DOI: https://doi.org/10.4414/smw.2020.20187
Bone tissue is simultaneously resorbed at millions of skeletal sites throughout the body and is replaced shortly afterwards. This continuous bone remodelling process is required to replace old bone tissue and to repair bone micro-cracks. In adults it has been estimated that 10% of bone is replaced every year and that this is crucial for maintenance of a healthy skeleton [1, 2]. Bone remodelling throughout life is strictly controlled through a complex network that controls the crosstalk between osteoblast and osteoclast cells. During bone renewal, small areas of bone are resorbed by osteoclasts, which in turn leads to osteoblast recruitment that fill these regions with new bone tissue. This coupling of osteoclast and osteoblast activity is termed the “basic multicellular unit” or BMU. Bone formation and bone resorption are coupled in the BMU through factors that are entrapped within the bone matrix and released upon its degradation by osteoclasts. The BMUs in trabecular and cortical bone show differences in their structure and how they remove and replace bone. In trabecular bone the BMUs are located on the bone surface and are covered by a thin canopy layer of elongated mesenchymal cells covering the whole bone remodelling area and separating it from the bone marrow. The BMUs of cortical bone are located inside the bone within cylinders that have a pointed “cutting zone” formed by osteoclasts [3]. Osteoblast precursors migrate along the abluminal surface of blood vessels [4] to form new bone along the cement line. Cement lines, visible by their reduced mineralisation [5], follow the scalloped surface created by osteoclasts and represents the interface between old and new bone.
Until recently, osteoclasts were described as initiating bone remodelling; however the trigger for this initiation was not known [3]. Evidence has accumulated in the last few years that osteocytes are indeed fact key initiators and drivers of bone remodelling.
Osteocytes are descendants of matrix-producing osteoblasts, encysted in cavities (lacunae) and are located deep inside the bone matrix. The exact life span of osteocytes is unknown; however, it is estimated that they can survive for decades if undisturbed in locations with a slow bone turnover rate [6]. Osteocytes are known to play a crucial role in sensing mechanical loading and regulation of calcium and phosphate homeostasis.
For decades it has been well known that osteocytes can remove and remodel their surrounding bone matrix structures. Osteocytes are able to demineralise their perilacunar/canalicular matrix, a mechanism defined as “osteocytic osteolysis”. It occurs during various physiological and pathophysiological conditions such as immobilisation, hyperparathyroidism and lactation, and has an important function in calcium homeostasis.
Qing et al. demonstrated that lactating mice have larger osteocyte lacunae than virgin controls to mobilise bone mineral. Seven days post-lactation new bone is formed around the lacunae and their size returns to virgin levels. Osteocytes seem to use similar molecular mechanisms to osteoclasts to remove mineralised bone matrix. They upregulate osteoclast markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K in response to elevated parathyroid hormone-related peptide (PTHrP) [7]. Additionally, they create an acidic environment in their lacunar-canalicular space through carbonic anhydrase 2 and the proton pumping vacuolar ATPases. However, they lack a sealed resorption pit and therefore it is not clear which mechanisms protect them from the acidic milieu [8, 9]. Further studies are needed to understand the precise mechanisms of osteocytic osteolysis and its differences from osteoclast-mediated bone resorption.
Recent studies have revealed that osteocytes control bone remodelling by communication with cells located in their surrounding environment. Each cell exhibits 40–100 dendritic extensions [10], which, by extending through the bone matrix in channels called canaliculi, form connections to neighbouring osteocytes, blood vessels and cells on the bone surface. Murine canaliculi are between 50 and 100 nm in diameter [11] and dendrites are connected to each other via gap junctions [12], building an active and responsive network inside the bone that links osteocytes to each other and to cells at the periosteal and endosteal surfaces as well as endothelial cells of the capillaries.
Whereas osteoblasts (and consequently osteocytes) originate from the mesenchymal lineage, osteoclasts are of haematopoietic origin and are derived from the monocyte/macrophage lineage. During osteoclast differentiation, the monocyte/macrophage-derived osteoclast precursors fuse with each other forming large multi-nucleated cells that are unique in their ability to degrade the bone matrix. Two osteoblast-derived factors, macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL), are essential for osteoclastogenesis, and the number of osteoclasts is severely reduced in mice in which the activity of either one of these factors is impaired [13–17]. RANKL signals via its receptor RANK, present on osteoclast precursors and mature osteoclasts. Loss-of-function mutations in either TNFSF11 or TNFRSF11A, the genes encoding RANKL and RANK, respectively, result in osteoclast-poor osteopetrosis characterised by increased bone mass due to defective osteoclast differentiation [18–21]. RANKL and M-CSF are also expressed by osteocytes supporting the generation of functional osteoclasts at sites of bone remodelling [22]. Interestingly, mice deficient in M-CSF display osteocyte defects as well as osteopetrosis [23]. This could be owing either to direct effects of M-CSF on the differentiation and/or survival of osteocytes, or to the absence of osteoclast- and/or macrophage-dependent signals. Nakashima et al. [24] demonstrated that purified osteocytes have a greater capacity to support osteoclastogenesis by release of RANKL than osteoblasts or bone marrow stromal cells. In agreement with this, it has been demonstrated that RANKL secreted by osteocytes is essential for bone remodelling whereas RANKL produced by osteoblasts is redundant [25]. The action of RANKL can be blocked by osteoprotegerin (OPG), a soluble decoy receptor that binds to RANKL, preventing it from binding to RANK [26], and the RANKL/OPG ratio is a key determinant of the degree to which osteoclast-mediated bone resorption occurs at a given site [27–29]. OPG expression by osteocytes was found to be highly increased upon ovariectomy, possibly as a protective or reparatory mechanism in response to increased bone loss [30].
In order to exert their function, osteoclasts adhere to the bone surface via an actin ring (or sealing zone) that surrounds a ruffled border, enabling them to create a local acidic resorptive microenvironment [21, 31]. Bone is a rich source of growth factors, which are embedded in the bone matrix and released upon osteoclast-mediated bone resorption [32]. One of these growth factors, which is highly present in the bone matrix and released and activated as a result of osteoclast activity is transforming growth factor-β (TGF-β) [33–35]. TGF-β is a pleiotropic cytokine controlling a wide variety of cellular responses during bone remodelling affecting, amongst others, the differentiation of both osteoclasts and osteoblasts. Importantly, TGF-β couples bone resorption and formation, since the active TGF-β1 released during osteoclast-mediated bone resorption induces migration of bone mesenchymal stem cells to the resorptive sites where they undergo osteoblastic differentiation in response to the signals provided by the local microenvironment generated by the osteoclasts [36]. Whereas osteoclasts recruit osteoblasts to sites of bone remodelling, the osteoclasts and their progenitors are thought to be directed to these locations in an osteocyte-dependent manner. Osteocytes undergo apoptosis as a result of microdamage or lack of mechanical stimulation due to disuse or weightlessness, which promotes recruitment and/or differentiation of osteoclast precursors to the sites where osteocyte apoptosis had occurred [37–39]. Increased expression of the osteoclast-promoting cytokines RANKL and vascular endothelial growth factor (VEGF) and decreased expression of OPG in osteocytes was found to coincide with up-regulation of caspase-3 and to be blocked by inhibiting osteocyte apoptosis [40–42]. The RANKL released from the apoptotic osteocytes subsequently controls osteoclast localisation and differentiation and bone resorption, followed by recruitment of osteoblast precursors [41, 42]. It is noteworthy that osteocyte-specific deletion of connexin 43, which mediates the communication between osteocytes and osteoblasts, is required for their survival, leading to an enrichment of osteoclasts in areas where osteocytes have undergone apoptosis and at the same time reduced their OPG expression levels [43]. Together, this evidence demonstrates that bone remodelling and the recruitment of osteoclast and osteoblast precursors is targeted to sites where osteocyte apoptosis has occurred by localised shifting of the RANKL/OPG ratio.
Osteocytes also regulate osteoblast mineralisation by targeting the Wnt signalling pathway through the release of bone anabolic Wnt proteins and their inhibitors the Dickkopf related protein 1 (DKK1) and sclerostin. Overexpression of Wnt1 in mice results in bone formation by increasing osteoblast number and activity [44]. Wnt inhibitor sclerostin and DKK1 are known to block Wnt proteins by binding LRP 5/6 receptors leading to increased bone volume and density [45–47].
Mesenchymal stromal cells (MSCs) are multipotent cells but do not fulfil all stem cell criteria [48]. MSCs can be isolated out of many tissues and the characteristics of these cells can differ; however, there are three minimum criteria to be fulfilled: they must be plastic-adherent under standard culture conditions; they must occupy the potential to differentiate into adipocytes, osteoblasts and chondroblasts in vitro; and they must express the surface markers CD105, CD73 and CD90 and lack haematopoietic markers including CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR [49].
At present, little is known about the influence of osteocytes on MSCs. Conditioned media from the osteocytic cell line MLO-Y4 increases the osteogenic differentiation potential of MSC compared with supernatant of the osteoblastic cell line MC3T3-E1 [50]. Moreover, co-cultures of osteocytes with MSCs leads to greater calcium deposition than osteoblasts cultured with MSCs [51], indicating an important role for MSC-osteocyte interactions on driving bone mineralisation. Despite the lack of knowledge concerning the soluble signals affecting osteogenic differentiation, osteocytes seem to have an active stimulatory impact on bone formation rates.
Osteocyte signalling is not only involved in osteogenic, but also in adipogenic differentiation of MSCs. Fairfield and colleagues found that sclerostin, an inhibitor of bone formation mainly released by osteocytes, induces adipogenesis in bone marrow-derived MSCs [52]. Knockout of the Sost gene (encoding sclerostin) or the pharmacological inhibition of sclerostin decreased bone marrow adipose tissue formation, demonstrating that the bone marrow niche is regulated by osteocytes in the extracellular matrix through sclerostin release. To our knowledge, there are currently no reports on the influence of osteocytes on chondrogenic differentiation of MSCs.
The importance of blood vessels in endochondral bone formation in the embryo has long been understood, with vascularisation of the bone primordium always preceding ossification. More recently the precise role of the blood vessel endothelial cells in guiding osteoblasts to sites of bone formation [4, 53] and regulating their maturation has been identified [54, 55]. In some instances it appears that the blood capillaries themselves can undergo mineralisation [56] and thus guide trabecular patterning.
In the context of trabecular bone modelling, the interaction of osteocytes with bone endothelial cells has not yet been investigated in detail; however, studies of the small bones of the ear has demonstrated that osteocyte lacunae align in parallel to bone capillaries, reminiscent of the patterning observed in the cortical bone osteons of larger mammals [53]. Indeed, analysis by scanning electron microscopy of human bone [57] and of optically cleared mouse long bones has demonstrated direct interaction of osteocyte canaliculi with endothelial cells and with the capillary-associated osteoclasts that are responsible for cutting the micro-canals that house these capillaries [58]. Given that osteocytes are the main producers of RANKL [24], it seems likely that osteocyte signalling guides the formation of transcortical capillaries by osteoclasts. Osteocytes also secrete prostaglandin E2 which has regulatory effects on angiogenesis and vascular permeability [59, 60], and therefore may also control bone oxygenation and metabolism.
In an initial study to assess the effect of inflammation on transcortical capillaries, Grϋneboom et al. [58] showed that chronic arthritis in mice increased transcortical vessel number within 62 days of arthritis onset, demonstrating that changes in the bone vascular system can be highly dynamic. This increase in vessel number was successfully blocked with bisphosphonate treatment, indicating the requirement of osteoclasts for new bone vessel formation. An increase in cortical canals has also been observed in patients with rheumatoid arthritis, in some cases very early in disease [61]. It remains to be seen whether the increase in bone capillaries is a driver of disease and damage in arthritis or secondary to the metabolic requirements of the inflamed tissue, however this represents an exciting and novel opportunity for future work.
The techniques now exist to visualise the bone in much higher detail that was previously possible. Advances in micro-computed tomography (microCT), including synchrotron-mediated microCT, are now able to give resolution below 1 μm [62, 63], allowing visualisation of cortical vessels. A particularly important development in this field is optical clearing of tissue which, for the first time has allowed 3D visualisation of immuno-labelled whole bones [64].
Although osteocytes are located deep inside the mineralised bone matrix, they do not exist isolated without connection to their environment. Osteocytes build a dense lacunar-canalicular network that affects bone remodelling by aiding communication between one cell and another. This network is also highly dynamic, osteocytes can extend and retract their dendrites [65] adapting to environmental changes to maintain bone homeostasis.
Patients with musculoskeletal disorders such as osteoporosis, rheumatoid arthritis, osteoarthritis and osteomyelitis suffer from imbalance of bone remodelling. It still remains largely unknown how the architecture of the osteocyte network is involved in this process and how this might influence bone loss and repair, especially under disease conditions. The importance of the osteocyte network during degenerative bone disorders will be discussed in the following sections.
Rheumatoid arthritis is a systemic autoimmune disease characterised by joint inflammation, pain, fatigue, and destruction of articular cartilage and bone. In particular, localised osteoclast-mediated bone resorption at the interface between inflammatory synovial tissue and subchondral bone is responsible for erosions seen in rheumatoid arthritis patients. The impact of osteocytes and their network on inflammatory bone loss due to the communication with adjacent osteoclasts in rheumatoid arthritis is not yet understood.
Recently, in vitro studies have demonstrated that stimulation of osteocytes with serum from patients with rheumatoid arthritis, containing elevated levels of inflammatory cytokines, increases the RANKL/OPG ratio, which leads subsequently to enhanced osteoclastogenesis and bone destruction [66]. Moreover, osteoblastogenesis is reduced as a result of enhanced expression of DKK1 and sclerostin by osteocytes, both negative regulators of bone regeneration. Pathak and co-authors found that osteocytes produce pro-inflammatory cytokines such as tumour necrosis-α (TNFα), interleukin (IL)-6 and IL-1β, key factors in rheumatoid arthritis pathogenesis, which are up-regulated by rheumatoid arthritis -serum treatment in vitro. Therefore osteocytes might provide a new therapeutic target to treat inflammatory bone loss in rheumatoid arthritis [67].
Osteoarthritis is a chronic, degenerative joint disease featuring articular cartilage degradation, osteophyte formation, muscle weakness and inflammation that has a number of reasons including mechanical joint instability, obesity, genetic pre-disposition and ageing. Osteoarthritis has been long considered as a primary disorder of articular cartilage; however, the impact of bone in the pathology of osteoarthritis is gaining increasing interest. It is commonly accepted that subchondral bone sclerosis is a hallmark of osteoarthritis [68, 69]. As a result of abnormal bone remodelling, subchondral bone is thickened and hyper-mineralised in osteoarthritis and these changes have been detected prior to any evidence of cartilage damage [70]. Moreover, phenotypic changes of osteocytes might be involved in the increase of bone volume and calcification: Studies from Jaiprakash et al. and Tate et al. demonstrated that the osteocyte bodies in subchondral bone from patients with osteoarthritis are rounder with fewer, more disorganised dendrites and decreased connectivity compared to healthy controls [71, 72]. They also identified reduced expression of sclerostin and increased expression of dentin matrix protein 1 (DMP1), a regulator of bone mineralisation, in osteoarthritis osteocytes that correlated with increased subchondral bone volume.
The importance of the osteocyte network for osteoarthritis development has recently been shown in a mechanically-induced post-traumatic osteoarthritis mouse model. The authors revealed that an intact osteocyte network in the subchondral bone contributes to the development of osteoarthritis, suggesting that targeting may ameliorate severity of the disease [73].
Although the role of osteocytes and their network in osteoarthritis remains to be elucidated, these current studies suggest a critical regulatory function of osteocytes in bone homeostasis and mineral metabolism during osteoarthritis development.
Osteomyelitis is characterised by inflammation of the bones and the surrounding tissues due to bacterial infections leading to bone deformations and destruction. This infection is very painful and not easy to treat, especially when it becomes chronic. Unfortunately, the rate of clinical failures in treatment is high, and the consequence is often loss of function and/or amputation [74, 75].
One of the main pathogens is Staphylococcus aureus, which persists in the bone by invading osteoblasts and osteoclasts, which provides protection from the immune system [76–78]. The internalisation of S. aureus by osteoblasts leads to decreased osteoblast activity characterised by reduced proliferation, increased apoptosis, decreased expression of osteogenic markers such as collagen type I, osteopontin, osteocalcin, and decreased alkaline phosphatase (ALP) activity [79–81]. Additionally, infected osteoblasts display an imbalanced RANKL/OPG ratio, shifting the bone remodelling process towards bone destruction. Osteoblasts infected by S. aureus show increased expression of RANKL and prostaglandin E2 (PGE2) [79, 81, 82]. PGE2 is a hormone-like molecule, which induces RANKL expression in an autocrine and paracrine manner via activation of the EP4 receptor on osteoblasts [83].
Osteoclasts and their precursors can also be infected by S. aureus. Infected precursors release pro-inflammatory cytokines that trigger osteoclastogenesis of uninfected cells, whereas the infection of mature osteoclasts lead to increased resorption activity and subsequent bone loss [78]. Of note, De Mesy Bentley et al. [84] demonstrated that in a murine mouse model of osteomyelitis S. aureus invades into the osteocyte lacunae, proliferates and migrates throughout the canaliculi network. Recently, they identified invasion of S. aureus in the osteocytic-canalicular system of an amputated limb from a patient suffering from diabetic foot ulcer and S. aureus chronic osteomyelitis [85]. These new findings reveal that microorganisms not only infect osteoclasts and osteoblasts, but are also able to migrate through the dense osteocyte network, providing a new mechanism to spread and sustain the infection.
Patients with osteoporosis display an imbalance between bone resorption and bone formation leading to bone loss and a higher fracture risk associated with an increase in morbidity and mortality. Osteoporosis is most commonly associated with ageing, affecting mostly postmenopausal women. It is assumed that the bone loss seen in these patients is linked to osteocyte death triggered by oestrogen deficiency [86]. Tatsumi et al. analysed osteocyte function in an osteocytes deletion model. This mouse model is based on a diphtheria toxin receptor-mediated cell knockout (TRECK) system under an osteocyte specific promoter. After a single injection of diphtheria toxin approximately 80% of osteocytes (but no osteoblasts) are deleted. This triggers TRAP+ osteoclast invasion into the cortical bone and recruitment of ALP+ osteoblasts to the intracortical cavities created by osteoclasts. Additionally, long-term effects such as decreased trabecular and cortical bone mass and microarchitecture deterioration occur, demonstrating a role for osteocytes in bone loss in osteoporosis [87].
Since the ability to sense and respond to mechanical load depends on many factors, such as shape of the cell and the cytoskeleton, it is likely that the mechanical stimulation of osteocytes in osteoporosis is affected [88]. Interestingly, osteocyte-ablated mice exhibited resistance to bone loss induced by limb-unloading [87]. Moreover, an ulnar-loading model demonstrated that enhanced loading reduces sclerostin release by osteocytes and promotes bone formation. On the contrary, unloading of the hind limbs lead to an upregulation of sost transcript [89] and subsequently inhibits bone formation. These results clearly demonstrate that osteocytes need mechanical load to sense and adapt to external stimuli.
The mechanical load on bone is amplified in the interstitial fluid flow, which is the movement of fluid throughout the lacunar-canaliculi system [90]. The resulting fluid shear stress in osteocytes controls the expression of signalling proteins that influences the differentiation or activity of osteoblasts and osteoclasts [89, 91, 92]. Ciani and colleagues showed enhanced fluid movement in the cancellous bone across the lacunar-canaliculi system in ovariectomised rats after mechanical stimulation [93]. In this model of postmenopausal osteoporosis, it could also be shown that oestrogen deficiency resulted in increased canaliculi size and lacunar-canalicular porosity [94]. This demonstrates that altered interstitial fluid flow in oestrogen-deficient rats is associated with an alteration of transmission signals. All together, these studies propose osteocytes as important mechanosensors regulating bone remodelling by releasing signal proteins to maintain bone homeostasis.
Therapeutics to treat bone loss have until recently focused on targeting osteoclasts. More recently drugs that promote osteoblast function have also been developed and the potential for targeting osteocyte activity is an exciting new avenue for therapy. Antiresorptive agents such as bisphosphonates are the most common medication to target osteoclast-mediated bone resorption in patients suffering from osteoporosis [95]. One major property of bisphosphonates is their ability to bind strongly to the bone mineral hydroxyapatite. Osteoclasts, during their resorption process, take up and internalise bisphosphonates from the bone surface. Bisphosphonates act by driving apoptosis of osteoclasts or through the inhibition of farnesyl pyrophosphate (FPP) synthase, preventing post-translational prenylation of GTPases that are required for the ruffled border formation of osteoclasts. As a consequence osteoclast resorption is inhibited [96, 97]. Another antiresorptive drug is denosumab, a monoclonal antibody against RANKL. However, treatment with bisphosphonates or RANKL inhibitors is able to prevent excessive bone resorption, and do not affect bone formation. One possible reason for that is that osteoclasts release crucial factors from the mineralised matrix such as active transforming growth factor-β (TGFβ) or insulin-like growth factor-1 (IGF-1) that are needed to enhance migration of mesenchymal stromal cell to the site of resorption to promote bone formation [36, 98]. Thus, new drugs that are able to inhibit bone resorption while increasing bone formation are needed.
Most recently, the potential for therapeutic-targeting of osteocytes has been realised with anabolic drugs such as anti-sclerostin antibodies for osteoporosis treatment coming onto the market. Sclerostin, mainly expressed by osteocytes, exerts its inhibitory actions on bone formation by negatively regulating the canonical Wnt signalling pathway, and therefore its inhibition is a very promising strategy to promote bone formation. Neutralising antibodies to sclerostin have been developed to treat patients with osteoporosis. Interestingly, these antibodies display dual effects by increasing bone formation as well as decreasing bone resorption. Phase III studies of the monoclonal anti-sclerostin antibody romosozumab (AMG785) have demonstrated positive results. In 2016, the FRAME study (Fracture study in postmenopausal women with osteoporosis) was published, in which 7180 postmenopausal women received a monthly dose of romosozumab or a placebo for one year, followed by denosumab treatment every 6 month for one year. After 12 and 24 months, the risk for new vertebral fractures decreased in the romosozumab group [99]. The FRAME study was followed by the ARCH study (Active Controlled Fracture Study in Postmenopausal women with osteoporosis at high risk), published in 2017. In this study, 4093 patients received monthly subcutaneous injections of romosozumab or weekly oral administration of alendronate, followed by alendronate only for another 12 months. The risk for new vertebral fractures was 48% lower in the romosozumab-to-alendronate group than in the alendronate-to-alendronate group; however, for the first time, an imbalance in cardiovascular adverse events in the romosozumab group compared with the alendronate only group was observed [100]. An explanation for this trend could be that sclerostin is constitutively expressed in the aorta [101] and is upregulated in foci of vascular and valvular calcification, but the function of sclerostin in cardiovascular tissue remains unknown [102, 103]. Unfortunately, because this study was not designed as a cardiovascular risk study it lacked a placebo-only control. Thus, further work is required to understand the cardiovascular risks of targeting sclerostin in these patients. The last phase III study, the BRIDGE study (placebo-controlled study evaluating the efficacy and safety of romosozumab in treating men with osteoporosis), tested romosozumab in men and was published in 2018. Two hundred and forty-five men were treated with romosozumab or placebo once a month for 1 year. The outcome of the study was significantly higher bone mineral density of the lumbar spine in the romosozumab group compared with placebo control. Again, there was an imbalance in adverse cardiovascular events in the romosozumab group [104]. In 2019, the US Food and Drugs Administration approved the drug romosozumab (trade name Evenity) for patients suffering from postmenopausal osteoporosis with high fracture risk. Excluded are women with a cardiovascular event one year before initiation of the treatment.
Despite their importance in controlling bone homeostasis, the precise function of osteocytes and the molecular mechanisms underpinning how osteocytes influence bone remodelling, especially under disease conditions, are far from clear. Several studies have revealed that osteocytes release signalling molecules that can either promote or impair osteoblastogenesis and osteoclastogenesis, and also seem to be important regulators for new blood vessel formation. It is likely that not only osteocytes themselves but also the pattern of the osteocyte lacunar-canalicular network plays a key role in maintaining healthy bone homeostasis. Advancing our understanding of osteocyte biology should prove useful in developing novel therapies to preserve osteocyte dendricity and viability thereby protecting against bone loss and maintaining bone health.
No financial support and no other potential conflict of interest relevant to this article was reported.
1 Parfitt AM . Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem. 1994;55(3):273–86. doi:.https://doi.org/10.1002/jcb.240550303
2 Manolagas SC . Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev. 2000;21(2):115–37.
3 Sims NA , Martin TJ . Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit. Bonekey Rep. 2014;3:481. doi:.https://doi.org/10.1038/bonekey.2013.215
4 Maes C , Kobayashi T , Selig MK , Torrekens S , Roth SI , Mackem S , et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell. 2010;19(2):329–44. doi:.https://doi.org/10.1016/j.devcel.2010.07.010
5 Burr DB , Schaffler MB , Frederickson RG . Composition of the cement line and its possible mechanical role as a local interface in human compact bone. J Biomech. 1988;21(11):939–45. doi:.https://doi.org/10.1016/0021-9290(88)90132-7
6 Dallas SL , Prideaux M , Bonewald LF . The osteocyte: an endocrine cell ... and more. Endocr Rev. 2013;34(5):658–90. doi:.https://doi.org/10.1210/er.2012-1026
7 Qing H , Ardeshirpour L , Pajevic PD , Dusevich V , Jähn K , Kato S , et al. Demonstration of osteocytic perilacunar/canalicular remodeling in mice during lactation. J Bone Miner Res. 2012;27(5):1018–29. doi:.https://doi.org/10.1002/jbmr.1567
8 Tsourdi E , Jähn K , Rauner M , Busse B , Bonewald LF . Physiological and pathological osteocytic osteolysis. J Musculoskelet Neuronal Interact. 2018;18(3):292–303.
9 Jähn K , Kelkar S , Zhao H , Xie Y , Tiede-Lewis LM , Dusevich V , et al. Osteocytes Acidify Their Microenvironment in Response to PTHrP In Vitro and in Lactating Mice In Vivo. J Bone Miner Res. 2017;32(8):1761–72. doi:.https://doi.org/10.1002/jbmr.3167
10 Beno T , Yoon YJ , Cowin SC , Fritton SP . Estimation of bone permeability using accurate microstructural measurements. J Biomech. 2006;39(13):2378–87. doi:.https://doi.org/10.1016/j.jbiomech.2005.08.005
11 Su M , Jiang H , Zhang P , Liu Y , Wang E , Hsu A , et al. Knee-loading modality drives molecular transport in mouse femur. Ann Biomed Eng. 2006;34(10):1600–6. doi:.https://doi.org/10.1007/s10439-006-9171-z
12 Palumbo C , Palazzini S , Marotti G . Morphological study of intercellular junctions during osteocyte differentiation. Bone. 1990;11(6):401–6. doi:.https://doi.org/10.1016/8756-3282(90)90134-K
13 Kodama H , Nose M , Niida S , Yamasaki A . Essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells. J Exp Med. 1991;173(5):1291–4. doi:.https://doi.org/10.1084/jem.173.5.1291
14 Lacey DL , Timms E , Tan HL , Kelley MJ , Dunstan CR , Burgess T , et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 1998;93(2):165–76. doi:.https://doi.org/10.1016/S0092-8674(00)81569-X
15 Simonet WS , Lacey DL , Dunstan CR , Kelley M , Chang MS , Lüthy R , et al. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 1997;89(2):309–19. doi:.https://doi.org/10.1016/S0092-8674(00)80209-3
16 Teitelbaum SL , Ross FP . Genetic regulation of osteoclast development and function. Nat Rev Genet. 2003;4(8):638–49. doi:.https://doi.org/10.1038/nrg1122
17 Yoshida H , Hayashi S , Kunisada T , Ogawa M , Nishikawa S , Okamura H , et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature. 1990;345(6274):442–4. doi:.https://doi.org/10.1038/345442a0
18 Guerrini MM , Sobacchi C , Cassani B , Abinun M , Kilic SS , Pangrazio A , et al. Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations. Am J Hum Genet. 2008;83(1):64–76. doi:.https://doi.org/10.1016/j.ajhg.2008.06.015
19 Nakagawa N , Kinosaki M , Yamaguchi K , Shima N , Yasuda H , Yano K , et al. RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem Biophys Res Commun. 1998;253(2):395–400. doi:.https://doi.org/10.1006/bbrc.1998.9788
20 Sobacchi C , Frattini A , Guerrini MM , Abinun M , Pangrazio A , Susani L , et al. Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL. Nat Genet. 2007;39(8):960–2. doi:.https://doi.org/10.1038/ng2076
21 Sobacchi C , Schulz A , Coxon FP , Villa A , Helfrich MH . Osteopetrosis: genetics, treatment and new insights into osteoclast function. Nat Rev Endocrinol. 2013;9(9):522–36. doi:.https://doi.org/10.1038/nrendo.2013.137
22 Zhao S , Kato Y , Zhang Y , Harris S , Ahuja SS , Bonewald LF . MLO-Y4 osteocyte-like cells support osteoclast formation and activation. J Bone Miner Res. 2002;17(11):2068–79. doi:.https://doi.org/10.1359/jbmr.2002.17.11.2068
23 Harris SE , MacDougall M , Horn D , Woodruff K , Zimmer SN , Rebel VI , et al. Meox2Cre-mediated disruption of CSF-1 leads to osteopetrosis and osteocyte defects. Bone. 2012;50(1):42–53. doi:.https://doi.org/10.1016/j.bone.2011.09.038
24 Nakashima T , Hayashi M , Fukunaga T , Kurata K , Oh-Hora M , Feng JQ , et al. Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat Med. 2011;17(10):1231–4. doi:.https://doi.org/10.1038/nm.2452
25 Xiong J , Onal M , Jilka RL , Weinstein RS , Manolagas SC , O’Brien CA . Matrix-embedded cells control osteoclast formation. Nat Med. 2011;17(10):1235–41. doi:.https://doi.org/10.1038/nm.2448
26 Hofbauer LC . Pathophysiology of RANK ligand (RANKL) and osteoprotegerin (OPG). Ann Endocrinol (Paris). 2006;67(2):139–41. doi:.https://doi.org/10.1016/S0003-4266(06)72569-0
27 Dougall WC . Molecular pathways: osteoclast-dependent and osteoclast-independent roles of the RANKL/RANK/OPG pathway in tumorigenesis and metastasis. Clin Cancer Res. 2012;18(2):326–35. doi:.https://doi.org/10.1158/1078-0432.CCR-10-2507
28 Dougall WC , Holen I , González Suárez E . Targeting RANKL in metastasis. Bonekey Rep. 2014;3:519. doi:.https://doi.org/10.1038/bonekey.2014.14
29 Kostenuik PJ . Osteoprotegerin and RANKL regulate bone resorption, density, geometry and strength. Curr Opin Pharmacol. 2005;5(6):618–25. doi:.https://doi.org/10.1016/j.coph.2005.06.005
30 Ikeda T , Utsuyama M , Hirokawa K . Expression profiles of receptor activator of nuclear factor kappaB ligand, receptor activator of nuclear factor kappaB, and osteoprotegerin messenger RNA in aged and ovariectomized rat bones. J Bone Miner Res. 2001;16(8):1416–25. doi:.https://doi.org/10.1359/jbmr.2001.16.8.1416
31 Teitelbaum SL . The osteoclast and its unique cytoskeleton. Ann N Y Acad Sci. 2011;1240(1):14–7. doi:.https://doi.org/10.1111/j.1749-6632.2011.06283.x
32 Hauschka PV , Mavrakos AE , Iafrati MD , Doleman SE , Klagsbrun M . Growth factors in bone matrix. Isolation of multiple types by affinity chromatography on heparin-Sepharose. J Biol Chem. 1986;261(27):12665–74.
33 Oreffo RO , Mundy GR , Seyedin SM , Bonewald LF . Activation of the bone-derived latent TGF beta complex by isolated osteoclasts. Biochem Biophys Res Commun. 1989;158(3):817–23. doi:.https://doi.org/10.1016/0006-291X(89)92795-2
34 Oursler MJ . Osteoclast synthesis and secretion and activation of latent transforming growth factor beta. J Bone Miner Res. 1994;9(4):443–52. doi:.https://doi.org/10.1002/jbmr.5650090402
35 Pfeilschifter J , Diel I , Scheppach B , Bretz A , Krempien R , Erdmann J , et al. Concentration of transforming growth factor beta in human bone tissue: relationship to age, menopause, bone turnover, and bone volume. J Bone Miner Res. 1998;13(4):716–30. doi:.https://doi.org/10.1359/jbmr.1998.13.4.716
36 Tang Y , Wu X , Lei W , Pang L , Wan C , Shi Z , et al. TGF-beta1-induced migration of bone mesenchymal stem cells couples bone resorption with formation. Nat Med. 2009;15(7):757–65. doi:.https://doi.org/10.1038/nm.1979
37 Clark WD , Smith EL , Linn KA , Paul-Murphy JR , Muir P , Cook ME . Osteocyte apoptosis and osteoclast presence in chicken radii 0-4 days following osteotomy. Calcif Tissue Int. 2005;77(5):327–36. doi:.https://doi.org/10.1007/s00223-005-0074-z
38 Aguirre JI , Plotkin LI , Stewart SA , Weinstein RS , Parfitt AM , Manolagas SC , et al. Osteocyte apoptosis is induced by weightlessness in mice and precedes osteoclast recruitment and bone loss. J Bone Miner Res. 2006;21(4):605–15. doi:.https://doi.org/10.1359/jbmr.060107
39 Al-Dujaili SA , Lau E , Al-Dujaili H , Tsang K , Guenther A , You L . Apoptotic osteocytes regulate osteoclast precursor recruitment and differentiation in vitro. J Cell Biochem. 2011;112(9):2412–23. doi:.https://doi.org/10.1002/jcb.23164
40 Kennedy OD , Laudier DM , Majeska RJ , Sun HB , Schaffler MB . Osteocyte apoptosis is required for production of osteoclastogenic signals following bone fatigue in vivo. Bone. 2014;64:132–7. doi:.https://doi.org/10.1016/j.bone.2014.03.049
41 Cabahug-Zuckerman P , Frikha-Benayed D , Majeska RJ , Tuthill A , Yakar S , Judex S , et al. Osteocyte Apoptosis Caused by Hindlimb Unloading is Required to Trigger Osteocyte RANKL Production and Subsequent Resorption of Cortical and Trabecular Bone in Mice Femurs. J Bone Miner Res. 2016;31(7):1356–65. doi:.https://doi.org/10.1002/jbmr.2807
42 Plotkin LI , Gortazar AR , Davis HM , Condon KW , Gabilondo H , Maycas M , et al. Inhibition of osteocyte apoptosis prevents the increase in osteocytic receptor activator of nuclear factor κB ligand (RANKL) but does not stop bone resorption or the loss of bone induced by unloading. J Biol Chem. 2015;290(31):18934–42. doi:.https://doi.org/10.1074/jbc.M115.642090
43 Bivi N , Condon KW , Allen MR , Farlow N , Passeri G , Brun LR , et al. Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation. J Bone Miner Res. 2012;27(2):374–89. doi:.https://doi.org/10.1002/jbmr.548
44 Joeng KS , Lee YC , Lim J , Chen Y , Jiang MM , Munivez E , et al. Osteocyte-specific WNT1 regulates osteoblast function during bone homeostasis. J Clin Invest. 2017;127(7):2678–88. doi:.https://doi.org/10.1172/JCI92617
45 Li J , Sarosi I , Cattley RC , Pretorius J , Asuncion F , Grisanti M , et al. Dkk1-mediated inhibition of Wnt signaling in bone results in osteopenia. Bone. 2006;39(4):754–66. doi:.https://doi.org/10.1016/j.bone.2006.03.017
46 Winkler DG , Sutherland MK , Geoghegan JC , Yu C , Hayes T , Skonier JE , et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J. 2003;22(23):6267–76. doi:.https://doi.org/10.1093/emboj/cdg599
47 Li X , Zhang Y , Kang H , Liu W , Liu P , Zhang J , et al. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem. 2005;280(20):19883–7. doi:.https://doi.org/10.1074/jbc.M413274200
48 Horwitz EM , Le Blanc K , Dominici M , Mueller I , Slaper-Cortenbach I , Marini FC , et al.; International Society for Cellular Therapy. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy. 2005;7(5):393–5. doi:.https://doi.org/10.1080/14653240500319234
49 Dominici M , Le Blanc K , Mueller I , Slaper-Cortenbach I , Marini F , Krause D , et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315–7. doi:.https://doi.org/10.1080/14653240600855905
50 Heino TJ , Hentunen TA , Väänänen HK . Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts. Exp Cell Res. 2004;294(2):458–68. doi:.https://doi.org/10.1016/j.yexcr.2003.11.016
51 Birmingham E , Niebur GL , McHugh PE , Shaw G , Barry FP , McNamara LM . Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche. Eur Cell Mater. 2012;23:13–27. doi:.https://doi.org/10.22203/eCM.v023a02
52 Fairfield H , Falank C , Harris E , Demambro V , McDonald M , Pettitt JA , et al. The skeletal cell-derived molecule sclerostin drives bone marrow adipogenesis. J Cell Physiol. 2018;233(2):1156–67. doi:.https://doi.org/10.1002/jcp.25976
53 Matsuo K , Kuroda Y , Nango N , Shimoda K , Kubota Y , Ema M , et al. Osteogenic capillaries orchestrate growth plate-independent ossification of the malleus. Development. 2015;142(22):3912–20. doi:.https://doi.org/10.1242/dev.123885
54 Kusumbe AP , Ramasamy SK , Adams RH . Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature. 2014;507(7492):323–8. doi:.. Correction in: Nature. 2014;513:574. https://doi.org/10.1038/nature13145
55 Langen UH , Pitulescu ME , Kim JM , Enriquez-Gasca R , Sivaraj KK , Kusumbe AP , et al. Cell-matrix signals specify bone endothelial cells during developmental osteogenesis. Nat Cell Biol. 2017;19(3):189–201. doi:.https://doi.org/10.1038/ncb3476
56 Ben Shoham A , Rot C , Stern T , Krief S , Akiva A , Dadosh T , et al. Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology. Development. 2016;143(21):3933–43. doi:.https://doi.org/10.1242/dev.139253
57 Prasadam I , Zhou Y , Du Z , Chen J , Crawford R , Xiao Y . Osteocyte-induced angiogenesis via VEGF-MAPK-dependent pathways in endothelial cells. Mol Cell Biochem. 2014;386(1-2):15–25. doi:.https://doi.org/10.1007/s11010-013-1840-2
58 Grüneboom A , Hawwari I , Weidner D , Culemann S , Müller S , Henneberg S , et al. A network of trans-cortical capillaries as mainstay for blood circulation in long bones. Nat Metab. 2019;1(2):236–50. doi:.https://doi.org/10.1038/s42255-018-0016-5
59 Birukova AA , Zagranichnaya T , Fu P , Alekseeva E , Chen W , Jacobson JR , et al. Prostaglandins PGE(2) and PGI(2) promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation. Exp Cell Res. 2007;313(11):2504–20. doi:.https://doi.org/10.1016/j.yexcr.2007.03.036
60 Pai R , Szabo IL , Soreghan BA , Atay S , Kawanaka H , Tarnawski AS . PGE(2) stimulates VEGF expression in endothelial cells via ERK2/JNK1 signaling pathways. Biochem Biophys Res Commun. 2001;286(5):923–8. doi:.https://doi.org/10.1006/bbrc.2001.5494
61 Werner D , Simon D , Englbrecht M , Stemmler F , Simon C , Berlin A , et al. Early Changes of the Cortical Micro-Channel System in the Bare Area of the Joints of Patients With Rheumatoid Arthritis. Arthritis Rheumatol. 2017;69(8):1580–7. doi:.https://doi.org/10.1002/art.40148
62 Lafage-Proust MH , Roche B , Langer M , Cleret D , Vanden Bossche A , Olivier T , et al. Assessment of bone vascularization and its role in bone remodeling. Bonekey Rep. 2015;4:662. doi:.https://doi.org/10.1038/bonekey.2015.29
63 Núñez JA , Goring A , Hesse E , Thurner PJ , Schneider P , Clarkin CE . Simultaneous visualisation of calcified bone microstructure and intracortical vasculature using synchrotron X-ray phase contrast-enhanced tomography. Sci Rep. 2017;7(1):13289. doi:.https://doi.org/10.1038/s41598-017-13632-5
64 Greenbaum A , Chan KY , Dobreva T , Brown D , Balani DH , Boyce R , et al. Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Sci Transl Med. 2017;9(387):eaah6518. doi:.https://doi.org/10.1126/scitranslmed.aah6518
65 Dallas SL , Bonewald LF . Dynamics of the transition from osteoblast to osteocyte. Ann N Y Acad Sci. 2010;1192(1):437–43. doi:.https://doi.org/10.1111/j.1749-6632.2009.05246.x
66 Pathak JL , Bravenboer N , Luyten FP , Verschueren P , Lems WF , Klein-Nulend J , et al. Mechanical loading reduces inflammation-induced human osteocyte-to-osteoclast communication. Calcif Tissue Int. 2015;97(2):169–78. doi:.https://doi.org/10.1007/s00223-015-9999-z
67 Pathak JL , Bakker AD , Luyten FP , Verschueren P , Lems WF , Klein-Nulend J , et al. Systemic Inflammation Affects Human Osteocyte-Specific Protein and Cytokine Expression. Calcif Tissue Int. 2016;98(6):596–608. doi:.https://doi.org/10.1007/s00223-016-0116-8
68 Burr DB , Gallant MA . Bone remodelling in osteoarthritis. Nat Rev Rheumatol. 2012;8(11):665–73. doi:.https://doi.org/10.1038/nrrheum.2012.130
69 Henrotin Y , Pesesse L , Sanchez C . Subchondral bone and osteoarthritis: biological and cellular aspects. Osteoporos Int. 2012;23(Suppl 8):847–51. doi:.https://doi.org/10.1007/s00198-012-2162-z
70 Newberry WN , Zukosky DK , Haut RC . Subfracture insult to a knee joint causes alterations in the bone and in the functional stiffness of overlying cartilage. J Orthop Res. 1997;15(3):450–5. doi:.https://doi.org/10.1002/jor.1100150319
71 Knothe Tate ML , Adamson JR , Tami AE , Bauer TW . The osteocyte. Int J Biochem Cell Biol. 2004;36(1):1–8. doi:.https://doi.org/10.1016/S1357-2725(03)00241-3
72 Jaiprakash A , Prasadam I , Feng JQ , Liu Y , Crawford R , Xiao Y . Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones: a possible pathological role in subchondral bone sclerosis. Int J Biol Sci. 2012;8(3):406–17. doi:.https://doi.org/10.7150/ijbs.4221
73 Staines KA , Ikpegbu E , Törnqvist AE , Dillon S , Javaheri B , Amin AK , et al. Conditional deletion of E11/podoplanin in bone protects against load-induced osteoarthritis. BMC Musculoskelet Disord. 2019;20(1):344. doi:.https://doi.org/10.1186/s12891-019-2731-9
74 Lew DP , Waldvogel FA . Osteomyelitis. Lancet. 2004;364(9431):369–79. doi:.https://doi.org/10.1016/S0140-6736(04)16727-5
75 Horst SA , Hoerr V , Beineke A , Kreis C , Tuchscherr L , Kalinka J , et al. A novel mouse model of Staphylococcus aureus chronic osteomyelitis that closely mimics the human infection: an integrated view of disease pathogenesis. Am J Pathol. 2012;181(4):1206–14. doi:.https://doi.org/10.1016/j.ajpath.2012.07.005
76 Bosse MJ , Gruber HE , Ramp WK . Internalization of bacteria by osteoblasts in a patient with recurrent, long-term osteomyelitis. A case report. J Bone Joint Surg Am. 2005;87(6):1343–7. doi:.https://doi.org/10.2106/00004623-200506000-00022
77 Reilly SS , Hudson MC , Kellam JF , Ramp WK . In vivo internalization of Staphylococcus aureus by embryonic chick osteoblasts. Bone. 2000;26(1):63–70. doi:.https://doi.org/10.1016/S8756-3282(99)00239-2
78 Trouillet-Assant S , Gallet M , Nauroy P , Rasigade JP , Flammier S , Parroche P , et al. Dual impact of live Staphylococcus aureus on the osteoclast lineage, leading to increased bone resorption. J Infect Dis. 2015;211(4):571–81. doi:.https://doi.org/10.1093/infdis/jiu386
79 Claro T , Widaa A , O’Seaghdha M , Miajlovic H , Foster TJ , O’Brien FJ , et al. Staphylococcus aureus protein A binds to osteoblasts and triggers signals that weaken bone in osteomyelitis. PLoS One. 2011;6(4):e18748. doi:.https://doi.org/10.1371/journal.pone.0018748
80 Sanchez CJ, Jr , Ward CL , Romano DR , Hurtgen BJ , Hardy SK , Woodbury RL , et al. Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro. BMC Musculoskelet Disord. 2013;14(1):187. doi:.https://doi.org/10.1186/1471-2474-14-187
81 Widaa A , Claro T , Foster TJ , O’Brien FJ , Kerrigan SW . Staphylococcus aureus protein A plays a critical role in mediating bone destruction and bone loss in osteomyelitis. PLoS One. 2012;7(7):e40586. doi:.https://doi.org/10.1371/journal.pone.0040586
82 Somayaji SN , Ritchie S , Sahraei M , Marriott I , Hudson MC . Staphylococcus aureus induces expression of receptor activator of NF-kappaB ligand and prostaglandin E2 in infected murine osteoblasts. Infect Immun. 2008;76(11):5120–6. doi:.https://doi.org/10.1128/IAI.00228-08
83 Kobayashi Y , Mizoguchi T , Take I , Kurihara S , Udagawa N , Takahashi N . Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1. J Biol Chem. 2005;280(12):11395–403. doi:.https://doi.org/10.1074/jbc.M411189200
84 de Mesy Bentley KL , Trombetta R , Nishitani K , Bello-Irizarry SN , Ninomiya M , Zhang L , et al. Evidence of Staphylococcus Aureus Deformation, Proliferation, and Migration in Canaliculi of Live Cortical Bone in Murine Models of Osteomyelitis. J Bone Miner Res. 2017;32(5):985–90. doi:.https://doi.org/10.1002/jbmr.3055
85 de Mesy Bentley KL , MacDonald A , Schwarz EM , Oh I . Chronic Osteomyelitis with Staphylococcus aureus Deformation in Submicron Canaliculi of Osteocytes: A Case Report. JBJS Case Connect. 2018;8(1):e8. doi:.https://doi.org/10.2106/JBJS.CC.17.00154
86 Tomkinson A , Reeve J , Shaw RW , Noble BS . The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone. J Clin Endocrinol Metab. 1997;82(9):3128–35. doi:.https://doi.org/10.1210/jc.82.9.3128
87 Tatsumi S , Ishii K , Amizuka N , Li M , Kobayashi T , Kohno K , et al. Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab. 2007;5(6):464–75. doi:.https://doi.org/10.1016/j.cmet.2007.05.001
88 Bacabac RG , Mizuno D , Schmidt CF , MacKintosh FC , Van Loon JJ , Klein-Nulend J , et al. Round versus flat: bone cell morphology, elasticity, and mechanosensing. J Biomech. 2008;41(7):1590–8. doi:.https://doi.org/10.1016/j.jbiomech.2008.01.031
89 Robling AG , Niziolek PJ , Baldridge LA , Condon KW , Allen MR , Alam I , et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J Biol Chem. 2008;283(9):5866–75. doi:.https://doi.org/10.1074/jbc.M705092200
90 Price C , Zhou X , Li W , Wang L . Real-time measurement of solute transport within the lacunar-canalicular system of mechanically loaded bone: direct evidence for load-induced fluid flow. J Bone Miner Res. 2011;26(2):277–85. doi:.https://doi.org/10.1002/jbmr.211
91 Klein-Nulend J , van der Plas A , Semeins CM , Ajubi NE , Frangos JA , Nijweide PJ , et al. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J. 1995;9(5):441–5. doi:.https://doi.org/10.1096/fasebj.9.5.7896017
92 You L , Temiyasathit S , Lee P , Kim CH , Tummala P , Yao W , et al. Osteocytes as mechanosensors in the inhibition of bone resorption due to mechanical loading. Bone. 2008;42(1):172–9. doi:.https://doi.org/10.1016/j.bone.2007.09.047
93 Ciani C , Sharma D , Doty SB , Fritton SP . Ovariectomy enhances mechanical load-induced solute transport around osteocytes in rat cancellous bone. Bone. 2014;59:229–34. doi:.https://doi.org/10.1016/j.bone.2013.11.026
94 Sharma D , Ciani C , Marin PA , Levy JD , Doty SB , Fritton SP . Alterations in the osteocyte lacunar-canalicular microenvironment due to estrogen deficiency. Bone. 2012;51(3):488–97. doi:.https://doi.org/10.1016/j.bone.2012.05.014
95 Ukon Y , Makino T , Kodama J , Tsukazaki H , Tateiwa D , Yoshikawa H , et al. Molecular-Based Treatment Strategies for Osteoporosis: A Literature Review. Int J Mol Sci. 2019;20(10):2557. doi:.https://doi.org/10.3390/ijms20102557
96 Halasy-Nagy JM , Rodan GA , Reszka AA . Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis. Bone. 2001;29(6):553–9. doi:.https://doi.org/10.1016/S8756-3282(01)00615-9
97 Itzstein C , Coxon FP , Rogers MJ . The regulation of osteoclast function and bone resorption by small GTPases. Small GTPases. 2011;2(3):117–30. doi:.https://doi.org/10.4161/sgtp.2.3.16453
98 Xian L , Wu X , Pang L , Lou M , Rosen CJ , Qiu T , et al. Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells. Nat Med. 2012;18(7):1095–101. doi:.https://doi.org/10.1038/nm.2793
99 Cosman F , Crittenden DB , Adachi JD , Binkley N , Czerwinski E , Ferrari S , et al. Romosozumab Treatment in Postmenopausal Women with Osteoporosis. N Engl J Med. 2016;375(16):1532–43. doi:.https://doi.org/10.1056/NEJMoa1607948
100 Saag KG , Petersen J , Brandi ML , Karaplis AC , Lorentzon M , Thomas T , et al. Romosozumab or Alendronate for Fracture Prevention in Women with Osteoporosis. N Engl J Med. 2017;377(15):1417–27. doi:.https://doi.org/10.1056/NEJMoa1708322
101 Didangelos A , Yin X , Mandal K , Baumert M , Jahangiri M , Mayr M . Proteomics characterization of extracellular space components in the human aorta. Mol Cell Proteomics. 2010;9(9):2048–62. doi:.https://doi.org/10.1074/mcp.M110.001693
102 Brandenburg VM , Kramann R , Koos R , Krüger T , Schurgers L , Mühlenbruch G , et al. Relationship between sclerostin and cardiovascular calcification in hemodialysis patients: a cross-sectional study. BMC Nephrol. 2013;14(1):219. doi:.https://doi.org/10.1186/1471-2369-14-219
103 Zhu D , Mackenzie NC , Millán JL , Farquharson C , MacRae VE . The appearance and modulation of osteocyte marker expression during calcification of vascular smooth muscle cells. PLoS One. 2011;6(5):e19595. doi:.https://doi.org/10.1371/journal.pone.0019595
104 Lewiecki EM , Blicharski T , Goemaere S , Lippuner K , Meisner PD , Miller PD , et al. A Phase III Randomized Placebo-Controlled Trial to Evaluate Efficacy and Safety of Romosozumab in Men With Osteoporosis. J Clin Endocrinol Metab. 2018;103(9):3183–93. doi:.https://doi.org/10.1210/jc.2017-02163
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