Elizabeth W. Bradley

While there are clear differences in bone accrual and maintenance between men and women due to hormonal regulation, the potential cell-intrinsic factors governing this difference are poorly understood. To investigate this, we performed bone marrow transplantation assays using male or female bone marrow transplantation into male hosts. Micro-CT analyses of lethally irradiated 8-week-old male mice transplanted with female bone marrow exhibited increased vertebral body trabecular and cortical bone mass. To identify cell types mediating these effects, we performed comparative single cell sequencing analyses and identified several CD11B ⁺ CD74 ⁺ myeloid cell clusters. Male donor BM chimeric mice had a higher proportion of Cd74-expressing clusters compared to female donor BM chimeric mice. We confirmed that males have greater numbers of bone marrow-resident CD11B ⁺ CD74 ⁺ cells under physiological conditions. Elevated expression of X-linked genes Tlr7 and KdmCa marked a population of CD11B ⁺ CD74 ⁺ cells. Lastly, we show that female CD11B ⁺ CD74 ⁺ cells exhibit diminished osteoclastogenesis. These findings demonstrate that male and female bone marrow-resident cells have cell-intrinsic differences that regulate bone mass and that CD74 marks a subset of osteoclast precursor–enriched myeloid populations.

Bone remodeling is regulated by circadian rhythm, yet the molecular mechanisms linking circadian clock components to osteoclast function remain largely undefined. We previously showed that osteoclast-specific deletion of Per1, a core circadian regulator, increased osteoclastogenesis and reduced bone mass in male mice, whereas deletion of Per2 had little effect. Here, we investigated Per1;Per2 conditional double KO (dKO) mice and unexpectedly found the opposite phenotype. In contrast to Per1 deficiency, dKO suppressed osteoclastogenesis and increased bone mass specifically in male mice, revealing previously unrecognized functional interactions between PER1 and PER2 in osteoclasts. Transcriptomic analyses showed that dKO osteoclasts preferentially downregulated innate immunity genes, including both positive and negative regulators of osteoclastogenesis. Chromatin immunoprecipitation and reporter assays further identified innate immunity genes as downstream targets of PER-dependent circadian regulation. These findings indicate that PER1 and PER2 regulate osteoclastogenesis through coordinated control of a network of immune genes with opposing effects on osteoclast differentiation, rather than acting as simple promoters or inhibitors of bone resorption. More broadly, this study establishes innate immunity as a mechanistic interface between the circadian clock and osteoclast function, providing a framework for understanding how circadian disruption influences bone remodeling and inflammatory bone diseases.

Although studies support disrupted bone remodelling within geriatric populations, mid‐life changes are understudied. To investigate this, we performed bone marrow transplantation assays using either 8‐ or 40‐week‐old mice. Micro‐CT analyses of lethally irradiated 8‐week‐old mice transplanted with 40‐week‐old bone marrow exhibited increased mid‐shaft femoral cortical bone mass and thickness. Intensive bone marrow regeneration mirrors hematopoietic development in that erythro‐myeloid progenitors (EMPs) first expand to support blood production before definitive hematopoietic stem cell (HSC) production. We hypothesized that reduced HSC capacity of 40‐week‐old bone marrow and compensatory expansion of EMPs may facilitate gains in cortical bone. Flow cytometry analyses revealed greater EMP to HSC ratios when mice were reconstituted with increasing percentages of middle‐aged bone marrow. To identify cell types mediating these effects, we performed comparative scRNA‐Seq analyses and identified CD11B ⁺ CD36 ⁺ myeloid cells exhibiting enriched expression of bone anabolic cytokines. Elevated levels of Wnt ligands, especially Wnt6, characterized these cells. In lineage tracing assays, CD11B ⁺ CD36 ⁺ cells were donor‐derived myeloid cells. In functional assays, we demonstrate that soluble factors produced by CD11B ⁺ CD36 ⁺ cells enhance osteogenesis. Moreover, CD11B/CD36/Wnt6 exquisitely mark anabolic macrophages within human bone marrow. These findings reveal a myeloid population present during midlife that enhances cortical bone.

Disruption of circadian rhythms predisposes shift workers to many chronic conditions, including osteoporosis. However, the effects of disrupted circadian rhythms on bone remodeling remain largely unknown. Here, we show that one of the core circadian regulators PER1 inhibits osteoclastogenesis by upregulating genes involved in inflammation. Conditional knockout (KO) of Per1 in osteoclasts and related cells resulted in decreased bone mass in the femurs of mice, along with increased osteoclasts and decreased osteoblasts. Osteoclastogenesis was also promoted by Per1 depletion in vitro with 16 downregulated inflammatory genes. Seven of these genes were known to promote or inhibit osteoclastogenesis depending on the stage of osteoclastogenesis and the presence or absence of infection. Knockdown of Nlrp3, Tlr8, or Tlr9 in the group of genes promoted osteoclastogenesis, mirroring the effects of Per1 KO and offering a mechanistic explanation for the Per1-mediated inhibition of osteoclastogenesis. These results were not observed following the KO of a paralog Per2. Per1 KO mice maintain general circadian rhythms, unlike arrhythmic Per1;Per2 double KO mice. This gives credence to Per1 as a selective target for therapeutic interventions without disrupting the circadian rhythms. This study uncovered a molecular link between a circadian regulator and osteoclastogenesis in the broader context of inflammatory reactions. Our findings may be mechanistically relevant to inflammatory bone diseases influenced by circadian rhythms, such as rheumatoid arthritis and osteoarthritis, as well as other bone diseases predisposed by chronic circadian disruption.

Bone remodeling is influenced by circadian rhythms as demonstrated by global gene expression patterns and the phenotypes of knockout mice of circadian regulators. However, the direct connections between circadian regulators and specific bone genes remain unclear. We previously found that a conditional knockout of Per1 , a central circadian regulator, in osteoclasts increased osteoclastogenesis and decreased bone mass, whereas Per2 knockout did not cause these phenotypes. Here, we extended the research to Per1 ; Per2 conditional double knockout mice and observed different phenotypes and underlying mechanisms from individual knockouts. In contrast to Per1 knockout, the double knockout decreased osteoclastogenesis and increased bone mass. This was accompanied by downregulation of genes involved in innate immunity, including several known promoters and inhibitors of osteoclastogenesis. Chromatin immunoprecipitation and reporter assay suggested direct regulation of some of them by PER proteins. These results indicate that PER1 and PER2 are critical regulators of osteoclastogenesis through balancing multiple competing activities of osteoclastogenesis, rather than acting as simple promoters or inhibitors of osteoclastogenesis. Regulation of innate immunity genes by circadian regulators is widely observed across other monocyte/macrophage lineages. Our results extend this common mechanism to osteoclasts with therapeutic potential to treat inflammatory bone diseases. Lay summary: Although circadian rhythms regulate bone remodeling, direct links between circadian regulators and bone genes remain unclear. We previously demonstrated that depletion of Per1 , a main circadian regulator, downregulates immunological genes, increases the number of osteoclasts, the main bone resorbing cells, and decreases bone mass in mice. Here, we showed the opposite effects with double depletion of Per1 and Per2 and identified a new set of downregulated immunological genes that promote or inhibit osteoclastogenesis. This study connects circadian rhythms to bone resorption through immunological genes with opposing activities in osteoclastogenesis, supporting therapeutic interventions targeting circadian regulators to treat inflammatory bone diseases.

Our understanding of the different developmental origins of osteoclast progenitors and their respective roles during homeostatic bone remodeling at different skeletal sites as well as their roles within bone regeneration and degenerative conditions is evolving. In this narrative review article, we summarize what is known about the developmental origins, anatomical sources, and markers of osteoclast progenitors. We touch on how osteoclast progenitors vary during different disease contexts, including periodontitis, rheumatoid arthritis, and osteoarthritis. In addition, we also characterize osteoclast progenitors that contribute to bone healing and define changes observed with advancing age. In this regard, we offer a critical review of gaps within our understanding and opportunities for future development within the field. Because of their diverse nature under different contexts, identifying and characterizing osteoclast progenitors may help to advance condition-specific therapies.

Germline and osteoblast-directed deletion of G protein-gated inwardly rectifying K+ channel 3 (Girk3) was recently shown to increase bone mass after 18 wk of age in male mice. Here, we show that germline Girk3 deletion also increases trabecular and cortical bone mass and increases the mechanical strength of the femur in female mice after 18 wk of age. Unlike male mice, however, osteoblast-directed Girk3 deletion using 2.3 kb-Col1a1-Cre does not increase bone mass in adult female mice. To discover mechanisms underlying high bone mass in female Girk3−/− mice, bulk RNA-sequencing was performed on 2-d-old calvarial bone, revealing lower expression of proinflammatory cytokines such as IL-1β and IL-6 in Girk3−/− mice. Accordingly, cytokines and chemokines are largely suppressed in the circulation of adult Girk3−/− mice compared to WT littermates. The cytokines GM-CSF, IL-1β, IL-2, and IL-9 are reduced in the serum of both male and female Girk3−/− mice, while eotaxin, IFNγ, MIP-1α, and others are sexually dimorphic. Histomorphometry reveals that osteoclast activity is modestly reduced in Girk3−/− bone, which is supported by in vitro osteoclast resorption assays. However, deletion of Girk3 in myeloid-lineage cells with LysM-Cre is not sufficient to recapitulate high bone mass in either male or female mice. Moreover, female Girk3−/− mice are not protected from ovariectomy-induced bone loss. Finally, single-cell screening using cytometry by time-of-flight in the BM revealed no differences in immune cell abundances due to global Girk3 deletion. Taken together, while Girk3 regulates inflammatory cytokine expression in the bone and serum, deletion of Girk3 in myeloid-lineage cells does not affect bone mass.

Disruption of circadian rhythms predisposes shift workers to many chronic conditions, including osteoporosis. However, the effects of disrupted circadian rhythms on bone remodeling remain largely unknown. Here, we show that one of the core circadian regulators PER1 inhibits osteoclastogenesis by upregulating genes involved in inflammation. The conditional knockout of Per1 in osteoclasts and related cells resulted in decreased bone mass in the femurs of mice, along with increased osteoclasts and decreased osteoblasts. Osteoclastogenesis was also promoted by Per1 depletion in vitro with 16 downregulated inflammatory genes. Seven of these genes were known to promote or inhibit osteoclastogenesis depending on the stage of osteoclastogenesis and the presence or absence of infection. Knockdown of Nlrp3 , Tlr8 , or Tlr9 in the group of genes promoted osteoclastogenesis, mirroring the effects of Per1 knockout and offering a mechanistic explanation for the Per1 -mediated inhibition of osteoclastogenesis. These results were not observed following the knockout of a paralog Per2 . Per1 knockout mice maintain general circadian rhythms, unlike arrhythmic Per1;Per2 double knockout mice. This gives credence to Per1 as a selective target for therapeutic interventions without disrupting the circadian rhythms. This study uncovered a molecular link between a circadian regulator and osteoclastogenesis in the broader context of inflammatory reactions. Our findings may be mechanistically relevant to inflammatory bone diseases influenced by circadian rhythms, such as rheumatoid arthritis and osteoarthritis, as well as other bone diseases predisposed by chronic circadian disruption. Lay summary: Disruption of circadian rhythms is a risk factor for many chronic diseases, including osteoporosis, among shift workers; however, underlying mechanisms remain largely unknown. In this study, the depletion of Per1 , a core circadian regulator, resulted in an increase in bone-resorbing osteoclasts and a decrease in bone mass in mice. These changes were accompanied by a decrease in the expression of inflammatory genes that promoted the formation of osteoclasts upon depletion. This study revealed a link between circadian rhythms and bone loss, with inflammatory genes serving as mediators, which could provide a basis for future therapeutic interventions.

Background/Objectives: In contrast to endochondral bone healing, the process of intramembranous bone regeneration is poorly understood. This limits our ability to repair and regenerate the craniofacial skeleton to either correct deformity or optimally heal tissues following injury. While there are several preclinical models of intramembranous regeneration within the craniofacial skeleton, some are not load bearing and others are technically challenging. The goal of this pilot study is therefore to describe a simple method for induction of cortical defects within the mandible that does not involve compounding injury to the surrounding tissues. Methods: Single cortex defects were generated in the mandible body of 8-week-old male and female mice. The extent of bone regeneration within the defect was characterized at days 0, 3, 14, and 28 following defect generation via micro-computed tomography and histology. Conclusions: Observed healing was predictable and reproducible and resulted in intramembranous bone formation. This model will help aid the understanding of intramembranous bone healing in load bearing bones (e.g., mandible) within the craniofacial skeleton