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Large group collaborative teaching approaches are rapidly gaining popularity in undergraduate medical education. The case-based collaborative Learning (CBCL) pedagogy was instituted for pre-clerkship teaching at Harvard Medical School in 2015 with subsequent implementation at other medical schools. CBCL emphasizes inductive reasoning, integrates basic and clinical sciences, stimulates curiosity, and fosters teamwork. Given the ongoing educational evolution, guidance on designing and facilitating collaborative learning sessions, such as CBCL may benefit faculty in their instructional design efforts. This perspective article describes strategies to create effective collaborative sessions using CBCL as an example. We reviewed the literature and summarized ten years of experience in CBCL teaching through the lens of contemporary theories of teaching and learning. The recommendations are organized into three main domains: Instructional Design, Facilitation, and Professional Transformation, each aligned with the theoretical principles of CBCL. The recommendations provide a conceptual model to assist faculty in designing engaging and effective class materials and support students' professional transformation during collaborative learning sessions., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2025.)

Introduction: This study assessed patient demographic factors associated with nonattendance for osteoporosis evaluation after being referred to our Bone Health Clinic (BHC), a hospital-based outpatient Fracture Liaison Service (FLS), for a fragility fracture. Methods: 507 patients sustaining a fragility fracture were referred to the BHC over a 39-month period. Retrospective chart review was conducted to capture osteoporosis evaluation attendance rates and demographic factors (age, gender, race, area deprivation index, insurance type, and fracture type). A post-fracture follow-up visit with either the BHC or another provider in which osteoporosis was noted in the assessment was considered attendance for osteoporosis evaluation. Nonattendance was determined at a cutoff of one year after the fracture date. Results: Of the 507 patients referred to the BHC following a fragility fracture, 177 patients attended osteoporosis evaluation with either the BHC or a primary care provider. Nonattendance was associated with older age ( p =0.0075), having private health insurance ( p =0.0434), and recent hip fracture ( p < 0.0001). Attendance was associated with having government health insurance ( p =0.0103). Conclusion: Inpatient evaluation and treatment for osteoporosis should be considered in patients who are older or have sustained a hip fracture as they may have more difficulty attending post-fracture appointments., Competing Interests: The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: T.S., J.E.C., C.H., S.J.E., and S.P.M. have no competing interest. J.F.C. has received a grant from Novartis for an investigator-initiated study and has participated in advisory boards at Ultragenyx, Alexion, and Kyowa Kirin. B.E.E. owns stock in Johnson and Johnson and Pfizer and is a consultant for Zimmer Biomet., (Copyright © 2024 Thany Seyok et al.)

Emerging evidence suggests a significant role of gut microbiome in bone health. Aging is well recognized as a crucial factor influencing the gut microbiome. In this study, we investigated whether age-dependent microbial change contributes to age-related bone loss in CB6F1 mice. The bone phenotype of 24-month-old germ-free (GF) mice was indistinguishable compared to their littermates colonized by fecal transplant at 1-month-old. Moreover, bone loss from 3 to 24-month-old was comparable between GF and specific pathogen-free (SPF) mice. Thus, GF mice were not protected from age-related bone loss. 16S rRNA gene sequencing of fecal samples from 3-month and 24-month-old SPF males indicated an age-dependent microbial shift with an alteration in energy and nutrient metabolism potential. An integrative analysis of 16S predicted metagenome function and LC-MS fecal metabolome revealed an enrichment of protein and amino acid biosynthesis pathways in aged mice. Microbial S-adenosyl methionine metabolism was increased in the aged mice, which has previously been associated with the host aging process. Collectively, aging caused microbial taxonomic and functional alteration in mice. To demonstrate the functional importance of young and old microbiome to bone, we colonized GF mice with fecal microbiome from 3-month or 24-month-old SPF donor mice for 1 and 8 months. The effect of microbial colonization on bone phenotypes was independent of the microbiome donors' age. In conclusion, our study indicates age-related bone loss occurs independent of gut microbiome., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1-34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11 + cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts., (© 2024. The Author(s).)

Heterogeneous nuclear ribonucleoprotein U (hnRNPU) is known to play multiple biological roles by regulating transcriptional expression, RNA splicing, RNA stability, and chromatin structure in a tissue-dependent manner. The role of hnRNPU in skeletal muscle development and maintenance has not been previously evaluated. In this study, skeletal muscle specific hnRNPU knock out mice is utilized and evaluated skeletal muscle mass and immune cell infiltration through development. By 4 weeks, muscle-specific hnRNPU knockout mice revealed Ly6C+ monocyte infiltration into skeletal muscle, which preceded muscle atrophy. Canonical NF-kB signaling is activated in a myofiber-autonomous manner with hnRNPU repression. Inducible hnRNPU skeletal muscle knockout mice further demonstrated that deletion of hnRNPU in adulthood is sufficient to cause muscle atrophy, suggesting that hnRNPU's role in muscle maintenance is not during development alone. Treatment with salirasib, to inhibit proliferation of immune cells, prevents muscle atrophy in muscle-specific hnRNPU knock out mice, indicating that immune cell infiltration plays causal role in muscle atrophy of hnRNPU knock out mice. Overall, the findings suggest that loss of hnRNPU triggers muscle inflammation and activates NF-κB signaling in a cell-autonomous manner, culminating in muscle atrophy., (© 2024 Wiley‐VCH GmbH.)

Background: Reduced viability in the deepest zones of osteochondral allografts (OCAs) can weaken the subchondral interface, potentially increasing the risk of failure. This reduction may result from nutritional imbalances due to uneven media distribution or interference from bone marrow elements., Purpose: To investigate whether culturing OCAs using a rotary shaker or removing the bone marrow elements would increase graft cellular viability., Study Design: Controlled laboratory study., Methods: Bovine osteochondral explants were stored for 28 days at 4°C under 3 different conditions (n = 6 explants per group): static (control group), rotary shaker at 150 rpm (shaker group), and static after removal of bone marrow elements using a Waterpik device (Waterpik group). Chondrocyte viability was assessed using live/dead staining across the entire tissue and in each zone (superficial, middle, deep). Subchondral bone viability was assessed using TUNEL (terminal deoxynucleotidal transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) staining to detect apoptotic cells., Results: Both shaker (64.2%; P = .010) and Waterpik (65.6%; P = .005) conditions showed significantly higher chondrocyte viability compared with control (49.8%). When samples were analyzed by zone, the shaker and Waterpik groups displayed higher cellular viability at the middle zone (shaker = 60.6%, P < .001; Waterpik = 56.1%, P < .001) and deep zone (shaker = 63.1%, P = .018; Waterpik = 61.5%, P = .025) than the control group (25.6% at middle zone; 32.8% at deep zone). Additionally, shaker (56.7%; P = .018) and Waterpik (51.4%; P = .007) groups demonstrated a lower percentage of apoptotic cells in subchondral bone compared with control (88.0%). No significant differences were observed between the shaker and Waterpik groups in any of the analyses., Conclusion: Both rotary shaking and removal of bone marrow elements during storage of osteochondral explants led to higher chondrocyte viability at the middle and deep zones of the graft compared with the static storage condition. Enhancing nutrition delivery to the graft could improve its quality, potentially improving outcomes of OCA transplantation., Clinical Relevance: The use of a rotary shaker or the removal of bone marrow elements may significantly improve the culture conditions, increasing graft viability and integrity after OCA storage., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: This project received support from the JRF Ortho Allograft Research Grant (award No. 007). J.F.C. has received consulting fees from Ultragenyx Pharmaceutical and Alexion and author royalty from Merck Sharp & Dohme. C.L. has received honoraria from Arthrosurface, Vericel, and JRF Ortho; consulting fees from Zimmer Biomet, Aastrom Biosciences, Sanofi-Aventis, Vericel, Flexion Therapeutics, JRF Ortho, and Samumed; and nonconsulting fees from Aesculap and Arthrosurface. G.M. has received consulting fees from JRF Ortho. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Background: The purpose of this study was to assess the impact of implementation of an outpatient fracture liaison service (FLS) on completion rates of dual-energy x-ray absorptiometry (DXA) and screening labs including 25-OH vitamin D and parathyroid hormone (PTH) in patients with upper extremity (UE) fragility fractures., Methods: At our institution, 367 patients were treated in 2014-2015 for UE fragility fractures of the distal radius and proximal humerus before implementation of our outpatient FLS and 395 patients in 2017-2018 after implementation. Retrospective chart review was conducted to identify completed DXA scans within 2 years of fracture treatment and completed 25-OH vitamin D and PTH labs within 1 year of fracture treatment., Results: There were no statistical differences in the demographics of patients treated for distal radial and proximal humeral fragility fractures during the 2014-2015 and 2017-2018 time periods. Implementation of the FLS resulted in a 9.9% increase ( P value = .021) in completed DXA scans within 2 years of fracture treatment. Completed 25-OH vitamin D and PTH labs saw a significant increase of 17.1% and 23.8%, respectively ( P values < .001)., Conclusions: Implementation of an outpatient FLS can help to improve osteoporosis evaluation with completed DXA scans and 25-OH vitamin D and PTH labs., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: TS, JEC, and SJE have no competing interest. JFC has received a grant from Novartis for an investigator-initiated study and is on the advisory board at Ultragenyx. BEE owns stock in Johnson & Johnson and Pfizer.

Objectives: Rare metabolic bone diseases can present with symptoms mimicking more common rheumatological conditions including spondyloarthritis, osteoarthritis, and fibromyalgia. Increasing awareness of these rare diseases within the rheumatology community is vital to ensure that affected patients are diagnosed and appropriately treated. The literature includes several reports of tumour-induced osteomalacia initially diagnosed as rheumatic disease, but other rare diseases such as X-linked hypophosphatemia (XLH) and hypophosphatasia (HPP) also deserve attention. Here, we describe two cases of adult patients incorrectly diagnosed with ankylosing spondylitis and osteoarthritis who, upon referral to a metabolic bone disease specialist, were subsequently diagnosed with XLH and HPP, respectively, profoundly altering their management., Methods: The cases were collected from Brigham and Women's Hospital, Boston, MA, USA, and Vanderbilt University Medical Center, Nashville, TN, USA., Results: Details of the patients' respective medical and family histories are presented, and the clinical and biochemical investigations undertaken to reach the correct diagnoses are described., Conclusion: Rheumatologists should be encouraged to think beyond common rheumatological diseases when faced with symptoms such as bone pain, muscle pain, and stiffness, especially when accompanied by manifestations including atraumatic fractures, poor dentition, and hearing loss. In cases where one of these rare diseases is suspected, referral to a metabolic bone disease specialist for confirmation of diagnosis is encouraged as effective treatment options have recently become available., Competing Interests: JFC has served as an advisory board member for Ultragenyx Pharmaceuticals and AbbVie, has participated in a focus group for Alexion Pharmaceuticals, and receives author royalties from UpToDate. SK is an employee of and holds stock in Ultragenyx Pharmaceuticals. KD is a clinical study investigator and has received consultancy fees and/or institutional research funding and/or grant support from Ultragenyx Pharmaceuticals, Alexion, AstraZeneca, and Kyowa Kirin. AOM reports no conflicts of interest., (Copyright © 2024 Julia F. Charles et al.)

At the end of gestation, fetal skeleton rapidly accumulates calcium, and bone development continues in offspring postnatally. To accommodate, maternal skeletal physiology is modulated in a serotonin-dependent manner. Selective serotonin reuptake inhibitors (SSRIs) are generally considered safe for treatment of major depressive disorder, postpartum depression, and other psychiatric illnesses during the peripartum period, but because serotonin affects bone remodeling, SSRIs are associated with decreased bone mass across all ages and sexes, and the impact of SSRIs during fetal and postnatal development has not been fully investigated. In the present study, our aim was to examine developmental fluoxetine exposure on offspring skeleton and to assess varying degrees of impact depending on dose and window of exposure in short-term and long-term contexts. We established that a low dose of lactational fluoxetine exposure caused a greater degree of insult to offspring bone than either a low dose during fetal and postpartum development or a high dose during lactation only in mice. We further discovered lasting impacts of developmental fluoxetine exposure, especially during lactation only, on adult bone and body composition. Herein, we provide evidence fluoxetine exposure during early development may have detrimental effects on the skeleton of offspring at weaning and into adulthood., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Objective: The objective is to update recommendations for prevention and treatment of glucocorticoid-induced osteoporosis (GIOP) for patients with rheumatic or nonrheumatic conditions receiving >3 months treatment with glucocorticoids (GCs) ≥2.5 mg daily., Methods: An updated systematic literature review was performed for clinical questions on nonpharmacologic, pharmacologic treatments, discontinuation of medications, and sequential therapy. Grading of Recommendations Assessment, Development and Evaluation approach was used to rate the certainty of evidence. A Voting Panel achieved ≥70% consensus on the direction (for or against) and strength (strong or conditional) of recommendations., Results: For adults beginning or continuing >3 months of GC treatment, we strongly recommend as soon as possible after initiation of GCs, initial assessment of fracture risks with clinical fracture assessment, bone mineral density with vertebral fracture assessment or spinal x-ray, and Fracture Risk Assessment Tool if ≥40 years old. For adults at medium, high, or very high fracture risk, we strongly recommend pharmacologic treatment. Choice of oral or intravenous bisphosphonates, denosumab, or parathyroid hormone analogs should be made by shared decision-making. Anabolic agents are conditionally recommended as initial therapy for those with high and very high fracture risk. Recommendations are made for special populations, including children, people with organ transplants, people who may become pregnant, and people receiving very high-dose GC treatment. New recommendations for both discontinuation of osteoporosis therapy and sequential therapies are included., Conclusion: This guideline provides direction for clinicians and patients making treatment decisions for management of GIOP. These recommendations should not be used to limit or deny access to therapies., (© 2023 American College of Rheumatology. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Purpose of Review: To provide a comprehensive overview of the inflammatory response following anterior cruciate ligament (ACL) injury and to highlight the relationship between specialized pro-resolving mediators (SPMs) and inflammatory joint conditions, emphasizing the therapeutic potential of modulating the post-injury resolution of inflammation to prevent posttraumatic osteoarthritis (PTOA)., Recent Findings: The inflammatory response triggered after joint injuries such as ACL tear plays a critical role in posttraumatic osteoarthritis development. Inflammation is a necessary process for tissue healing, but unresolved or overactivated inflammation can lead to chronic diseases. SPMs, a family of lipid molecules derived from essential fatty acids, have emerged as active players in the resolution of inflammation and tissue repair. While their role in other inflammatory conditions has been studied, their relationship with PTOA remains underexplored. Proinflammatory mediators contribute to cartilage degradation and PTOA pathogenesis, while anti-inflammatory and pro-resolving mediators may have chondroprotective effects. Therapies aimed at suppressing inflammation in PTOA have limitations, as inflammation is crucial for tissue healing. SPMs offer a pro-resolving response without causing immunosuppression, making them a promising therapeutic option. The known onset date of PTOA makes it amenable to early interventions, and activating pro-resolving pathways may provide new possibilities for preventing PTOA progression. Harnessing the pro-resolving potential of SPMs may hold promise for preventing PTOA and restoring tissue homeostasis and function after joint injuries., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Postpartum depression affects many individuals after parturition, and selective serotonin reuptake inhibitors (SSRIs) are often used as the first-line treatment; however, both SSRIs and lactation are independently associated with bone loss due to the role of serotonin in bone remodeling. Previously, we have established that administration of the SSRI fluoxetine during the peripartal period results in alterations in long-term skeletal characteristics. In the present study, we treated mice with either a low or high dose of fluoxetine during lactation to determine the consequences of the perturbation of serotonin signaling during this time period on the dam skeleton. We found that lactational fluoxetine exposure affected both cortical and trabecular parameters, altered gene expression and circulating markers of bone turnover, and affected mammary gland characteristics, and that these effects were more pronounced in the dams that were exposed to the low dose of fluoxetine in comparison to the high dose. Fluoxetine treatment during the postpartum period in rodents had short term effects on bone that were largely resolved 3 months post-weaning. Despite the overall lack of long-term insult to bone, the alterations in serotonin-driven lactational bone remodeling raises the question of whether fluoxetine is a safe option for the treatment of postpartum depression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fricke, Krajco, Perry, Brettingen, Wake, Charles and Hernandez.)

The peripartal period is marked by alterations in calcium metabolism to accommodate for embryonic skeletal mineralization and support bone development of offspring in early life, and serotonin plays a critical role in modulating peripartal bone remodeling. Selective serotonin reuptake inhibitors (SSRIs) are commonly used as first-line treatment for psychiatric illness during pregnancy and the postpartum period and considered safe for maternal use during this time frame. In order to evaluate the effect of peripartal alterations of the serotonergic system on maternal skeletal physiology, we treated dams with the SSRI fluoxetine during gestation only, lactation only, or during the entire peripartal period. Overall, we found a low dose of fluoxetine during gestation only had minimal impacts on maternal bone at weaning, but there were implications on maternal skeleton at weaning when dams were exposed during lactation only or during the entire peripartal period. We found that these effects were differential between female mice dosed lactationally or peripartally, and there were also impacts on maternal mammary gland at weaning in both of these groups. Though SSRIs are largely considered safe maternally during the peripartal period, this study raises the question whether safety of SSRIs, specifically fluoxetine, during the peripartal period should be reevaluated., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background: Upper extremity (UE) fragility fractures are common and strong predictors of subsequent fractures. To investigate the relative importance of an UE fragility fracture in determining future fracture risk, we conducted a cross-sectional study to compare future fracture risk between patients presenting for osteoporosis evaluation after an UE fragility fracture and a similarly aged cohort of patients without an UE fracture., Methods: In all, 129 UE fracture patients seen in our bone health clinic (BHC) and 114 non-fracture UE fracture patients seen in an UE clinic completed clinic intake surveys assessing for fracture risk factors. Prefracture fracture risk (PFFR) and fracture risk assessment tool (FRAX) scores estimated the future fracture risks at the timepoint before and after the UE fragility fracture event, respectively. The primary study outcome was the 10-year risk of future fracture., Results: The 10-year probability of major osteoporotic and hip fractures were significantly higher among the BHC group when estimated with FRAX. When estimated with PFFR score, there was no difference in the 10-year probability of hip fracture between the groups. Prevalence of secondary osteoporosis and glucocorticoid use was higher in the BHC group, and prevalence of rheumatoid arthritis was higher in the UE clinic group., Conclusions: This study underscores the importance of an UE fragility fracture in determining the risk of future fracture. A fragility fracture of the UE should be considered a sentinel event and physicians who evaluate these patients should recognize them as a high-risk group for future hip fracture., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: TS, JEC, and SE have no competing interest. JFC has received a grant from Novartis for an investigator-initiated study and is on the advisory board at Ultragenyx. BE owns stock in Johnson and Johnson and Pfizer.

Cartilage tissue exhibits early degenerative changes with onset of osteoarthritis (OA). Early diagnosis is critical as there is only a narrow time window during which therapeutic intervention can reverse disease progression. Computed tomography (CT) has been considered for cartilage imaging as a tool for early OA diagnosis by introducing radio-opaque contrast agents like ioxaglate (IOX) into the joint. IOX, however, is anionic and thus repelled by negatively charged cartilage glycosaminoglycans (GAGs) that hinders its intra-tissue penetration and partitioning, resulting in poor CT attenuation. This is further complicated by its short intra-tissue residence time owing to rapid clearance from joints, which necessitates high doses causing toxicity concerns. Here we engineer optimally charged cationic contrast agents based on cartilage negative fixed charge density by conjugating cartilage targeting a cationic peptide carrier (CPC) and multi-arm avidin nanoconstruct (mAv) to IOX, such that they can penetrate through the full thickness of cartilage within 6 h using electrostatic interactions and elicit similar CT signal with about 40× lower dose compared to anionic IOX. Their partitioning and distribution correlate strongly with spatial GAG distribution within healthy and early- to late-stage arthritic bovine cartilage tissues at 50-100× lower doses than other cationic contrast agents used in the current literature. The use of contrast agents at low concentrations also allowed for delineation of cartilage from subchondral bone as well as other soft tissues in rat tibial joints. These contrast agents are safe to use at current doses, making CT a viable imaging modality for early detection of OA and staging of its severity.

Still's disease is a severe inflammatory syndrome characterized by fever, skin rash and arthritis affecting children and adults. Patients with Still's disease may also develop macrophage activation syndrome, a potentially fatal complication of immune dysregulation resulting in cytokine storm. Here we show that mTORC1 (mechanistic target of rapamycin complex 1) underpins the pathology of Still's disease and macrophage activation syndrome. Single-cell RNA sequencing in a murine model of Still's disease shows preferential activation of mTORC1 in monocytes; both mTOR inhibition and monocyte depletion attenuate disease severity. Transcriptomic data from patients with Still's disease suggest decreased expression of the mTORC1 inhibitors TSC1/TSC2 and an mTORC1 gene signature that strongly correlates with disease activity and treatment response. Unrestricted activation of mTORC1 by Tsc2 deletion in mice is sufficient to trigger a Still's disease-like syndrome, including both inflammatory arthritis and macrophage activation syndrome with hemophagocytosis, a cellular manifestation that is reproduced in human monocytes by CRISPR/Cas-mediated deletion of TSC2. Consistent with this observation, hemophagocytic histiocytes from patients with macrophage activation syndrome display prominent mTORC1 activity. Our study suggests a mechanistic link of mTORC1 to inflammation that connects the pathogenesis of Still's disease and macrophage activation syndrome., (© 2022. The Author(s).)

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Increasing evidence points to the involvement of group IIA secreted phospholipase A 2 (sPLA 2 -IIA) in pathologies characterized by abnormal osteoclast bone-resorption activity. Here, the role of this moonlighting protein has been deepened in the osteoclastogenesis process driven by the RANKL cytokine in RAW264.7 macrophages and bone-marrow derived precursor cells from BALB/cJ mice. Inhibitors with distinct selectivity toward sPLA 2 -IIA activities and recombinant sPLA 2 -IIA (wild-type or catalytically inactive forms, full-length or partial protein sequences) were instrumental to dissect out sPLA 2 -IIA function, in conjunction with reduction of sPLA 2 -IIA expression using small-interfering-RNAs and precursor cells from Pla2g2a knock-out mice. The reported data indicate sPLA 2 -IIA participation in murine osteoclast maturation, control of syncytium formation and resorbing activity, by mechanisms that may be both catalytically dependent and independent. Of note, these studies provide a more complete understanding of the still enigmatic osteoclast multinucleation process, a crucial step for bone-resorbing activity, uncovering the role of sPLA 2 -IIA interaction with a still unidentified receptor to regulate osteoclast fusion through p38 SAPK activation. This could pave the way for the design of specific inhibitors of sPLA 2 -IIA binding to interacting partners implicated in osteoclast syncytium formation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mangini, D’Angelo, Vinciguerra, Payré, Lambeau, Balestrieri, Charles and Mariggiò.)

Myeloid cell mediated mechanisms regulate synovial joint inflammation. IL-34, a macrophage (Mø) growth and differentiation molecule, is markedly expressed in neutrophil and Mø-rich arthritic synovium. IL-34 engages a newly identified independent receptor, protein-tyrosine phosphatase, receptor-type, zeta (PTPRZ), that we find is expressed by Mø. As IL-34 is prominent in rheumatoid arthritis, we probed for the IL-34 and PTPRZ-dependent myeloid cell mediated mechanisms central to arthritis using genetic deficient mice in K/BxN serum-transfer arthritis. Unanticipatedly, we now report that IL-34 and PTPRZ limited arthritis as intra-synovial pathology and bone erosion were more severe in IL-34 and PTPRZ KO mice during induced arthritis. We found that IL-34 and PTPRZ: (i) were elevated, bind, and induce downstream signaling within the synovium in arthritic mice and (ii) were upregulated in the serum and track with disease activity in rheumatoid arthritis patients. Mechanistically, IL-34 and PTPRZ skewed Mø toward a reparative phenotype, and enhanced Mø clearance of apoptotic neutrophils, thereby decreasing neutrophil recruitment and intra-synovial neutrophil extracellular traps. With fewer neutrophils and neutrophil extracellular traps in the synovium, destructive inflammation was restricted, and joint pathology and bone erosion diminished. These novel findings suggest that IL-34 and PTPRZ-dependent mechanisms in the inflamed synovium limit, rather than promote, inflammatory arthritis., (© 2022. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.)

Compositional and functional alterations to the gut microbiota during aging are hypothesized to potentially impact our health. Thus, determining aging-specific gut microbiome alterations is critical for developing microbiome-based strategies to improve health and promote longevity in the elderly. In this study, we performed a meta-analysis of publicly available 16S rRNA gene sequencing data from studies investigating the effect of aging on the gut microbiome in mice. Aging reproducibly increased gut microbial alpha diversity and shifted the microbial community structure in mice. We applied the bioinformatic tool PICRUSt2 to predict microbial metagenome function and established a random forest classifier to differentiate between microbial communities from young and old hosts and to identify aging-specific metabolic features. In independent validation data sets, this classifier achieved an area under the receiver operating characteristic curve (AUC) of 0.75 to 0.97 in differentiating microbiomes from young and old hosts. We found that 50% of the most important predicted aging-specific metabolic features were involved in carbohydrate metabolism. Furthermore, fecal short-chain fatty acid (SCFA) concentrations were significantly decreased in old mice, and the expression of the SCFA receptor Gpr41 in the colon was significantly correlated with the relative abundances of gut microbes and microbial carbohydrate metabolic pathways. In conclusion, this study identified aging-specific alterations in the composition and function of the gut microbiome and revealed a potential relationship between aging, microbial carbohydrate metabolism, fecal SCFA, and colonic Gpr41 expression. IMPORTANCE Aging-associated microbial alteration is hypothesized to play an important role in host health and longevity. However, investigations regarding specific gut microbes or microbial functional alterations associated with aging have had inconsistent results. We performed a meta-analysis across 5 independent studies to investigate the effect of aging on the gut microbiome in mice. Our analysis revealed that aging increased gut microbial alpha diversity and shifted the microbial community structure. To determine if we could reliably differentiate the gut microbiomes from young and old hosts, we established a random forest classifier based on predicted metagenome function and validated its performance against independent data sets. Alterations in microbial carbohydrate metabolism and decreased fecal short-chain fatty acid (SCFA) concentrations were key features of aging and correlated with host colonic expression of the SCFA receptor Gpr41 . This study advances our understanding of the impact of aging on the gut microbiome and proposes a hypothesis that alterations in gut microbiota-derived SCFA-host GPR41 signaling are a feature of aging.

Women mobilize up to 10% of their bone mass during lactation to provide milk calcium. About 8%-13% of mothers use selective serotonin reuptake inhibitors (SSRI) to treat peripartum depression, but SSRIs independently decrease bone mass. Previously, peripartal use of the SSRI fluoxetine reduced maternal bone mass sustained post-weaning and reduced offspring bone length. To determine whether these effects were fluoxetine-specific or consistent across SSRI compounds, we examined maternal and offspring bone health using the most prescribed SSRI, sertraline. C57BL/6 mice were given 10 mg/kg/day sertraline, from the beginning of pregnancy through the end of lactation. Simultaneously, we treated nulliparous females on the same days as the primiparous groups, resulting in age-matched nulliparous groups. Dams were euthanized at lactation day 10 (peak lactation, n = 7 vehicle; n = 9 sertraline), lactation day 21 (weaning, n = 9 vehicle; n = 9 sertraline), or 3m post-weaning (n = 10 vehicle; n = 10 sertraline) for analysis. Offspring were euthanized at peak lactation or weaning for analysis. We determined that peripartum sertraline treatment decreased maternal circulating calcium concentrations across the treatment period, which was also seen in nulliparous treated females. Sertraline reduced the bone formation marker, procollagen 1 intact N-terminal propeptide, and tended to reduce maternal BV/TV at 3m post-weaning but did not impact maternal or offspring bone health otherwise. Similarly, sertraline did not reduce nulliparous female bone mass. However, sertraline reduced immunofluorescence staining of the tight junction protein, zona occludens in the mammary gland, and altered alveoli morphology, suggesting sertraline may accelerate mammary gland involution. These findings indicate that peripartum sertraline treatment may be a safer SSRI for maternal and offspring bone rather than fluoxetine., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Leucine-rich repeat containing G-protein-coupled receptor 6 (LGR6) is a marker of osteoprogenitor cells and is dynamically expressed during in vitro osteodifferentation of mouse and human mesenchymal stem cells (MSCs). While the Lgr6 genomic locus has been associated with osteoporosis in human cohorts, the precise molecular function of LGR6 in osteogenesis and maintenance of bone mass are not yet known. In this study, we performed in vitro Lgr6 knockdown and overexpression experiments in murine osteoblastic cells and find decreased Lgr6 levels results in reduced osteoblast proliferation, differentiation, and mineralization. Consistent with these data, overexpression of Lgr6 in these cells leads to significantly increased proliferation and osteodifferentiation. To determine whether these findings are recapitulated in vivo, we performed microCT and ex vivo osteodifferentiation analyses using our newly generated CRISPR-Cas9 mediated Lgr6 mouse knockout allele (Lgr6-KO). We find that ex vivo osteodifferentiation of Lgr6-KO primary MSCs is significantly reduced, and 8 week-old Lgr6-KO mice have less trabecular bone mass as compared to Lgr6 wildtype controls, indicating that Lgr6 is necessary for normal osteogenesis and bone mass. Towards mechanism, we analyzed in vitro signaling in the context of two LGR6 ligands, RSPO2 and MaR1. We find that RSPO2 stimulates LGR6-mediated WNT/β-catenin signaling whereas MaR1 stimulates LGR6-mediated cAMP activity, suggesting two ligand-dependent functions for LGR6 receptor signaling during osteogenesis. Collectively, this study reveals that Lgr6 is necessary for wildtype levels of proliferation and differentiation of osteoblasts, and achieving normal bone mass., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Osteoporosis is caused by an imbalance of osteoclasts and osteoblasts, occurring in close proximity to hematopoietic cells in the bone marrow. Recurrent somatic mutations that lead to an expanded population of mutant blood cells is termed clonal hematopoiesis of indeterminate potential (CHIP). Analyzing exome sequencing data from the UK Biobank, we found CHIP to be associated with increased incident osteoporosis diagnoses and decreased bone mineral density. In murine models, hematopoietic-specific mutations in Dnmt3a, the most commonly mutated gene in CHIP, decreased bone mass via increased osteoclastogenesis. Dnmt3a-/- demethylation opened chromatin and altered activity of inflammatory transcription factors. Bone loss was driven by proinflammatory cytokines, including Irf3-NF-κB-mediated IL-20 expression from Dnmt3a mutant macrophages. Increased osteoclastogenesis due to the Dnmt3a mutations was ameliorated by alendronate or IL-20 neutralization. These results demonstrate a novel source of osteoporosis-inducing inflammation., Competing Interests: Disclosures: A. Niroula reported grants from the Knut and Alice Wallenberg Foundation outside the submitted work. A. Bick reported personal fees from Foresite Labs outside the submitted work. J. Pirruccello reported personal fees from Maze Therapeutics outside the submitted work. M. Agrawal reported personal fees from German Accelerator Life Sciences outside the submitted work, and is the co-founder of and holds equity in iuvando Health. None of these are related to the submitted work. D.P. Kiel reported grants from Amgen, Radius Health, and Solarea Bio; and "other" from Solarea Bio, Pfizer, and Wolters Kluwer outside the submitted work. J.B. Richards reported personal fees from GlaxoSmithKline and Deerfield Capital; grants from Biogen and Eli Lilly; and non-financial support from 5 Prime Sciences outside the submitted work. J.B. Richards has served as an advisor to GlaxoSmithKline and Deerfield Capital. His institution has received investigator-initiated grant funding from Eli Lilly, GlaxoSmithKline and Biogen for projects unrelated to this research. He is the founder of 5 Prime Sciences. M.N. Wein reported grants from Radius Health and Galapagos NV, and "other" from Relation Therapeutics outside the submitted work. S. Jaiswal reported personal fees from AVRO Bio, Novartis, Genentech, and Foresite Labs outside the submitted work. P. Natarajan reported grants from Apple, AstraZeneca, Boston Scientific, and Novartis; personal fees from Apple, AstraZeneca, Genentech, Novartis, Blackstone Life Sciences, and Foresite Labs; and "other" from Vertex outside the submitted work. B.L. Ebert reported grants from Celgene, Novartis, Deerfield, and Calico; and personal fees from Exo Therapeutics, Skyhawk Therapeutics, Neomorph Therapeutics, and TenSixteen Bio outside the submitted work. No other disclosures were reported., (© 2021 Kim et al.)

IL-1β is a proinflammatory mediator with roles in innate and adaptive immunity. Here we show that IL-1β contributes to autoimmune arthritis by inducing osteoclastogenic capacity in Tregs. Using mice with joint inflammation arising through deficiency of the IL-1 receptor antagonist (Il1rn-/-), we observed that IL-1β blockade attenuated disease more effectively in early arthritis than in established arthritis, especially with respect to bone erosion. Protection was accompanied by a reduction in synovial CD4+Foxp3+ Tregs that displayed preserved suppressive capacity and aerobic metabolism but aberrant expression of RANKL and a striking capacity to drive RANKL-dependent osteoclast differentiation. Both Il1rn-/- Tregs and wild-type Tregs differentiated with IL-1β accelerated bone erosion upon adoptive transfer. Human Tregs exhibited analogous differentiation, and corresponding RANKLhiFoxp3+ T cells could be identified in rheumatoid arthritis synovial tissue. Together, these findings identify IL-1β-induced osteoclastogenic Tregs as a contributor to bone erosion in arthritis.

The thiazolidinediones (TZDs) are ligands of PPARγ that improve insulin sensitivity, but their use is limited by significant side effects. Recently, we demonstrated a mechanism wherein TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPARγ at serine 273. However, the role of this modification hasn't been tested genetically. Here we demonstrate that mice encoding an allele of PPARγ that cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm insulin sensitivity. RNA-seq in these mice reveals reduced expression of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find Gdf3 inhibits BMP signaling and insulin signaling in vitro. Together, these results highlight the diabetogenic role of PPARγ S273 phosphorylation and focus attention on a putative target, Gdf3., Competing Interests: Declaration of Interests E.D.R. is a paid consultant of Novartis. B.B.K. consults for Alterna and serves on the Scientific Advisory Board of Janssen Pharmaceuticals. A.S.B. and B.M.S. hold a patent related to this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.

Evolution is replete with reuse of genes in different contexts, leading to multifunctional roles of signaling factors during development. Here, we explore osteoclast regulation during skeletal development through analysis of colony-stimulating factor 1 receptor ( csf1r ) function in the zebrafish. A primary role of Csf1r signaling is to regulate the proliferation, differentiation and function of myelomonocytic cells, including osteoclasts. We demonstrate the retention of two functional paralogues of csf1r in zebrafish. Mutant analysis indicates that the paralogues have shared, non-redundant roles in regulating osteoclast activity during the formation of the adult skeleton. csf1ra , however, has adopted unique roles in pigment cell patterning not seen in the second paralogue. We identify a unique noncoding element within csf1ra of fishes that is sufficient for controlling gene expression in pigment cells during development. As a role for Csf1r signaling in pigmentation is not observed in mammals or birds, it is likely that the overlapping roles of the two paralogues released functional constraints on csf1ra , allowing the signaling capacity of Csf1r to serve a novel function in the evolution of pigment pattern in fishes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Osteoporosis, often called a silent disease, is a systemic condition of bone as a result of loss of bone mass and deterioration of its microarchitecture. The result is weakened bone, leading to an increased risk of fragility fractures. An estimated 9 million osteoporotic fractures occur every year worldwide. However, the true incidence of osteoporotic fractures is unknown because many are undetected. Astoundingly, this epidemic equates to an osteoporotic fracture every 3 seconds. Orthopaedic surgeons need to not only treat these fractures but also understand the underlying pathogenesis and risk factors to help prevent them. The management of osteoporosis is a critical part of musculoskeletal care. We must be familiar with the tools to assess osteoporosis and the treatments available, including risks and benefits. This review article is intended to deliver a review of the vast literature and provide the orthopaedic surgeon with the essential information necessary to manage the current osteoporosis epidemic.

G protein-coupled receptor 137b (GPR137b) is an orphan seven-pass transmembrane receptor of unknown function. In mouse, Gpr137b is highly expressed in osteoclasts in vivo and is upregulated during in vitro differentiation. To elucidate the role that GPR137b plays in osteoclasts, we tested the effect of GPR137b deficiency on osteoclast maturation and resorbing activity. We used CRISPR/Cas9 gene editing in mouse-derived ER-Hoxb8 immortalized myeloid progenitors to generate GPR137b-deficient osteoclast precursors. Decreasing Gpr137b in these precursors led to increased osteoclast differentiation and bone resorption activity. To explore the role of GPR137b during skeletal development, we generated zebrafish deficient for the ortholog gpr137ba. Gpr137ba-deficient zebrafish are viable and fertile and do not display overt morphological defects as adults. However, analysis of osteoclast function in gpr137ba -/- mutants demonstrated increased bone resorption. Micro-computed tomography evaluation of vertebral bone mass and morphology demonstrated that gpr137ba-deficiency altered the angle of the neural arch, a skeletal site with high osteoclast activity. Vital staining of gpr137ba -/- fish with calcein and alizarin red indicated that bone formation in the mutants is also increased, suggesting high bone turnover. These results identify GPR137b as a conserved negative regulator of osteoclast activity essential for normal resorption and patterning of the skeleton. Further, these data suggest that coordination of osteoclast and osteoblast activity is a conserved process among vertebrates and may have similar regulation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Serum bone turnover markers show diurnal variation in humans, suggesting that circadian rhythms contribute to normal bone physiology. This conclusion is corroborated by bone phenotypes in mice with genetic disruption of the circadian molecular clock mechanism, for instance via deletion of the transcription factor Brain and Muscle Arntl-like 1 (Bmal1). To dissect the contribution of circadian molecular clocks in individual bone cell types, we generated mice with conditional deletion of Bmal1 in osteoclasts (Ctsk-cre) and in mesenchymal cells of the limbs (Prx1-cre). We report that deletion of Bmal1 in osteoclasts had no effect on trabecular or cortical bone parameters in vivo or on osteoclast differentiation in vitro. In contrast, Bmal1 f/f .Prx1-cre mice had significantly less trabecular and cortical bone than Bmal1 f/f littermate controls, recapitulating the bone phenotype of Bmal1 germline deficient mice. The number of osteoblast precursors in the bone marrow of Bmal1 f/f .Prx1-cre mice was similar to wild-type controls, while the in vitro differentiation capacity of Bmal1-deficient osteoblast precursors, measured as induction of alkaline phosphatase activity, was significantly lower. Despite this, serum procollagen type 1 N-terminal propeptide (P1NP), a measure of bone formation in vivo, was higher in Bmal1 f/f .Prx1-cre mice than in Bmal1 f/f mice. Consistent with a high bone turnover state in the mutant mice, the bone resorption marker serum C-terminal telopeptides of Type I collagen (CTX-I) was also elevated, and Bmal1 f/f .Prx1-cre mice had a higher number of tartrate resistant acid phosphatase (TRAP) positive osteoclasts than Bmal1 f/f controls. These results demonstrate that adult bone mass in mice is controlled by the intrinsic circadian molecular clock in mesenchymal cells but not osteoclasts. The effect of the mesenchymal cell clock on bone turnover appears to involve osteoblast-osteoclast cross-talk., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bone-resorbing osteoclasts (OCs) are derived from myeloid precursors (MPs). Several transcription factors are implicated in OC differentiation and function; however, their hierarchical architecture and interplay are not well known. Analysis for enriched motifs in PU.1 and MITF chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) data from differentiating OCs identified eomesodermin (EOMES) as a potential novel binding partner of PU.1 and MITF at genes critical for OC differentiation and function. We were able to demonstrate using co-immunoprecipitation and sequential ChIP analysis that PU.1, MITF, and EOMES are in the same complex and present as a complex at OC genomic loci. Furthermore, EOMES knockdown in MPs led to osteopetrosis associated with decreased OC differentiation and function both in vitro and in vivo. Although EOMES is associated with embryonic development and other hematopoietic lineages, this is the first study demonstrating the requirement of EOMES in the myeloid compartment., (Copyright © 2019 Medical University of South Carolina. Published by Elsevier Inc. All rights reserved.)

Selective Serotonin Reuptake Inhibitors (SSRIs) such as fluoxetine are widely prescribed to pregnant and breastfeeding women, yet the effects of peripartum SSRI exposure on neonatal bone are not known. In adult populations, SSRI use is associated with compromised bone health, and infants exposed to in utero SSRIs have a smaller head circumference and are shorter, suggesting possible effects on longitudinal growth. Yet no study to date has examined the effects of peripartum SSRIs on long bone growth or mass. We used microCT to determine the outcomes of in utero and lactational SSRI exposure on C57BL6 pup bone microarchitecture. We found that peripartum exposure to 20 mg/kg fluoxetine reduced femoral bone mineral density and bone volume fraction, negatively impacted trabecular and cortical parameters, and resulted in shorter femurs on postnatal day 21. Although SSRIs are considered the first-choice antidepressant for pregnant and lactating women due to a low side effect profile, SSRI exposure may compromise fetal and neonatal bone development.

Long-term effects of breastfeeding on maternal bone are not fully understood. Excessive maternal bone loss stimulated by serotonin signaling during lactation may increase bone fragility later in life. We hypothesized that inhibiting nonneuronal serotonin activity by feeding a small-molecule inhibitor of the rate-limiting enzyme in serotonin synthesis [tryptophan hydroxylase 1 (TPH1)] would preserve maternal bone postweaning without affecting neonatal bone. Chow supplemented with the small-molecule TPH1 inhibitor LP778902 (~100 mg/kg) or control chow was fed to C57BL/6 dams throughout pregnancy and lactation, and blood was collected on days 1 and 21 of lactation. Dams returned to a common diet postweaning and were aged to 3 or 9 mo postweaning. Pups were euthanized at weaning. The effect of TPH1 inhibition on dam and pup femoral bone was determined by micro-computed tomography. Peripartum dietary supplementation with LP778902 decreased maternal serum serotonin concentrations ( P = 0.0007) and reduced bone turnover, indicated by serum NH 2 -terminal propeptide of type I collagen ( P = 0.01) and COOH-terminal collagen cross-links ( P = 0.02) concentrations, on day 21 of lactation. Repressed bone turnover from TPH1 inhibition was not associated with structural changes in maternal femur at 3 or 9 mo postweaning. By contrast, neonates exposed to peripartum LP778902 demonstrated differences in trabecular and cortical femoral bone compared with pups from control dams, with fewer ( P = 0.02) and thinner ( P = 0.001) trabeculae as well as increased trabecular spacing ( P = 0.04). Additionally, cortical porosity was increased ( P = 0.007) and cortical tissue mineral density was decreased ( P = 0.005) in pups of LP778902-treated dams. Small-molecule TPH1 inhibitors should be carefully considered in pregnant and lactating women, given potential risks to neonatal bone development.

Selective serotonin reuptake inhibitors (SSRIs) have been linked to osteopenia and fracture risk; however, their long-term impact on bone health is not well understood. SSRIs are widely prescribed to pregnant and breastfeeding women who might be at particular risk of bone pathology because lactation is associated with considerable maternal bone loss. We used microCT and molecular approaches to test whether the SSRI fluoxetine, administered to C57BL/6 mice from conception through the end of lactation, causes persistent maternal bone loss. We found that peripartum fluoxetine increases serum calcium and reduces circulating markers of bone formation during lactation but does not affect osteoclastic resorption. Peripartum fluoxetine exposure also enhances mammary gland endocrine function during lactation by increasing synthesis of serotonin and PTHrP, a hormone that liberates calcium for milk synthesis and reduces bone mineral volume. Peripartum fluoxetine exposure reduces the trabecular bone volume fraction at 3 months after weaning. These findings raise new questions about the long-term consequences of peripartum SSRI use on maternal health.

Microbiota and their hosts have coevolved for millions of years. Microbiota are not only critical for optimal development of the host under normal physiological growth, but also important to ensure proper host development during nutrient scarcity or disease conditions. A large body of research has begun to detail the mechanism(s) of how microbiota cooperate with the host to maintain optimal health status. One crucial host pathway recently demonstrated to be modulated by microbiota is that of the growth factor insulin like growth factor 1 (IGF-1). Gut microbiota are capable of dynamically modulating circulating IGF-1 in the host, with the majority of data suggesting that microbiota induce host IGF-1 synthesis to influence growth. Microbiota-derived metabolites such as short chain fatty acids are sufficient to induce IGF-1. Whether microbiota induction of IGF-1 is mediated by the difference in growth hormone expression or the host sensitivity to growth hormone is still under investigation. This review summarizes the current data detailing the interaction between gut microbiota, IGF-1 and host development.

Genome-wide association studies (GWASs) have been instrumental in understanding complex phenotypic traits. However, they have rarely been used to understand lineage-specific pathways and functions that contribute to the trait. In this study, by integrating lineage-specific enhancers from mesenchymal and myeloid compartments with bone mineral density loci, we were able to segregate osteoblast- and osteoclast (OC)-specific functions. Specifically, in OCs, a PU.1-dependent transcription factor (TF) network was revealed. Deletion of PU.1 in OCs in mice resulted in severe osteopetrosis. Functional genomic analysis indicated PU.1 and MITF orchestrated a TF network essential for OC differentiation. Several of these TFs were regulated by cooperative binding of PU.1 with BRD4 to form superenhancers. Further, PU.1 is essential for conformational changes in the superenhancer region of Nfatc1. In summary, our study demonstrates that combining GWASs with genome-wide binding studies and model organisms could decipher lineage-specific pathways contributing to complex disease states., Competing Interests: The authors declare that they have no conflict of interest.

Inactivating mutations in the ubiquitously expressed membrane trafficking component GMAP-210 (encoded by Trip11 ) cause achondrogenesis type 1A (ACG1A). ACG1A is surprisingly tissue specific, mainly affecting cartilage development. Bone development is also abnormal, but as chondrogenesis and osteogenesis are closely coupled, this could be a secondary consequence of the cartilage defect. A possible explanation for the tissue specificity of ACG1A is that cartilage and bone are highly secretory tissues with a high use of the membrane trafficking machinery. The perinatal lethality of ACG1A prevents investigating this hypothesis. We therefore generated mice with conditional Trip11 knockout alleles and inactivated Trip11 in chondrocytes, osteoblasts, osteoclasts and pancreas acinar cells, all highly secretory cell types. We discovered that the ACG1A skeletal phenotype is solely due to absence of GMAP-210 in chondrocytes. Mice lacking GMAP-210 in osteoblasts, osteoclasts and acinar cells were normal. When we inactivated Trip11 in primary chondrocyte cultures, GMAP-210 deficiency affected trafficking of a subset of chondrocyte-expressed proteins rather than globally impairing membrane trafficking. Thus, GMAP-210 is essential for trafficking specific cargoes in chondrocytes but is dispensable in other highly secretory cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The mechanisms underlying the systemic effects mediated by gut microbiota are under active investigation. In addition to local, direct effects of gut microbiota on the host, metabolic products from microbiota may act peripherally, reaching distal organs through the circulation. In our recent publication we demonstrated that gut microbiota influence bone remodeling distally, promoting both bone resorption and formation. We proposed that these effects are mediated, at least in part, by the induction of insulin like growth factor (IGF-1) by the microbiota metabolite short chain fatty acids (SCFA). Here we explore additional mechanisms by which microbial metabolites could directly or indirectly alter host bone remodeling. We discuss whether SCFA directly modulate bone resorption by their actions on osteoclasts, and test the possibility that serotonin is another gut microbiota derived long-distance mediator of effects on bone remodeling. A detailed understanding of the mechanisms of microbiota effect on bone remodeling could help establish potential therapeutic strategies to promote bone health.

The zebrafish is a powerful experimental model to investigate the genetic and morphologic basis of vertebrate development. Analysis of skeletogenesis in this fish is challenging as a result of the small size of the developing and adult zebrafish. Many of the bones of small fishes such as the zebrafish and medaka are quite thin, precluding many standard assays of bone quality and morphometrics commonly used on bones of larger animals. Microcomputed tomography (microCT) is a common imaging technique used for detailed analysis of the skeleton of the zebrafish and determination of mutant phenotypes. However, the utility of this modality for analysis of the zebrafish skeleton, and the effect of inherent variation among individual zebrafish, including variables such as sex, age and strain, is not well understood. Given the increased use and accessibility of microCT, we set out to define the sensitivity of microCT methods in developing and adult zebrafish. We assessed skeletal shape and density measures in the developing vertebrae and parasphenoid of the skull base. We found most skeletal variables are tightly correlated to standard length, but that at later growth stages (>3months) there are age dependent effects on some skeletal measures. Further we find modest strain but not sex differences in skeletal measures. These data suggest that the appropriate control for assessing mutant phenotypes should be age and strain matched, ideally a wild-type sibling. By analyzing two mutants exhibiting skeletal dysplasia, we show that microCT imaging can be a sensitive method to quantify distinct skeletal parameters of adults. Finally, as developing zebrafish skeletons remain difficult to resolve by radiographic means, we define a contrast agent specific for bone that enhances resolution at early stages, permitting detailed morphometric analysis of the forming skeleton. This increased capability for detection extends the use of this imaging modality to leverage the zebrafish model to understand the development causes of skeletal dysplasias., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose of Review: The gut microbiota can be considered a hidden organ that plays essential roles in host homeostasis. Exploration of the effects of microbiota on bone has just begun. Complimentary studies using germ-free mice, antibiotic, and probiotic treatments reveal a complicated relationship between microbiota and bone. Here, we review recent reports addressing the effect of gut microbiota on bone health, discuss potential reasons for discrepant findings, and explore potential mechanisms for these effects., Recent Findings: Manipulation of microbiota by colonization of germ-free mice, antibiotics, or probiotic supplementation significantly alters bone remodeling, bone development and growth, as well as bone mechanical strength. Different experimental models reveal context-dependent effects of gut microbiota on bone. By examining phenotypic effects, experimental context, and proposed mechanisms, revealed by recent reports, we hope to provide comprehensive and fresh insights into the many facets of microbiota and bone interactions.

Monocytes are derived from hematopoietic stem cells through a series of intermediate progenitor stages, but the factors that regulate this process are incompletely defined. Using a Ccr2/Cx 3 cr1 dual-reporter system to model murine monocyte ontogeny, we conducted a small-molecule screen that identified an essential role of mechanistic target of rapamycin complex 1 (mTORC1) in the development of monocytes and other myeloid cells. Confirmatory studies using mice with inducible deletion of the mTORC1 component Raptor demonstrated absence of mature circulating monocytes, as well as disruption in neutrophil and dendritic cell development, reflecting arrest of terminal differentiation at the granulocyte-monocyte progenitor stage. Conversely, excess activation of mTORC1 through deletion of the mTORC1 inhibitor tuberous sclerosis complex 2 promoted spontaneous myeloid cell development and maturation. Inhibitor studies and stage-specific expression profiling identified failure to down-regulate the transcription factor Myc by the mTORC1 target ribosomal S6 kinase 1 (S6K1) as the mechanistic basis for disrupted myelopoiesis. Together, these findings define the mTORC1-S6K1-Myc pathway as a key checkpoint in terminal myeloid development., (Copyright © 2017, American Association for the Advancement of Science.)

Objective: Proinflammatory molecules promote osteoclast-mediated bone erosion by up-regulating local RANKL production. However, recent evidence suggests that combinations of cytokines, such as tumor necrosis factor (TNF) plus interleukin-6 (IL-6), induce RANKL-independent osteoclastogenesis. The purpose of this study was to better understand TNF/IL-6-induced osteoclast formation and to determine whether RANK is absolutely required for osteoclastogenesis and bone erosion in murine inflammatory arthritis., Methods: Myeloid precursors from wild-type (WT) mice or mice with either germline or conditional deletion of Rank, Nfatc1, Dap12, or Fcrg were treated with either RANKL or TNF plus IL-6. Osteoprotegerin, anti-IL-6 receptor (anti-IL-6R), and hydroxyurea were used to block RANKL, the IL-6R, and cell proliferation, respectively. Clinical scoring, histologic assessment, micro-computed tomography, and quantitative polymerase chain reaction (qPCR) were used to evaluate K/BxN serum-transfer arthritis in WT and RANK-deleted mice. Loss of Rank was verified by qPCR and by osteoclast cultures., Results: TNF/IL-6 generated osteoclasts in vitro that resorbed mineralized tissue through a pathway dependent on IL-6R, NFATc1, DNAX-activation protein 12, and cell proliferation, but independent of RANKL or RANK. Bone erosion and osteoclast formation were reduced, but not absent, in arthritic mice with inducible deficiency of RANK. TNF/IL-6, but not RANKL, induced osteoclast formation in bone marrow and synovial cultures from animals deficient in Rank. Multiple IL-6 family members (IL-6, leukemia inhibitory factor, oncostatin M) were up-regulated in the synovium of arthritic mice., Conclusion: The persistence of bone erosion and synovial osteoclasts in Rank-deficient mice, and the ability of TNF/IL-6 to induce osteoclastogenesis, suggest that more than one cytokine pathway exists to generate these bone-resorbing cells in inflamed joints., (© 2016, American College of Rheumatology.)