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1. Germ‐Free C57BL /6 Mice Have Increased Bone Mass and Altered Matrix Properties but Not Decreased Bone Fracture Resistance

   https://doi.org/10.1002/jbmr.4835  

   Vahidi, Ghazal ; Moody, Maya ; Welhaven, Hope D. ; Davidson, Leah ; Rezaee, Taraneh ; Behzad, Ramina ; Karim, Lamya ; Roggenbeck, Barbara A. ; Walk, Seth T. ; Martin, Stephen A. ; et al ( June 2023 , Journal of Bone and Mineral Research)

   The gut microbiome impacts bone mass, which implies a disruption to bone homeostasis. However, it is not yet clear how the gut microbiome affects the regulation of bone mass and bone quality. We hypothesized that germ‐free (GF) mice have increased bone mass and decreased bone toughness compared with conventionally housed mice. We tested this hypothesis using adult (20‐ to 21‐week‐old) C57BL/6J GF and conventionally raised female and male mice (n = 6–10/group). Trabecular microarchitecture and cortical geometry were measured from micro–CT of the femur distal metaphysis and cortical midshaft. Whole‐femur strength and estimated material properties were measured using three‐point bending and notched fracture toughness. Bone matrix properties were measured for the cortical femur by quantitative back‐scattered electron imaging and nanoindentation, and, for the humerus, by Raman spectroscopy and fluorescent advanced glycation end product (fAGE) assay. Shifts in cortical tissue metabolism were measured from the contralateral humerus. GF mice had reduced bone resorption, increased trabecular bone microarchitecture, increased tissue strength and decreased whole‐bone strength that was not explained by differences in bone size, increased tissue mineralization and fAGEs, and altered collagen structure that did not decrease fracture toughness. We observed several sex differences in GF mice, most notably for bone tissue metabolism. Malemore »GF mice had a greater signature of amino acid metabolism, and female GF mice had a greater signature of lipid metabolism, exceeding the metabolic sex differences of the conventional mice. Together, these data demonstrate that the GF state in C57BL/6J mice alters bone mass and matrix properties but does not decrease bone fracture resistance. © 2023 The Authors.Journal of Bone and Mineral Researchpublished by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

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2. Relations Between Bone Quantity, Microarchitecture, and Collagen Cross‐links on Mechanics Following In Vivo Irradiation in Mice

   https://doi.org/10.1002/jbm4.10545  

   Pendleton, Megan M. ; Emerzian, Shannon R. ; Sadoughi, Saghi ; Li, Alfred ; Liu, Jennifer W. ; Tang, Simon Y. ; O'Connell, Grace D. ; Sibonga, Jean D. ; Alwood, Joshua S. ; Keaveny, Tony M. ( September 2021 , JBMR Plus)

   Humans are exposed to ionizing radiation via spaceflight or cancer radiotherapy, and exposure from radiotherapy is known to increase risk of skeletal fractures. Although irradiation can reduce trabecular bone mass, alter trabecular microarchitecture, and increase collagen cross‐linking, the relative contributions of these effects to any loss of mechanical integrity remain unclear. To provide insight, while addressing both the monotonic strength and cyclic‐loading fatigue life, we conducted total‐body, acute, gamma‐irradiation experiments on skeletally mature (17‐week‐old) C57BL/6J male mice (n = 84). Mice were administered doses of either 0 Gy (sham), 1 Gy (motivated by cumulative exposures from a Mars mission), or 5 Gy (motivated by clinical therapy regimens) with retrieval of the lumbar vertebrae at either a short‐term (11‐day) or long‐term (12‐week) time point after exposure. Micro‐computed tomography was used to assess trabecular and cortical quantity and architecture, biochemical composition assays were used to assess collagen quality, and mechanical testing was performed to evaluate vertebral compressive strength and fatigue life. At 11 days post‐exposure, 5 Gy irradiation significantly reduced trabecular mass (p < 0.001), altered microarchitecture (eg, connectivity densityp < 0.001), and increased collagen cross‐links (p < 0.001). Despite these changes, vertebral strength (p = 0.745) and fatigue life (p = 0.332) remained unaltered. At 12 weeks after 5 Gy exposure, the trends in trabecular bone persisted; in addition,more »regardless of irradiation, cortical thickness (p < 0.01) and fatigue life (p < 0.01) decreased. These results demonstrate that the highly significant effects of 5 Gy total‐body irradiation on the trabecular bone morphology and collagen cross‐links did not translate into detectable effects on vertebral mechanics. The only mechanical deficits observed were associated with aging. Together, these vertebral results suggest that for spaceflight, irradiation alone will likely not alter failure properties, and for radiotherapy, more investigations that include post‐exposure time as a positive control and testing of both failure modalities are needed to determine the cause of increased fracture risk. © 2021 The Authors.JBMR Pluspublished by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

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3. Loss of Fgfr1 and Fgfr2 in Scleraxis‐lineage cells leads to enlarged bone eminences and attachment cell death

   https://doi.org/10.1002/dvdy.600  

   Wernlé, Kendra K. ; Sonnenfelt, Michael A. ; Leek, Connor C. ; Ganji, Elahe ; Sullivan, Anna Lia ; Offutt, Claudia ; Shuff, Jordan ; Ornitz, David M. ; Killian, Megan L. ( May 2023 , Developmental Dynamics)

   Tendons and ligaments attach to bone are essential for joint mobility and stability in vertebrates. Tendon and ligament attachments (ie, entheses) are found at bony protrusions (ie, eminences), and the shape and size of these protrusions depend on both mechanical forces and cellular cues during growth. Tendon eminences also contribute to mechanical leverage for skeletal muscle. Fibroblast growth factor receptor (FGFR) signaling plays a critical role in bone development, andFgfr1andFgfr2are highly expressed in the perichondrium and periosteum of bone where entheses can be found.

   We used transgenic mice for combinatorial knockout ofFgfr1and/orFgfr2in tendon/attachment progenitors (ScxCre) and measured eminence size and shape. Conditional deletion of both, but not individual,Fgfr1andFgfr2inScxprogenitors led to enlarged eminences in the postnatal skeleton and shortening of long bones. In addition,Fgfr1/Fgfr2double conditional knockout mice had more variation collagen fibril size in tendon, decreased tibial slope, and increased cell death at ligament attachments. These findings identify a role for FGFR signaling in regulating growth and maintenance of tendon/ligament attachments and the size and shape of bony eminences.
4. Deletion of Fibroblast growth factor 9 globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments

   https://doi.org/10.1002/dvdy.383  

   Leek, Connor C. ; Soulas, Jaclyn M. ; Bhattacharya, Iman ; Ganji, Elahe ; Locke, Ryan C. ; Smith, Megan C. ; Bhavsar, Jaysheel D. ; Polson, Shawn W. ; Ornitz, David M. ; Killian, Megan L. ( June 2021 , Developmental Dynamics)

   The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon‐bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion offibroblast growth factor 9(Fgf9), the DT size is notably enlarged. In this study, we explored the tissue‐specific regulation of DT size using both global and targeted deletion ofFgf9.

   We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss ofFgf9. Loss ofFgf9during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development inFgf9^nullattachments. We then showed that targeted deletion ofFgf9in skeletal muscle leads to postnatal enlargement of the DT.

   Taken together, we discovered thatFgf9may play an influential role in muscle‐bone cross‐talk during embryonic and postnatal development.
5. Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait

   https://doi.org/10.1111/joa.13120  

   Saers, Jaap P. P. ; Ryan, Timothy M. ; Stock, Jay T. ( November 2019 , Journal of Anatomy)

   Trabecular bone structure in adulthood is a product of a process of modelling during ontogeny and remodelling throughout life. Insight into ontogeny is essential to understand the functional significance of trabecular bone structural variation observed in adults. The complex shape and loading of the human calcaneus provides a natural experiment to test the relationship between trabecular morphology and locomotor development. We investigated the relationship between calcaneal trabecular bone structure and predicted changes in loading related to development of gait and body size in growing children. We sampled three main trabecular regions of the calcanei using micro‐computed tomography scans of 35 individuals aged between neonate to adult from the Norris Farms #36 site (1300 AD, USA) and from Cambridge (1200–1500 AD, UK). Trabecular properties were calculated in volumes of interest placed beneath the calcaneocuboid joint, plantar ligaments, and posterior talar facet. At birth, thin trabecular struts are arranged in a dense and relatively isotropic structure. Bone volume fraction strongly decreases in the first year of life, whereas anisotropy and mean trabecular thickness increase. Dorsal compressive trabecular bands appear around the onset of bipedal walking, although plantar tensile bands develop prior to predicted propulsive toe‐off. Bone volume fraction and anisotropy increase untilmore »the age of 8, when gait has largely matured. Connectivity density gradually reduces, whereas trabeculae gradually thicken from birth until adulthood. This study demonstrates that three different regions of the calcaneus develop into distinct adult morphologies through varying developmental trajectories. These results are similar to previous reports of ontogeny in human long bones and are suggestive of a relationship between the mechanical environment and trabecular bone architecture in the human calcaneus during growth. However, controlled experiments combined with more detailed biomechanical models of gait maturation are necessary to establish skeletal markers linking growth to loading. This has the potential to be a novel source of information for understanding loading levels, activity patterns, and perhaps life history in the fossil record.

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