Genetic variation influences the skeletal response to hindlimb unloading in the eight founder strains of the diversity outbred mouse population.

During disuse, mechanical unloading causes extensive bone loss, decreasing bone volume and strength. Variations in bone mass and risk of osteoporosis are influenced by genetics; however, it remains unclear how genetic variation affects the skeletal response to unloading. We previously found that genetic variation affects the musculoskeletal response to 3 weeks of immobilization in the 8 Jackson Laboratory J:DO founder strains: C57Bl/6J, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ. Hindlimb unloading (HLU) is the best model for simulating local and systemic contributors of disuse and therefore may have a greater impact on bones than immobilization. We hypothesized that genetic variation would affect the response to HLU across the eight founder strains. Mice of each founder strain were placed in HLU for 3 weeks, and the femurs and tibias were analyzed. There were significant HLU and mouse strain interactions on body weight, femur trabecular BV/TV, and femur ultimate force. This indicates that unloading only caused significant catabolic effects in some mouse strains. C57BL/6 J mice were most affected by unloading while other strains were more protected. There were significant HLU and mouse strain interactions on gene expression of genes encoding bone metabolism genes in the tibia. This indicates that unloading only caused significant effects on bone metabolism genes in some mouse strains. Different mouse strains respond to HLU differently, and this can be explained by genetic differences. These results suggest the outbred J:DO mice will be a powerful model for examining the effects of genetics on the skeletal response to HLU.

Genetic diversity modulates the physical and transcriptomic response of skeletal muscle to simulated microgravity in male mice.

Developments in long-term space exploration necessitate advancements in countermeasures against microgravity-induced skeletal muscle loss. Astronaut data shows considerable variation in muscle loss in response to microgravity. Previous experiments suggest that genetic background influences the skeletal muscle response to unloading, but no in-depth analysis of genetic expression has been performed. Here, we placed eight, male, inbred founder strains of the diversity outbred mice (129S1/SvImJ, A/J, C57BL/6J, CAST/EiJ, NOD/ShiLtJ, NZO/HILtJ, PWK/PhJ, and WSB/EiJ) in simulated microgravity (SM) via hindlimb unloading for three weeks. Body weight, muscle morphology, muscle strength, protein synthesis marker expression, and RNA expression were collected. A/J and CAST/EiJ mice were most susceptible to SM-induced muscle loss, whereas NOD/ShiLtJ mice were the most protected. In response to SM, A/J and CAST/EiJ mice experienced reductions in body weight, muscle mass, muscle volume, and muscle cross-sectional area. A/J mice had the highest number of differentially expressed genes (68) and associated gene ontologies (328). Downregulation of immunological gene ontologies and genes encoding anabolic immune factors suggest that immune dysregulation contributes to the response of A/J mice to SM. Several muscle properties showed significant interactions between SM and mouse strain and a high degree of heritability. These data imply that genetic background plays a role in the degree of muscle loss in SM and that more individualized programs should be developed for astronauts to protect their skeletal muscles against microgravity on long-term missions.

Connexin 43 and cell culture substrate differentially regulate OCY454 osteocytic differentiation and signaling to primary bone cells.

Connexin 43 (Cx43), the predominate gap junction protein in bone, is essential for intercellular communication and skeletal homeostasis. Previous work suggests that osteocyte-specific deletion of Cx43 leads to increased bone formation and resorption; however, the cell-autonomous role of osteocytic Cx43 in promoting increased bone remodeling is unknown. Recent studies using three-dimensional (3D) culture substrates in OCY454 cells suggest that 3D cultures may offer increased bone remodeling factor expression and secretion, such as sclerostin and receptor activator of nuclear factor-κB ligand (RANKL). In this study, we compared culturing OCY454 osteocytes on 3D Alvetex scaffolds with traditional 2D tissue culture, both with [wild-type (WT)] and without Cx43 (Cx43 KO). Conditioned media from OCY454 cell cultures were used to determine soluble signaling to differentiate primary bone marrow cells into osteoblasts and osteoclasts. OCY454 cells cultured on 3D portrayed a mature osteocytic phenotype, relative to cells on 2D, shown by increased osteocytic gene expression and reduced cell proliferation. In contrast, OCY454 differentiation based on these same markers was not affected by Cx43 deficiency in 3D. Interestingly, increased sclerostin secretion was found in 3D cultured WT cells compared with that of Cx43 KO cells. Conditioned media from Cx43 KO cells promoted increased osteoblastogenesis and osteoclastogenesis, with maximal effects from 3D cultured Cx43 KO cells. These results suggest that Cx43 deficiency promotes increased bone remodeling in a cell-autonomous manner with minimal changes in osteocyte differentiation. Finally, 3D cultures appear better suited to study mechanisms from Cx43-deficient OCY454 osteocytes in vitro due to their ability to promote osteocyte differentiation, limit proliferation, and increase bone remodeling factor secretion.NEW & NOTEWORTHY 3D cell culture of OCY454 cells promoted increased differentiation compared with traditional 2D culture. Although Cx43 deficiency did not affect OCY454 differentiation, it resulted in increased signaling, promoting osteoblastogenesis and osteoclastogenesis. Our results suggest that Cx43 deficiency promotes increased bone remodeling in a cell-autonomous manner with minimal changes in osteocyte differentiation. Also, 3D cultures appear better suited to study mechanisms in Cx43-deficient OCY454 osteocytes.

Connexin 43 and Cell Culture Substrate Differentially Regulate OCY454 Osteocytic Differentiation and Signaling to Primary Bone Cells.

Connexin 43 (Cx43), the predominate gap junction protein in bone, is essential for intercellular communication and skeletal homeostasis. Previous work suggests osteocyte-specific deletion of Cx43 leads to increased bone formation and resorption, however the cell-autonomous role of osteocytic Cx43 in promoting increased bone remodeling is unknown. Recent studies using 3D culture substrates in OCY454 cells suggest 3D cultures may offer increased bone remodeling factor expression and secretion, such as sclerostin and RANKL. In this study, we compared culturing OCY454 osteocytes on 3D Alvetex scaffolds to traditional 2D tissue culture, both with (WT) and without Cx43 (Cx43 KO). Conditioned media from OCY454 cell cultures was used to determine soluble signaling to differentiate primary bone marrow stromal cells into osteoblasts and osteoclasts. OCY454 cells cultured on 3D portrayed a mature osteocytic phenotype, relative to cells on 2D, shown by increased osteocytic gene expression and reduced cell proliferation. In contrast, OCY454 differentiation based on these same markers was not affected by Cx43 deficiency in 3D. Interestingly, increased sclerostin secretion was found in 3D cultured WT cells compared to Cx43 KO cells. Conditioned media from Cx43 KO cells promoted increased osteoblastogenesis and increased osteoclastogenesis, with maximal effects from 3D cultured Cx43 KO cells. These results suggest Cx43 deficiency promotes increased bone remodeling in a cell autonomous manner with minimal changes in osteocyte differentiation. Finally, 3D cultures appear better suited to study mechanisms from Cx43-deficient OCY454 osteocytes in vitro due to their ability to promote osteocyte differentiation, limit proliferation, and increase bone remodeling factor secretion. New and Noteworthy: 3D cell culture of OCY454 cells promoted increased differentiation compared to traditional 2D culture. While Cx43 deficiency did not affect OCY454 differentiation, it resulted in increased signaling, promoting osteoblastogenesis and osteoclastogenesis. Our results suggest Cx43 deficiency promotes increased bone remodeling in a cell autonomous manner with minimal changes in osteocyte differentiation. Also, 3D cultures appear better suited to study mechanisms in Cx43-deficient OCY454 osteocytes.

Similarities Between Disuse and Age-Induced Bone Loss.

Disuse and aging are known risk factors associated with low bone mass and quality deterioration, resulting in increased fracture risk. Indeed, current and emerging evidence implicate a large number of shared skeletal manifestations between disuse and aging scenarios. This review provides a detailed overview of current preclinical models of musculoskeletal disuse and the clinical scenarios they seek to recapitulate. We also explore and summarize the major similarities between bone loss after extreme disuse and advanced aging at multiple length scales, including at the organ/tissue, cellular, and molecular level. Specifically, shared structural and material alterations of bone loss are presented between disuse and aging, including preferential loss of bone at cancellous sites, cortical thinning, and loss of bone strength due to enhanced fragility. At the cellular level bone loss is accompanied, during disuse and aging, by increased bone resorption, decreased formation, and enhanced adipogenesis due to altered gap junction intercellular communication, WNT/ß-catenin and RANKL/OPG signaling. Major differences between extreme short-term disuse and aging are discussed, including anatomical specificity, differences in bone turnover rates, periosteal modeling, and the influence of subject sex and genetic variability. The examination also identifies potential shared mechanisms underlying bone loss in aging and disuse that warrant further study such as collagen cross-linking, advanced glycation end products/receptor for advanced glycation end products (AGE-RAGE) signaling, reactive oxygen species (ROS) and nuclear factor κB (NF-κB) signaling, cellular senescence, and altered lacunar-canalicular connectivity (mechanosensation). Understanding the shared structural alterations, changes in bone cell function, and molecular mechanisms common to both extreme disuse and aging are paramount to discovering therapies to combat both age-related and disuse-induced osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Update on the effects of microgravity on the musculoskeletal system.

With the reignited push for manned spaceflight and the development of companies focused on commercializing spaceflight, increased human ventures into space are inevitable. However, this venture would not be without risk. The lower gravitational force, known as microgravity, that would be experienced during spaceflight significantly disrupts many physiological systems. One of the most notably affected systems is the musculoskeletal system, where exposure to microgravity causes both bone and skeletal muscle loss, both of which have significant clinical implications. In this review, we focus on recent advancements in our understanding of how exposure to microgravity affects the musculoskeletal system. We will focus on the catabolic effects microgravity exposure has on both bone and skeletal muscle cells, as well as their respective progenitor stem cells. Additionally, we report on the mechanisms that underlie bone and muscle tissue loss resulting from exposure to microgravity and then discuss current countermeasures being evaluated. We reveal the gaps in the current knowledge and expound upon how current research is filling these gaps while also identifying new avenues of study as we continue to pursue manned spaceflight.