, the studies focusing on estrogen’s indirect osteoprotective effects, which mean that secondary effects of estrogen’s target tissue can regulate bone metabolism, have been developed.

It has been reported that bone-resorbing osteoclasts can be differentiated by stimulations of inflammatory cytokines as well as RANKL. At this point, Pacifici and colleagues focused estrogen’s effects on immune cells and they clarified that estrogen can repress the production of inflammatory cytokines, such as TNFα, IL-1, 6, and 7. In the estrogen-deficient status, immune cells can produce abundant inflammatory cytokines and facilitate osteoclast differentiation and bone resorption followed by bone loss [15].

In addition, Zaidi and colleagues clarified the function of follicle stimulating hormone (FSH), whose concentration is increased postmenopausal status, for bone metabolism. FSH can positively regulate osteoclast differentiation, that is, not only deficiency of estrogen but also increased FSH accelerates osteoclastic bone resorption and progress osteoporosis [16]. In fact, it has been reported that serum levels of FSH negatively correlate with bone mass in a clinical situation [17].

Estrogen exerts its effects in various target tissues/organs. This means that there can be diverse secondary/indirect effects of estrogen for controlling bone mass.

As mentioned above, estrogen can indirectly regulate bone metabolism via the immune system and brain. However, is there no direct effect of estrogen in bone mass regulation? Indeed, systemic ERαKO did not display osteoporotic phenotype due to abnormal negative feedback loop of estrogen synthesis. To overcome this issue, cell type specific conditional knockout mice have been generated using Cre/loxP system, where mice lack ERα gene only in bone cells and were not affected by endocrine disorders like systemic ERαKO.