The primary cilium is a solitary, sensory organelle with many roles in bone development, maintenance, and function. In the osteogenic cell lineage, including skeletal stem cells, osteoblasts, and osteocytes, the primary cilium plays a vital role in the regulation of bone formation, and this has made it a promising pharmaceutical target to maintain bone health. While the role of the primary cilium in the osteogenic cell lineage has been increasingly characterized, little is known about the potential impact of targeting the cilium in relation to osteoclasts, a hematopoietic cell responsible for bone resorption. The objective of this study was to determine whether osteoclasts have a primary cilium and to investigate whether or not the primary cilium of macrophages, osteoclast precursors, serves a functional role in osteoclast formation. Using immunocytochemistry, we showed the macrophages have a primary cilium, while osteoclasts lack this organelle. Furthermore, we increased macrophage primary cilia incidence and length using fenoldopam mesylate and found that cells undergoing such treatment showed a significant decrease in the expression of osteoclast markers tartrate-resistant acid phosphatase, cathepsin K, and c-Fos, as well as decreased osteoclast formation. This work is the first to show that macrophage primary cilia resorption may be a necessary step for osteoclast differentiation. Since primary cilia and preosteoclasts are responsive to fluid flow, we applied fluid flow at magnitudes present in the bone marrow to differentiating cells and found that osteoclastic gene expression by macrophages was not affected by fluid flow mechanical stimulation, suggesting that the role of the primary cilium in osteoclastogenesis is not a mechanosensory one. The primary cilium has been suggested to play a role in bone formation, and our findings indicate that it may also present a means to regulate bone resorption, presenting a dual benefit of developing ciliary-targeted pharmaceuticals for bone disease.

Bone metastases are a common cause of suffering in breast and prostate cancer patients, however, the interaction between bone cells and cancer cells is poorly understood. Using a series of co-culture, conditioned media, human cancer spheroid, and organ-on-a-chip experiments, this study reveals that osteocytes suppress cancer cell proliferation and increase migration via tumor necrosis factor alpha (TNF-α) secretion. This action is regulated by osteocyte primary cilia and associated intraflagellar transport protein 88 (IFT88). Furthermore, it shows that cancer cells block this mechanism by secreting transforming growth factor beta (TGF-β), which disrupts osteocyte cilia and IFT88 gene expression. This bi-directional crosstalk signaling between osteocytes and cancer cells is common to both breast and prostate cancer. This study also proposes that osteocyte inhibition of cancer cell proliferation decreases as cancer cells increase, producing more TGF-β. Hence, a positive feedback loop develops accelerating metastatic tumor growth. These findings demonstrate the importance of cancer cell-osteocyte signaling in regulating breast and prostate bone metastases and support the development of therapies targeting this pathway.

Bone cells actively respond to mechanical stimuli to direct bone formation, yet there is no current treatment strategy for conditions of low bone mass and osteoporosis designed to target the inherent mechanosensitivity of bone. Our group has previously identified the primary cilium as a critical mechanosensor within bone, and that pharmacologically targeting the primary cilium with fenoldopam can enhance osteocyte mechanosensitivity. Here, we demonstrate that potentiating osteocyte mechanosensing with fenoldopam in vitro promotes pro-osteogenic paracrine signaling to osteoblasts. Conversely, impairing primary cilia formation and the function of key ciliary mechanotransduction proteins attenuates this intercellular signaling cascade. We then utilize an in vivo model of load-induced bone formation to demonstrate that fenoldopam treatment sensitizes bones of both healthy and osteoporotic mice to mechanical stimulation. Furthermore, we show minimal adverse effects of this treatment and demonstrate that prolonged treatment biases trabecular bone adaptation. This work is the first to examine the efficacy of targeting primary cilia-mediated mechanosensing to enhance bone formation in osteoporotic animals. © 2022 American Society for Bone and Mineral Research (ASBMR).

Breast and prostate cancers preferentially metastasise to bone tissue, with metastatic lesions forming in the skeletons of most patients. On arriving in bone tissue, disseminated tumour cells enter a mechanical microenvironment that is substantially different to that of the primary tumour and is largely regulated by bone cells. Osteocytes, the most ubiquitous bone cell type, orchestrate healthy bone remodelling in response to physical exercise. However, the effects of mechanical loading of osteocytes on cancer cell behaviour is still poorly understood. The aim of this study was to characterise the effects of osteocyte mechanical stimulation on the behaviour of breast and prostate cancer cells. To replicate an osteocyte-controlled environment, this study treated breast (MDA-MB-231 and MCF-7) and prostate (PC-3 and LNCaP) cancer cell lines with conditioned media from MLO-Y4 osteocyte-like cells exposed to mechanical stimulation in the form of fluid shear stress. We found that osteocyte paracrine signalling acted to inhibit metastatic breast and prostate tumour growth, characterised by reduced proliferation and invasion and increased migration. In breast cancer cells, these effects were largely reversed by mechanical stimulation of osteocytes. In contrast, conditioned media from mechanically stimulated osteocytes had no effect on prostate cancer cells. To further investigate these interactions, we developed a microfluidic organ-chip model using the Emulate platform. This new organ-chip model enabled analysis of cancer cell migration, proliferation and invasion in the presence of mechanical stimulation of osteocytes by fluid shear stress, resulting in increased invasion of breast and prostate cancer cells. These findings demonstrate the importance of osteocytes and mechanical loading in regulating cancer cell behaviour and the need to incorporate these factors into predictive in vitro models of bone metastasis.

Increases in mechanical loading can enhance the addition of new bone, altering geometry and density such that bones better withstand higher forces. Bone-forming osteoblasts have long been thought to originate from progenitors, but the exact source is yet to be identified. Previous studies indicate osteogenic precursors arise from Prx1-expressing progenitors during embryonic development and adult fracture repair. However, it is unknown whether this cell population is also a source for mechanically induced active osteoblasts. We first identified that Prx1 is expressed in skeletally mature mouse periosteum, a thin tissue covering the surface of the bone that is rich in osteoprogenitors. We then traced Prx1 progenitor lineage using a transgenic mouse model carrying both a Prx1-driven tamoxifen-inducible Cre and a ROSA-driven lacZ reporter gene. Cells that expressed Prx1 when compressive axial loading was applied were detected within the cortical bone days after stimulation, indicating osteocytes are of Prx1-expressing cell origin. In addition, we evaluated how these cells sense and respond to physical stimulation in vivo by disrupting their primary cilia, which are antenna-like sensory organelles known to enhance mechanical and chemical signaling kinetics. Although Prx1-driven primary cilium disruption did not affect osteoblast recruitment to the bone surface, the relative mineral apposition and bone formation rates were decreased by 53% and 34%, respectively. Thus, this cell population contributes to load-induced bone formation, and primary cilia are needed for a complete response. Interestingly, Prx1-expressing progenitors are easily extracted from periosteum and are perhaps an attractive alternative to marrow stem cells for bone tissue regeneration strategies.

Bone adapts to physical forces and this process is dependent on osteocyte mechanotransduction. One way osteocytes sense mechanical stimulation is through the primary cilium, a sensory organelle that triggers intracellular signaling cascades in response to fluid shear. Our lab previously determined that flow-induced ciliary Ca2+ influx and changes in cytosolic cAMP levels are critical for osteogenesis. We also identified two proteins important for osteocyte mechanotransduction: transient receptor potential vanilloid 4 (TRPV4) and adenylyl cyclase 6 (AC6). Interestingly, disrupting the Ca2+-binding ability of these proteins results in loss of function. Although knockdowns of TRPV4 and AC6 disrupt osteogenesis, there is no definitive evidence linking them to Ca2+/cAMP dynamics that facilitate osteocyte mechanotransduction. We therefore transfected MLO-Y4 osteocytes with AC3/6 and TRPV4 overexpression plasmids that fail to interact with Ca2+ and observed the response to fluid shear. Indeed, mutant groups exhibited adverse changes in cAMP and lower mRNA expression of an osteogenic marker, COX-2, at the onset of flow. This pattern persisted for AC3 and TRPV4 but we detected no difference in AC6 at longer exposure to flow. These results suggest TRPV4 and ACs mediate Ca2+/cAMP dynamics that are important for osteocyte mechanotransduction. These mechanisms are potential targets for therapeutics to combat bone loss and should be investigated further.

Although Prx1 (also known as PRRX1)-expressing cells and their primary cilia are critical for embryonic development, they have yet to be studied in the context of postnatal skeletogenesis owing to the lethality of mouse models. A tamoxifen-inducible Prx1 model has been developed, and we determined that expression directed by this promoter is highly restricted to the cambium layers in the periosteum and perichondrium after birth. To determine the postnatal role of these cambium layer osteochondroprogenitors (CLOPs) and their primary cilia, we developed models to track the fate of CLOPs (Prx1CreER-GFP;Rosa26tdTomato) and selectively disrupt their cilia (Prx1CreER-GFP;Ift88fl/fl). Our tracking studies revealed that CLOPs populate cortical and trabecular bone, the growth plate and secondary ossification centers during the normal program of postnatal skeletogenesis. Furthermore, animals lacking CLOP cilia exhibit stunted limb growth due to disruptions in endochondral and intramembranous ossification. Histological examination indicates that growth is stunted due to limited differentiation, proliferation and/or abnormal hypertrophic differentiation in the growth plate. Collectively, our results suggest that CLOPs are programmed to rapidly populate distant tissues and produce bone via a primary cilium-mediated mechanism in the postnatal skeleton.

The original article [1] contained two minor errors affecting the labelling of Fig. 3d and Figs. 6b & 6c; these errors have now been corrected in the respective figures in the original article.

The role of computational modeling for biomechanics research and related clinical care will be increasingly prominent. The biomechanics community has been developing computational models routinely for exploration of the mechanics and mechanobiology of diverse biological structures. As a result, a large array of models, data, and discipline-specific simulation software has emerged to support endeavors in computational biomechanics. Sharing computational models and related data and simulation software has first become a utilitarian interest, and now, it is a necessity. Exchange of models, in support of knowledge exchange provided by scholarly publishing, has important implications. Specifically, model sharing can facilitate assessment of reproducibility in computational biomechanics and can provide an opportunity for repurposing and reuse, and a venue for medical training. The community's desire to investigate biological and biomechanical phenomena crossing multiple systems, scales, and physical domains, also motivates sharing of modeling resources as blending of models developed by domain experts will be a required step for comprehensive simulation studies as well as the enhancement of their rigor and reproducibility. The goal of this paper is to understand current perspectives in the biomechanics community for the sharing of computational models and related resources. Opinions on opportunities, challenges, and pathways to model sharing, particularly as part of the scholarly publishing workflow, were sought. A group of journal editors and a handful of investigators active in computational biomechanics were approached to collect short opinion pieces as a part of a larger effort of the IEEE EMBS Computational Biology and the Physiome Technical Committee to address model reproducibility through publications. A synthesis of these opinion pieces indicates that the community recognizes the necessity and usefulness of model sharing. There is a strong will to facilitate model sharing, and there are corresponding initiatives by the scientific journals. Outside the publishing enterprise, infrastructure to facilitate model sharing in biomechanics exists, and simulation software developers are interested in accommodating the community's needs for sharing of modeling resources. Encouragement for the use of standardized markups, concerns related to quality assurance, acknowledgement of increased burden, and importance of stewardship of resources are noted. In the short-term, it is advisable that the community builds upon recent strategies and experiments with new pathways for continued demonstration of model sharing, its promotion, and its utility. Nonetheless, the need for a long-term strategy to unify approaches in sharing computational models and related resources is acknowledged. Development of a sustainable platform supported by a culture of open model sharing will likely evolve through continued and inclusive discussions bringing all stakeholders at the table, e.g., by possibly establishing a consortium.

ABSTRACT The primary cilium is a solitary, antenna‐like sensory organelle with many important roles in cartilage and bone development, maintenance, and function. The primary cilium's potential role as a signaling nexus in the growth plate makes it an attractive therapeutic target for diseases and disorders associated with bone development and maintenance. Many signaling pathways that are mediated by the cilium—such as Hh, Wnt, Ihh/PTHrP, TGFβ, BMP, FGF, and Notch—are also known to influence endochondral ossification, primarily by directing growth plate formation and chondrocyte behavior. Although a few studies have demonstrated that these signaling pathways can be directly tied to the primary cilium, many pathways have yet to be evaluated in context of the cilium. This review serves to bridge this knowledge gap in the literature, as well as discuss the cilium's importance in the growth plate's ability to sense and respond to chemical and mechanical stimuli. Furthermore, we explore the importance of using the appropriate mechanism to study the cilium in vivo and suggest IFT88 deletion is the best available technique. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:533–545, 2018.

The primary cilium is a mechanosensor in a variety of mammalian cell types, initiating and directing intracellular signalling cascades in response to external stimuli. When primary cilia formation is disrupted, cells have diminished mechanosensitivity and an abrogated response to mechanical stimulation. Due to this important role, we hypothesised that increasing primary cilia length would enhance the downstream response and therefore, mechanosensitivity. To test this hypothesis, we increased osteocyte primary cilia length with fenoldopam and lithium and found that cells with longer primary cilia were more mechanosensitive. Furthermore, fenoldopam treatment potentiated adenylyl cyclase activity and was able to recover primary cilia form and sensitivity in cells with impaired cilia. This work demonstrates that modulating the structure of the primary cilium directly impacts cellular mechanosensitivity. Our results implicate cilium length as a potential therapeutic target for combating numerous conditions characterised by impaired cilia function.

Primary cilia are immotile, microtubule-based organelles extending from the surface of nearly every mammalian cell. Mechanical stimulation causes deflection of the primary cilium, initiating downstream signaling cascades to the rest of the cell. The cilium forms a unique subcellular microdomain, and defects in ciliary protein composition or physical structure have been associated with a myriad of human pathologies. In this review, we discuss the importance of ciliary mechanotransduction at the cell and tissue level, and how furthering our molecular understanding of primary cilia mechanobiology may lead to therapeutic strategies to treat human diseases.

Cellular mechanosensation is critical in diseases responsible for enormous human suffering including atherosclerosis, osteoarthritis, cancer, and osteoporosis. Nonetheless, very little is understood about the molecular mechanisms of mechanotransduction outside of a small number of specialized sensory cells. Primary cilia are solitary linear cellular extensions that extend from the surface of virtually all cells. For decades, the biologic function of these enigmatic structures was elusive, however, recent evidence suggests an emerging picture in which the primary cilium functions as a complex nexus where both physical and chemical extracellular signals are sensed and responses coordinated.In our laboratory we have shown that primary cilia act as mechanical sensors in bone and that conditional deletion of primary cilia lead to mechanosensing defects. Recently, we developed a novel combined experimental/modeling approach to determine the mechanical properties of primary cilia. We found a wide variety of previously unreported deformation modes including smooth bending and rigid‐body rotations. This suggests that the mechanics of both the cilium shaft and basal anchorage are important to understanding deflection patterns. Interestingly, both the cilium itself and its anchorage to the microtubule cytoskeleton alter their structure in response to physical loading, suggesting structural adaptation or “remodeling”. We have also developed novel molecular biology tools to elucidate the details of mechanically activated ciliary signaling pathways. For example, we have created a cilia‐directed biosensor that has allowed us to distinguish intraciliary from intracellular calcium signaling. We have also developed a method for distinguishing the roles of the cytoplasmic and ciliary pools of proteins that are found in both compartments. In summary, primary cilia are non‐linear, richly varied, mechanical structures (biomechanics) as well as structurally adaptive (mechanobiology). Simultaneously they are a biochemical microdomain where signaling events are catalyzed, enhanced, and integrated.Support or Funding InformationSupported by NIH AR45989 and AR54156 and New York State Stem Cell Grant N089‐210

The primary cilium is an extracellular organelle that transduces mechanical signals into intracellular signaling cascades. The cilium is accompanied by a dense collection of stretch-activated channels and proteins involved in mechanotransduction, which form a highly sensitive microdomain at the cell surface that serves to amplify detected stimuli. The putative stress buildup at the base of the cilium activates components in this microdomain to trigger signaling via second messengers such as calcium and cyclic AMP. Defects in the structure of the cilium and mutations in ciliary proteins disrupt the cilium's ability to detect and transduce mechanical stimuli. This dysfunction at the cellular and molecular level translates to the organ and tissue level, resulting in disastrous diseases and syndromes spanning multiple organs. This chapter discusses the mechanisms for primary cilium-mediated mechanotransduction, the use of computational models to characterize cilium mechanics, modifications in cilium structure and composition to tune mechanosensitivity, and animal models created to explore cilium-based health complications.

It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitmentin vitro In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cellsin vivo Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitmentin vivoand that the primary cilium contributes to this process.-Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism.

Mechanotransduction is an essential cellular function in a variety of tissues including bone, kidney, and endothelia. The primary cilium is a single immotile organelle protruding from the surface of these cells, and has repeatedly been demonstrated as a critical mechanotransducer in these cell types. When the primary cilium is impaired, these cells have an abrogated response to mechanical stimulation. Here, we demonstrate a method to enhance cellular mechanotransduction, by increasing primary cilia length. To elongate primary cilia, we treated MLO-Y4 osteocytes with fenoldopam, lithium chloride, or vehicle control for 16 hours. We then subjected these cells to oscillatory fluid flow for 1 hour at 1 Hz and 1 Pa wall shear stress. Immediately following flow, cells were lysed, and mRNA expression was analyzed. Cells with longer cilia displayed increased expression of osteogenic markers cyclooxygenase-2 and osteopontin, compared to vehicle control, suggesting that these cells are more mechanosensitive. To discern the role of fenoldopam on cilia length from other cellular processes, we treated cells with IFT88 siRNA_IFT88 is critical for primary cilia formation_which resulted shorter cilia and an abrogated response to fluid flow. Fenoldopam was able to restore cilia length in cells with impaired cilia formation, and rescued flow-induced osteogenic signaling. Together, these data suggest that cells with longer cilia are more mechanosensitive, and that cellular mechanotransduction can easily be modulated by pharmacologically lengthening primary cilia. Primary cilia-mediated mechanotransduction is a critical function in an array of cell types, and numerous diseases, such as polycystic kidney disease and Bardet-Biedl Syndrome, are characterized by impaired cilia function. This work suggests a potential therapeutic strategy to combat such conditions.