Aging impairs the osteocytic regulation of collagen integrity and bone quality.

Poor bone quality is a major factor in skeletal fragility in elderly individuals. The molecular mechanisms that establish and maintain bone quality, independent of bone mass, are unknown but are thought to be primarily determined by osteocytes. We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling (PLR), which maintains bone material properties. We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFß signaling (TßRIIocy-/-) that suppresses PLR. The control aged bone displayed decreased TGFß signaling and PLR, but aging did not worsen the existing PLR suppression in male TßRIIocy-/- bone. This relationship impacted the behavior of collagen material at the nanoscale and tissue scale in macromechanical tests. The effects of age on bone mass, density, and mineral material behavior were independent of osteocytic TGFß. We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFß-dependent maintenance of collagen integrity.

Effect of Passaging on Bovine Chondrocyte Gene Expression and Engineered Cartilage Production.

Tissue engineering strategies show great potential for repairing osteochondral defects in osteoarthritic joints; however, these approaches often rely on passaging cells multiple times to obtain enough cells to produce functional tissue. Unfortunately, monolayer expansion culture causes chondrocyte dedifferentiation, which is accompanied by a phenotypical and morphological shift in chondrocyte properties that leads to a reduction in the quality of de novo cartilage produced. Thus, the objective of this study was to evaluate transcriptional variations during in vitro expansion culture and determine how differences in cell phenotype from monolayer expansion alter development of functional engineered cartilage. We used an unbiased approach to explore genome-wide transcriptional differences in chondrocyte phenotype at passage 1 (P1), P3, and P5, and then seeded cells into hydrogel scaffolds at P3 and P5 to assess cells' abilities to produce cartilaginous extracellular matrix in three dimensional (3D). We identified distinct phenotypic differences, specifically for genes related to extracellular organization and cartilage development. Both P3 and P5 chondrocytes were able to produce chondrogenic tissue in 3D, with P3 cells producing matrix with greater compressive properties and P5 cells secreting matrix with higher glycosaminoglycan/DNA and collagen/DNA ratios. Furthermore, we identified 24 genes that were differentially expressed with passaging and enriched in human osteoarthritis (OA) genome-wide association studies, thereby prioritizing them as functionally relevant targets to improve protocols that recapitulate functional healthy cartilage with cells from adult donors. Specifically, we identified novel genes, such as TMEM190 and RAB11FIP4, which were enriched with human hip OA and may play a role in chondrocyte dedifferentiation. This work lays the foundation for several pathways and genes that could be modulated to enhance the efficacy for chondrocyte culture for tissue regeneration, which could have transformative impacts for cell-based cartilage repair strategies.

Priming chondrocytes during expansion alters cell behavior and improves matrix production in 3D culture.

OBJECTIVE: Cartilage tissue engineering strategies that use autologous chondrocytes require in vitro expansion of cells to obtain enough cells to produce functional engineered tissue. However, chondrocytes dedifferentiate during expansion culture, limiting their ability to produce chondrogenic tissue and their utility for cell-based cartilage repair strategies. The current study identified conditions that favor cartilage production and the mechanobiological mechanisms responsible for these benefits. DESIGN: Chondrocytes were isolated from juvenile bovine knee joints and cultured with (primed) or without (unprimed) a growth factor cocktail. Gene expression, cell morphology, cell adhesion, cytoskeletal protein distribution, and cell mechanics were assessed. Following passage 5, cells were embedded into agarose hydrogels to evaluate functional properties of engineered cartilage. RESULTS: Priming cells during expansion culture altered cell phenotype and chondrogenic tissue production. Unbiased ribonucleic acid-sequencing analysis suggested, and experimental studies confirmed, that growth factor priming delays dedifferentiation associated changes in cell adhesion and cytoskeletal organization. Priming also overrode mechanobiological pathways to prevent chondrocytes from remodeling their cytoskeleton to accommodate the stiff, monolayer microenvironment. Passage 1 primed cells deformed less and had lower yes associated protein 1 activity than unprimed cells. Differences in cell adhesion, morphology, and cell mechanics between primed and unprimed cells were mitigated by passage 5. CONCLUSIONS: Priming suppresses mechanobiologic cytoskeletal remodeling to prevent chondrocyte dedifferentiation, resulting in more cartilage-like tissue-engineered constructs.

Type 2 diabetes impairs annulus fibrosus fiber deformation and rotation under disc compression in the University of California Davis type 2 diabetes mellitus (UCD-T2DM) rat model.

Understanding the biomechanical behavior of the intervertebral disc is crucial for studying disease mechanisms and developing tissue engineering strategies for managing disc degeneration. We used synchrotron small-angle X-ray scattering to investigate how changes to collagen behavior contribute to alterations in the disc's ability to resist compression. Coccygeal motion segments from 6-month-old lean Sprague-Dawley rats ( n=7) and diabetic obese University of California Davis type 2 diabetes mellitus (UCD-T2DM) rats ( n=6, diabetic for 68±7 days) were compressed during simultaneous synchrotron scanning to measure collagen strain at the nanoscale (beamline 7.3.3 of the Advanced Light Source). After compression, the annulus fibrosus was assayed for nonenzymatic cross-links. In discs from lean rats, resistance to compression involved two main energy-dissipation mechanisms at the nanoscale: (1) rotation of the two groups of collagen fibrils forming the annulus fibrosus and (2) straightening (uncrimping) and stretching of the collagen fibrils. In discs from diabetic rats, both mechanisms were significantly impaired. Specifically, diabetes reduced fibril rotation by 31% and reduced collagen fibril strain by 30% (compared to lean discs). The stiffening of collagen fibrils in the discs from diabetic rats was consistent with a 31% higher concentration of nonenzymatic cross-links and with evidence of earlier onset plastic deformations such as fibril sliding and fibril-matrix delamination. These findings suggest that fibril reorientation, stretching, and straightening are key deformation mechanisms that facilitate whole-disc compression, and that type 2 diabetes impairs these efficient and low-energy elastic deformation mechanisms, thereby altering whole-disc behavior and inducing the earlier onset of plastic deformation.

Genetic and Gene Expression Resources for Osteoporosis and Bone Biology Research.

PURPOSE OF REVIEW: The integration of data from multiple genomic assays from humans and non-human model organisms is an effective approach to identify genes involved in skeletal fragility and fracture risk due to osteoporosis and other conditions. This review summarizes genome-wide genetic variation and gene expression data resources relevant to the discovery of genes contributing to skeletal fragility and fracture risk. RECENT FINDINGS: Genome-wide association studies (GWAS) of osteoporosis-related traits are summarized, in addition to gene expression in bone tissues in humans and non-human organisms, with a focus on rodent models related to skeletal fragility and fracture risk. Gene discovery approaches using these genomic data resources are described. We also describe the Musculoskeletal Knowledge Portal (MSKKP) that integrates much of the available genomic data relevant to fracture risk. The available genomic resources provide a wealth of knowledge and can be analyzed to identify genes related to fracture risk. Genomic resources that would fill particular scientific gaps are discussed.

Deep coverage and quantification of the bone proteome provides enhanced opportunities for new discoveries in skeletal biology and disease.

Dysregulation of cell signaling in chondrocytes and in bone cells, such as osteocytes, osteoblasts, osteoclasts, and an elevated burden of senescent cells in cartilage and bone, are implicated in osteoarthritis (OA). Mass spectrometric analyses provides a crucial molecular tool-kit to understand complex signaling relationships in age-related diseases, such as OA. Here we introduce a novel mass spectrometric workflow to promote proteomic studies of bone. This workflow uses highly specialized steps, including extensive overnight demineralization, pulverization, and incubation for 72 h in 6 M guanidine hydrochloride and EDTA, followed by proteolytic digestion. Analysis on a high-resolution Orbitrap Eclipse and Orbitrap Exploris 480 mass spectrometer using Data-Independent Acquisition (DIA) provides deep coverage of the bone proteome, and preserves post-translational modifications, such as hydroxyproline. A spectral library-free quantification strategy, directDIA, identified and quantified over 2,000 protein groups (with ≥ 2 unique peptides) from calcium-rich bone matrices. Key components identified were proteins of the extracellular matrix (ECM), bone-specific proteins (e.g., secreted protein acidic and cysteine rich, SPARC, and bone sialoprotein 2, IBSP), and signaling proteins (e.g., transforming growth factor beta-2, TGFB2), and lysyl oxidase homolog 2 (LOXL2), an important protein in collagen crosslinking. Post-translational modifications (PTMs) were identified without the need for specific enrichment. This includes collagen hydroxyproline modifications, chemical modifications for collagen self-assembly and network formation. Multiple senescence factors were identified, such as complement component 3 (C3) protein of the complement system and many matrix metalloproteinases, that might be monitored during age-related bone disease progression. Our innovative workflow yields in-depth protein coverage and quantification strategies to discover underlying biological mechanisms of bone aging and to provide tools to monitor therapeutic interventions. These novel tools to monitor the bone proteome open novel horizons to investigate bone-specific diseases, many of which are age-related.

miR181a/b-1 controls osteocyte metabolism and mechanical properties independently of bone morphology.

Bone derives its ability to resist fracture from bone mass and quality concurrently; however, many questions about the molecular mechanisms controlling bone quality remain unanswered, limiting the development of diagnostics and therapeutics. Despite the increasing evidence on the importance of miR181a/b-1 in bone homeostasis and disease, whether and how osteocyte-intrinsic miR181a/b-1 controls bone quality remains elusive. Osteocyte-intrinsic deletion of miR181a/b-1 in osteocytes in vivo resulted in compromised overall bone mechanical behavior in both sexes, although the parameters affected by miR181a/b-1 varied distinctly based on sex. Furthermore, impaired fracture resistance in both sexes was unexplained by cortical bone morphology, which was altered in female mice and intact in male mice with miR181a/b-1-deficient osteocytes. The role of miR181a/b-1 in the regulation of osteocyte metabolism was apparent in bioenergetic testing of miR181a/b-1-deficient OCY454 osteocyte-like cells and transcriptomic analysis of cortical bone from mice with osteocyte-intrinsic ablation of miR181a/b-1. Altogether, this study demonstrates the control of osteocyte bioenergetics and the sexually dimorphic regulation of cortical bone morphology and mechanical properties by miR181a/b-1, hinting at the role of osteocyte metabolism in the regulation of mechanical behavior.

Zoledronic acid improves bone quality and muscle function in a high bone turnover state.

SUMMARY: Zoledronic acid (ZA) prevents muscle weakness in mice with bone metastases; however, its role in muscle weakness in non-tumor-associated metabolic bone diseases and as an effective treatment modality for the prevention of muscle weakness associated with bone disorders, is unknown. We demonstrate the role of ZA-treatment on bone and muscle using a mouse model of accelerated bone remodeling, which represents the clinical manifestation of non-tumor associated metabolic bone disease. ZA increased bone mass and strength and rescued osteocyte lacunocanalicular organization. Short-term ZA treatment increased muscle mass, whereas prolonged, preventive treatment improved muscle mass and function. In these mice, muscle fiber-type shifted from oxidative to glycolytic and ZA restored normal muscle fiber distribution. By blocking TGFß release from bone, ZA improved muscle function, promoted myoblast differentiation and stabilized Ryanodine Receptor-1 calcium channel. These data demonstrate the beneficial effects of ZA in maintaining bone health and preserving muscle mass and function in a model of metabolic bone disease. Context and significance: TGFß is a bone regulatory molecule which is stored in bone matrix, released during bone remodeling, and must be maintained at an optimal level for the good health of the bone. Excess TGFß causes several bone disorders and skeletal muscle weakness. Reducing excess TGFß release from bone using zoledronic acid in mice not only improved bone volume and strength but also increased muscle mass, and muscle function. Progressive muscle weakness coexists with bone disorders, decreasing quality of life and increasing morbidity and mortality. Currently, there is a critical need for treatments improving muscle mass and function in patients with debilitating weakness. Zoledronic acid's benefit extends beyond bone and could also be useful in treating muscle weakness associated with bone disorders.

Senescence and Inflammation: Summary of a Gerontological Society of America and National Institute on Aging-Sponsored Symposium.

The National Institute on Aging sponsored a symposium at the Gerontological Society of America (GSA) annual meeting in Indianapolis, Indiana, to discuss recent discoveries related to senescent and inflammatory mechanisms in aging and disease. Consistent with the 2022 Biological Sciences GSA program led by Dr. Rozalyn Anderson, the symposium featured early-stage investigators and a leader in the field of geroscience research. Cell senescence and immune interactions coordinate homeostatic and protective programming throughout the life span. Dysfunctional communication in this exchange eventuates in inflammation-related compositional changes in aged tissues, including propagation of the senescence-associated secretory phenotype and accumulation of senescent and exhausted immune cells. Presentations in this symposium explored senescent and immune-related dysfunction in aging from diverse viewpoints and featured emerging cellular and molecular methods. A central takeaway from the event was that the use of new models and approaches, including single-cell -omics, novel mouse models, and 3D culture systems, is revealing dynamic properties and interactions of senescent and immune cell fates. This knowledge is critical for devising new therapeutic approaches with important translational relevance.

Molecular and Cellular Crosstalk between Bone and Brain: Accessing Bidirectional Neural and Musculoskeletal Signaling during Aging and Disease.

Molecular omics technologies, including proteomics, have enabled the elucidation of key signaling pathways that mediate bidirectional communication between the brain and bone tissues. Here we provide a brief summary of the clinical and molecular evidence of the need to study the bone-brain axis of cross-tissue cellular communication. Clear clinical and molecular evidence suggests biological interactions and similarities between bone and brain cells. Here we review the current mass spectrometric techniques for studying brain and bone diseases with an emphasis on neurodegenerative diseases and osteoarthritis/osteoporosis, respectively. Further study of the bone-brain axis on a molecular level and evaluation of the role of proteins, neuropeptides, osteokines, and hormones in molecular pathways linked to bone and brain diseases is critically needed. The use of mass spectrometry and other omics technologies to analyze these cross-tissue signaling events and interactions will help us better understand disease progression and comorbidities and potentially identify new pathways and targets for therapeutic interventions. Proteomic measurements are particularly favorable for investigating the role of signaling and secreted and circulating analytes and identifying molecular and metabolic pathways implicated in age-related diseases.

Bone-cartilage crosstalk informed by aging mouse bone transcriptomics and human osteoarthritis genome-wide association studies.

Subchondral bone participates in crosstalk with articular cartilage to maintain joint homeostasis, and disruption of either tissue results in overall joint degeneration. Among the subchondral bone changes observed in osteoarthritis (OA), subchondral bone plate (SBP) thickening has a time-dependent relationship with cartilage degeneration and has recently been shown to be regulated by osteocytes. Here, we evaluate the effect of age on SBP thickness and cartilage degeneration in aging mice. We find that SBP thickness significantly increases by 18-months of age, corresponding temporally with increased cartilage degeneration. To identify factors in subchondral bone that may participate in bone cartilage crosstalk or OA, we leveraged mouse transcriptomic data from one joint tissue compartment - osteocyte-enriched bone - to search for enrichment with human OA in UK Biobank and Arthritis Research UK Osteoarthritis Genetics (arcOGEN) GWAS using the mouse2human (M2H, www.mouse2human.org) strategy. Genes differentially expressed in aging mouse bone are significantly enriched for human OA, showing joint site-specific (knee vs. hip) relationships, exhibit temporal associations with age, and unique gene clusters are implicated in each type of OA. Application of M2H identifies genes with known and unknown functions in osteocytes and OA development that are clinically associated with human OA. Altogether, this work prioritizes genes with a potential role in bone/cartilage crosstalk for further mechanistic study based on their association with human OA in GWAS.

Deep learning models to map osteocyte networks can successfully distinguish between young and aged bone.

Osteocytes, the most abundant and mechanosensitive cells in bone tissue, play a pivotal role in bone homeostasis and mechano-responsiveness, orchestrating the intricate balance between bone formation and resorption under daily activity. Studying osteocyte connectivity and understanding their intricate arrangement within the lacunar canalicular network (LCN) is essential for unraveling bone physiology. This is particularly true as our bones age, which is associated with decreased integrity of the osteocyte network, disrupted mass transport, and lower sensitivity to the mechanical stimuli that allow the skeleton to adapt to changing demands. Much work has been carried out to investigate this relationship, often involving high resolution microscopy of discrete fragments of this network, alongside advanced computational modelling of individual cells. However, traditional methods of segmenting and measuring osteocyte connectomics are time-consuming and labour-intensive, often hindered by human subjectivity and limited throughput. In this study, we explore the application of deep learning and computer vision techniques to automate the segmentation and measurement of osteocyte connectomics, enabling more efficient and accurate analysis. We compare several state-of-the-art computer vision models (U-Nets and Vision Transformers) to successfully segment the LCN, finding that an Attention U-Net model can accurately segment and measure 81.8% of osteocytes and 42.1% of dendritic processes, when compared to manual labelling. While further development is required, we demonstrate that this degree of accuracy is already sufficient to distinguish between bones of young (2 month old) and aged (36 month old) mice, as well as capturing the degeneration induced by genetic modification of osteocytes. By harnessing the power of these advanced technologies, further developments can unravel the complexities of osteocyte networks in unprecedented detail, revolutionising our understanding of bone health and disease.

At the Crux of Joint Crosstalk: TGFß Signaling in the Synovial Joint.

PURPOSE OF REVIEW: The effect of the transforming growth factor beta (TGFß) signaling pathway on joint homeostasis is tissue-specific, non-linear, and context-dependent, representing a unique complexity in targeting TGFß signaling in joint disease. Here we discuss the variety of mechanisms that TGFß signaling employs in the synovial joint to maintain healthy joint crosstalk and the ways in which aberrant TGFß signaling can result in joint degeneration. RECENT FINDINGS: Osteoarthritis (OA) epitomizes a condition of disordered joint crosstalk in which multiple joint tissues degenerate leading to overall joint deterioration. Synovial joint tissues, such as subchondral bone, articular cartilage, and synovium, as well as mesenchymal stem cells, each demonstrate aberrant TGFß signaling during joint disease, whether by excessive or suppressed signaling, imbalance of canonical and non-canonical signaling, a perturbed mechanical microenvironment, or a distorted response to TGFß signaling during aging. The synovial joint relies upon a sophisticated alliance among each joint tissue to maintain joint homeostasis. The TGFß signaling pathway is a key regulator of the health of individual joint tissues, and the subsequent interaction among these different joint tissues, also known as joint crosstalk. Dissecting the sophisticated function of TGFß signaling in the synovial joint is key to therapeutically interrogating the pathway to optimize overall joint health.

Osteoarthritis Pathophysiology: Therapeutic Target Discovery may Require a Multifaceted Approach.

Molecular understanding of osteoarthritis (OA) has greatly increased through careful analysis of tissue samples, preclinical models, and large-scale agnostic "-omic" studies. There is broad acceptance that systemic and biomechanical signals affect multiple tissues of the joint, each of which could potentially be targeted to improve patient outcomes. In this review six experts in different aspects of OA pathogenesis provide their independent view on what they believe to be good tractable approaches to OA target discovery. We conclude that molecular discovery has been high but future transformative studies require a multidisciplinary holistic approach to develop therapeutic strategies with high clinical efficacy.

Prioritization of Genes Relevant to Bone Fragility Through the Unbiased Integration of Aging Mouse Bone Transcriptomics and Human GWAS Analyses.

Identifying new genetic determinants of bone mineral density (BMD) and fracture promises to yield improved diagnostics and therapies for bone fragility. However, prioritizing candidate genes from genome-wide screens can be challenging. To overcome this challenge, we prioritized mouse genes that are differentially expressed in aging mouse bone based on whether their human homolog is associated with human BMD and/or fracture. Unbiased RNA-seq analysis of young and old male C57BL/6 mouse cortical bone identified 1499, 1685, and 5525 differentially expressed genes (DEGs) in 1, 2, and 2.5-year-old bone, relative to 2-month-old bone, respectively. Gene-based scores for heel ultrasound bone mineral density (eBMD) and fracture were estimated using published genome-wide association studies (GWAS) results of these traits in the UK Biobank. Enrichment analysis showed that mouse bone DEG sets for all three age groups, relative to young bone, are significantly enriched for eBMD, but only the oldest two DEG sets are enriched for fracture. Using gene-based scores, this approach prioritizes among thousands of DEGs by a factor of 5- to 100-fold, yielding 10 and 21 genes significantly associated with fracture in the two oldest groups of mouse DEGs. Though these genes were not the most differentially expressed, they included Sost, Lrp5, and others with well-established functions in bone. Several others have, as yet, unknown roles in the skeleton. Therefore, this study accelerates identification of new genetic determinants of bone fragility by prioritizing a clinically relevant and experimentally tractable number of candidate genes for functional analysis. Finally, we provide a website (www.mouse2human.org) to enable other researchers to easily apply our strategy. © 2022 American Society for Bone and Mineral Research (ASBMR).

Altered canalicular remodeling associated with femur fracture in mice.

We previously showed that femur fracture in mice caused a reduction in bone volume at distant skeletal sites within 2 weeks post-fracture. Osteocytes also have the ability to remodel their surrounding bone matrix through perilacunar/canalicular remodeling (PLR). If PLR is altered systemically following fracture, this could affect bone mechanical properties and increase fracture risk at all skeletal sites. In this study, we investigated whether lacunar-canalicular microstructure and the rate of PLR are altered in the contralateral limb following femoral fracture in mice. We hypothesized that femoral fracture would accelerate PLR by 2 weeks postfracture, followed by partial recovery by 4 weeks. We used histological evaluation and high-resolution microcomputed tomography to quantify the morphology of the lacunar-canalicular network at the contralateral tibia, and we used quantitative real-time polymerase chain reaction (RT-PCR) and RNA-seq to measure the expression of PLR-associated genes in the contralateral femur. We found that at both 2 and 4 weeks postfracture, canalicular width was significantly increased by 18.6% and 16.6%, respectively, in fractured mice relative to unfractured controls. At 3 days and 4 weeks post-fracture, we observed downregulation of PLR-associated genes; RNA-seq analysis at 3 days post-fracture showed a deceleration of bone formation and mineralization in the contralateral limb. These data demonstrate notable canalicular changes following fracture that could affect bone mechanical properties. These findings expand our understanding of systemic effects of fracture and how biological and structural changes at distant skeletal sites may contribute to increased fracture risk following an acute injury.

Mechanosensitive miR-100 coordinates TGFß and Wnt signaling in osteocytes during fluid shear stress.

Organism scale mechanical forces elicit cellular scale changes through coordinated regulation of multiple signaling pathways. The mechanisms by which cells integrate signaling to generate a unified biological response remains a major question in mechanobiology. For example, the mechanosensitive response of bone and other tissues requires coordinated signaling by the transforming growth factor beta (TGFß) and Wnt pathways through mechanisms that are not well-defined. Here we report a new microRNA-dependent mechanism that mediates mechanosensitive crosstalk between TGFß and Wnt signaling in osteocytes exposed to fluid shear stress (FSS). From 60 mechanosensitive microRNA (miRs) identified by small-RNAseq, miR100 expression is suppressed by in vivo hindlimb loading in the murine tibia and by cellular scale FSS in OCY454 cells. Though FSS activates both TGFß and Wnt signaling in osteocytes, only TGFß represses miR-100 expression. miR-100, in turn, antagonizes Wnt signaling by targeting and inhibiting expression of Frizzled receptors (FZD5/FZD8). Accordingly, miR-100 inhibition blunts FSS- and TGFß-inducible Wnt signaling. Therefore, our results identify FSS-responsive miRNAs in osteocytes, including one that integrates the mechanosensitive function of two essential signaling pathways in the osteoanabolic response of bone to mechanical load.

Disrupted osteocyte connectivity and pericellular fluid flow in bone with aging and defective TGF-ß signaling.

Skeletal fragility in the elderly does not simply result from a loss of bone mass. However, the mechanisms underlying the concurrent decline in bone mass, quality, and mechanosensitivity with age remain unclear. The important role of osteocytes in these processes and the age-related degeneration of the intricate lacunocanalicular network (LCN) in which osteocytes reside point to a primary role for osteocytes in bone aging. Since LCN complexity severely limits experimental dissection of these mechanisms in vivo, we used two in silico approaches to test the hypothesis that LCN degeneration, due to aging or an osteocyte-intrinsic defect in transforming growth factor beta (TGF-ß) signaling (TßRIIocy-/-), is sufficient to compromise essential osteocyte responsibilities of mass transport and exposure to mechanical stimuli. Using reconstructed confocal images of bone with fluorescently labeled osteocytes, we found that osteocytes from aged and TßRIIocy-/- mice had 33 to 45% fewer, and more tortuous, canaliculi. Connectomic network analysis revealed that diminished canalicular density is sufficient to impair diffusion even with intact osteocyte numbers and overall LCN architecture. Computational fluid dynamics predicts that the corresponding drop in shear stress experienced by aged or TßRIIocy-/- osteocytes is highly sensitive to canalicular surface area but not tortuosity. Simulated expansion of the osteocyte pericellular space to mimic osteocyte perilacunar/canalicular remodeling restored predicted shear stress for aged osteocytes to young levels. Overall, these models show how loss of LCN volume through LCN pruning may lead to impaired fluid dynamics and osteocyte exposure to mechanostimulation. Furthermore, osteocytes emerge as targets of age-related therapeutic efforts to restore bone health and function.

Fluid shear stress generates a unique signaling response by activating multiple TGFß family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFß) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone-forming and bone-resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFß signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFß signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFß target gene expression, even in the absence of added TGFß. Indeed, relative to treatment with TGFß, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFß receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFß family R-Smads by stimulating multimerization and concurrently activating several TGFß and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFß and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

Mechanosensitive Control of Articular Cartilage and Subchondral Bone Homeostasis in Mice Requires Osteocytic Transforming Growth Factor ß Signaling.

OBJECTIVE: Transforming growth factor ß (TGFß) signaling plays a complex tissue-specific and nonlinear role in osteoarthritis (OA). This study was conducted to determine the osteocytic contributions of TGFß signaling to OA. METHODS: To identify the role of osteocytic TGFß signaling in joint homeostasis, we used 16-week-old male mice (n = 9-11 per group) and female mice (n = 7-11 per group) with an osteocyte-intrinsic ablation of TGFß receptor type II (TßRIIocy-/- mice) and assessed defects in cartilage degeneration, subchondral bone plate (SBP) thickness, and SBP sclerostin expression. To further investigate these mechanisms in 16-week-old male mice, we perturbed joint homeostasis by subjecting 8-week-old mice to medial meniscal/ligamentous injury (MLI), which preferentially disrupts the mechanical environment of the medial joint to induce OA. RESULTS: In all contexts, independent of sex, genotype, or medial or lateral joint compartment, increased SBP thickness and SBP sclerostin expression were spatially associated with cartilage degeneration. Male TßRIIocy-/- mice, but not female TßRIIocy-/- mice, had increased cartilage degeneration, increased SBP thickness, and higher levels of SBP sclerostin compared with control mice (all P < 0.05), demonstrating that the role of osteocytic TGFß signaling on joint homeostasis is sexually dimorphic. With changes in joint mechanics following injury, control mice had increased SBP thickness, subchondral bone volume, and SBP sclerostin expression (all P < 0.05). TßRIIocy-/- mice, however, were insensitive to subchondral bone changes with injury, suggesting that mechanosensation at the SBP requires osteocytic TGFß signaling. CONCLUSION: Our results provide new evidence that osteocytic TGFß signaling is required for a mechanosensitive response to injury, and that osteocytes control SBP homeostasis to maintain cartilage health, identifying osteocytic TGFß signaling as a novel therapeutic target for OA.