What’s New in Musculoskeletal Basic Science

Musculoskeletal basic science provides a foundation for clinical knowledge while fostering innovation in the management of orthopaedic conditions. In the past year, several outstanding articles have been published that advance our understanding of neural system function and its crosstalk with immunity in tissue healing and regulation of bone homeostasis, revealing new roles of mechanotransduction in bone and critically assessing vitamin D supplementation in schoolchildren (6 to 13 years of age). Novel research was generated on maintaining muscle strength and function, establishing the role of synovium in chronic inflammatory joint pathology, and the effects of aging on the musculoskeletal system. These publications provide new insight into the physiologic and pathologic processes underlying orthopaedic conditions and critically assess routine clinical practices with high-level evidence. All 20 musculoskeletal science articles chosen for this review were published in extremely prestigious, high-impact-factor journals. Somatic and Autonomic Innervation Physical activity requires precisely coordinated participation of the somatic nervous system controlling skeletal muscles and the autonomic nervous system enabling synchronous responses from the inner organs. Although somatic and autonomic innervations have separate anatomic representations, the nature of their coordinated interactions remains elusive. Zhang et al.1 discovered that spinal-projecting neurons in the rostral ventromedial medulla (rVMM) simultaneously coregulate both somatic motor and autonomic sympathetic activity in different bodily functional states. Using adeno-associated virus (AAV) retrograde tracing and optogenetic mapping, spinal-projecting neurons located in the gigantocellular reticular nucleus alpha (GiA) and ventral part (GiV) of the rVMM exhibited axon collaterals connecting the motor and sympathetic spinal neurons. These spinal-projecting neurons exhibited excitatory or inhibitory regulation of skeletal muscle tone concurrently with sympathetic tone (blood pressure, heart rate). Active states such as exercise exhibited spinal-projecting neuron excitation causing high motor and sympathetic tones, whereas quiescent states such as sleep showed spinal-projecting neuron inhibition yielding skeletal muscle atonia and sympathetic hypoactivity. This study considerably advances our knowledge on neuronal circuitry governing coordinated motor and sympathetic responses during different functional states. Neuroimmune Modulation of Tissue Healing Nociceptive sensory neurons are important immunoregulators exerting protective or damaging effects via neurogenic inflammation. The neuroimmune interactions during tissue healing following an injury are poorly understood. Lu et al.2 demonstrated that ablation of the Nav1.8 voltage-gated sodium channels in nociceptive neurons in the dorsal root ganglia (DRG) impaired healing of full-thickness skin wounds and regeneration of volumetric muscle loss. Transgenic Nav1.8cre/Rosa26DTA mice lacking Nav1.8 exhibit impaired sensory axon ingrowth into granulation tissue, which coincides with local downregulation of calcitonin gene-related peptide (CGRP). CGRP released by the Nav1.8+ neurons enables skin and muscle healing by interacting with CD11b+ myeloid cells via receptor activity-modifying protein 1 (RAMP1). CGRP upregulates thrombospondin-1 (TSP-1) in neutrophils and monocytes/macrophages to inhibit their recruitment and enhance efferocytosis and M2 polarization. Mice with diabetic neuropathy (Leprdb/db), similar to Nav1.8-deficient mice, exhibit reduced CGRP expression in sensory neurons and impaired tissue-healing, which can be mitigated with exogenous local CGRP delivery. The study demonstrated that tissue-healing involves complex CGRP-mediated neuroimmune interactions and suggested potential new treatment options. Regulation of Bone Homeostasis A demand for high calcium levels in breast milk during lactation causes abrupt bone loss. In mechanistic studies, Babey et al.3 identified cellular communication network factor 3 (CCN3) as a potent osteoanabolic hormone released in the early postpartum period throughout lactation. CCN3 is secreted from the kisspeptin-positive neurons of the arcuate nucleus (ARCKISS1+) in the brain. Parabiosis of female wild-type mice and female mice overexpressing CCN3 (Esr1Nkx2-1-cre) revealed the same high bone mass. Further, human stem cells exposed to CCN3 showed enhanced osteogenic differentiation and matrix mineralization, regardless of cell donor age and sex. Systemic CCN3 delivery augmented the bone phenotype in ovariectomized mice and reversed the skeletal sequelae of a chronic high-fat diet known to compromise ARCKISS1+ neurons. Finally, localized CCN3 delivery enhanced femoral osteotomy healing in aged female and male mice. Knocking down Ccn3 in the arcuate nucleus leads to excessive bone loss during lactation, further compounded by a low-calcium diet. The study identified maternal CCN3 as a potent brain-derived, osteoprotective hormone released during lactation and delineated its osteoanabolic properties. Osteoclastogenesis controls bone turnover, although several pathways regulating the process remain unclear. Colocalization of osteoclast precursors with eosinophils in bone marrow suggests their interaction. Andreev et al.4 discovered that eosinophils regulate osteoclast activity in both normal and pathologic bone conditions. Eosinophil-deficient ΔdblGATA knockout mice exhibit a marked reduction in bone mass due to increased osteoclastogenesis. Conversely, eosinophilia in transgenic interleukin (IL)-5 mice strongly inhibits osteoclast differentiation and activity by altering their transcriptional profile. Eosinophil peroxidase (EPX) was identified as a regulator of osteoclast formation and function by downregulating mitochondrial reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) in osteoclast precursors. EPX treatment ameliorated bone loss induced by inflammatory arthritis and estrogen deprivation. In humans, the number and the activity of eosinophils highly correlated with bone mass in healthy participants and patients with rheumatoid arthritis. This study revealed a novel function of eosinophils to regulate bone homeostasis and underscored the role of innate immunity in the process. Mechanotransduction in Development, Health, and Disease Postnatally, mechanical signals dynamically regulate skeletal homeostasis and its adaptive changes; however, the role of mechanical cues in embryonic skeletal development is largely unknown. Collins et al.5 established the essential role of Hippo signaling and its downstream transcription factors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) in fetal murine osteoblasts and their coordinated interactions with endothelial cells during embryonic endochondral ossification to form bone. In transgenic mice, selective YAP/TAZ deletion in osteogenic osterix (Osx)-expressing cells causes loss in mechanoresponsivness due to alteration of gene expression required for cell cytoskeleton rearrangement to sense mechanical stimuli. YAP/TAZ spatially couple Osx+ osteoblast precursors with endothelial cells of sprouting vessels to initiate cartilage hypertrophy and remodeling at the endochondral ossification centers, thereby mechanoregulating bone formation in murine developing limbs. Single-cell transcriptomics revealed a specific population of vessel-associated osteoblast precursors, and YAP/TAZ deletion in these cells downregulated C-X-C motif chemokine 12, which mediates vascular morphogenesis. Ex vivo bioreactor-based mechanical stimulation of explanted embryonic (E15.5) mouse hindlimbs increased mineralization, and YAP/TAZ deletion abrogated load-induced osteogenesis at the ossification sites. The study reveals a key role of YAP/TAZ in mechanosignaling during embryonic bone development, with potential implications for skeletal pathologies associated with fetal akinesia or hypokinesia and uterine crowding. Daylight is the primary entrainer of circadian rhythms, pacing and resetting the neuronal signals in the brain. Food intake, ambient temperature, and stress can uncouple circadian rhythms at autonomous tissue and organ levels. In a murine mechanistic study, Dudek et al.6 demonstrated that cyclic mechanical loading and resultant tissue osmolarity changes can reset the circadian rhythm phase and amplitude in femoral head articular cartilage and intervertebral disc devoid of innervation and vascularity. Using treadmill exercise in PER2::Luc reporter mice, they established that hyperosmolarity alone can effectively shift and reset the internal clock in young and aging skeletal phenotypes by modulating the expression of specific circadian rhythm genes. Transcriptomic and biochemical analyses revealed the PLD2-mTORC2-AKT-GSK3β molecular pathway as a convergent mechanism mediating both the loading-induced and hyperosmolarity-induced clock entrainments. These results demonstrate a key role of cartilage mechanoresponsiveness in synchronizing skeletal circadian rhythms and suggest cartilage osmolar intervention options. Altered mechanoresponsiveness was implicated in bone fragility in type-2 diabetes mellitus due to detrimental effects of a high-glucose and high-fat diet on osteocytes. Shao et al.7 demonstrated that unilateral in vivo tibial cyclic loading (1,200 cycles per day for 2 weeks) fails to improve bone mass, microarchitecture, and strength in genetically spontaneous (KK-Ay) and high-fat-diet combined with streptozotocin (HFD/STZ)-induced mice with type-2 diabetes mellitus. The lack of bone mechanoresponsiveness was directly associated with attenuated Ca2+ oscillatory dynamics in osteocytes mediated by a reduced expression of the SERCA2 pump. This process involves peroxisome proliferator-activated receptor α (PPARα), leading to fewer and weaker Ca2+ spikes with prolonged Ca2+ uptake in osteocytes with the type-2 diabetes mellitus phenotype. The loading-dependent Ca2+ oscillations cause immediate actin network contractions, thereby modulating osteocyte secretory activities (RANKL, OPG, SOST). Conversely, treatment with istaroxime, a SERCA2 agonist, improves bone mechanoresponsiveness by rescuing osteocyte Ca2+ dynamics in both type-2 diabetes mellitus models. Similarly, conditional osteocyte-specific SERCA2 overexpression (SERCA2flox/flox; DMP1-Cre) mitigated the type-2 diabetes mellitus-related suppression of bone mechanoresponsiveness. The study provided a mechanistic insight into compromised bone mechanoresponsiveness in type-2 diabetes mellitus and identified osteocyte Ca2+ signatures as druggable targets. Bone and Its Blood Supply Transcortical vessels connect the endosteum with periosteum to permit molecule and cell trafficking between the inner medullary and external bone compartments across the osteocyte-populated cortex. Liao et al.8 revealed a unique role of osteocytes, which, via rich direct dendritic connections with transcortical vessels, transfer mitochondria to the endothelial cells to protect them from oxidative stress, and promote their proliferation and migration, thereby enhancing transcortical vessel formation. The ablation of osteocytes in transgenic (Dmp1Cre-DTAki/wt) mice exhibits substantial reduction in transcortical vessel density and connectivity, resulting from significant downexpression of angiogenic genes (Vegfc, Slit3, Notch3, Notch4). The osteocyte-endothelium transfer of Dendra2-labeled mitochondria was visualized in vitro and in vivo and was required for transcortical vessel formation and functionality. The metabolic effects of the endothelial mitochondria uptake involved induction of D-sphingosine catalyzed by sphingosine kinase 1 (SPHK1). In vivo delivery of osteocyte-derived mitochondria or D-sphingosine (2 mg/kg for 7 days) enhanced intact femoral bone mass, density, and microarchitecture, and augmented diaphyseal defect healing in mice. These findings identify a unique mitochondria transfer regulating transcortical vessel formation and homeostasis. The pathways orchestrating synchronous new vessel formation and osteogenic stem cell recruitment and migration during bone-healing lack thorough understanding. In a dynamic imaging study, Bixel et al.9 employed intravital multiphoton microscopy to longitudinally surveil vascularization in murine calvarial defect healing and determined that initial microvessel (CD31+ Emcn+) formation did not spatially or temporally associate with osteoprogenitors (Osx+ Runx2+) emerging from the periosteum. Second-harmonic generation imaging permitted simultaneous visualization of angiogenesis and osteogenesis in double-transgenic Flk1-GFP and SP7-mCherry reporter mice. The early-formed vessels did not connect with the adjacent uninjured vessels, but rather with the outer periosteal and meningeal vasculature, and the blood flow velocities were highly variable and independent of the vessel diameter. Compared with femoral fracture-healing, the uncoupling of angiogenesis from osteogenesis was unique to calvarial healing. In contrast to femoral fracture-healing, the angiogenesis-related and hypoxia-related genes (Hif1a, Vegfa, Angpt2) were only slightly expressed in calvarial healing, and surprisingly, although they profoundly altered vascular ingrowth, endothelial Notch and vascular endothelial growth factor (VEGF) signaling did affect calvarial healing. The study represented a pioneering effort in sequential intravital imaging of the entire process of calvarial bone repair at a multiscale level and delineated highly dynamic relationships between angiogenesis and osteogenesis. Vitamin D Supplementation in Schoolchildren Vitamin D is a hormone promoting bone mineralization, specifically in the growing skeleton. Severe and prolonged vitamin D deficiency causes rickets and requires vitamin D therapy; however, routine vitamin D supplementation in children lacks evidence. Recently, 2 sister subtrials were reported, both nested within 2 separate main Phase-3 randomized clinical trials (RCTs). In the subtrial by Ganmaa et al.10 involving a secondary analysis of the outcomes of an RCT conducted in Mongolia, 8,851 schoolchildren (6 to 13 years of age) were randomized to receive weekly oral 14,000-IU vitamin D3 (4,418 schoolchildren) or placebo (4,433 schoolchildren) for 3 years and fracture incidence was assessed. At baseline, 31.9% of the schoolchildren were vitamin D-deficient (<25 nmol/L), 63.5% were vitamin D-insufficient (25 to 50 nmol/L), and only 4.4% were vitamin D-sufficient (>50 nmol/L). At the trial end point, schoolchildren in the vitamin D group had normalized vitamin D levels (mean, 72.1 nmol/L), whereas, in the placebo group, vitamin D status remained insufficient (26.1 nmol/L); nevertheless, fracture incidence was similar in the vitamin D group (6.4%) compared with the placebo group (6.1%). Alterations in blood parathyroid hormone (PTH), alkaline phosphatase (ALP), and minerals were noted only in vitamin D-deficient children. The subtrial by Middelkoop et al.11 was nested within the ViDiKids trial and assessed the effect of weekly oral 10,000-IU vitamin D3 compared with placebo for 3 years on bone mineral content (BMC) and serum calcium, vitamin D3, and PTH levels, bone remodeling markers (ALP, C-terminal telopeptide of type I collagen [CTX], procollagen type I N-propeptide [P1NP]), and fracture incidence in 450 Black African schoolchildren (6 to 11 years of age) from South Africa. At baseline, 5.8% of participating schoolchildren were vitamin D-insufficient (<50 nmol/L), and, at the trial end point, serum vitamin D3 was higher in the vitamin D group compared with the placebo group (mean difference, 39.9 nmol/L), whereas serum PTH was lower (mean difference, 0.55 pmol/L). There were no differences in BMC by dual x-ray absorptiometry (DXA) (whole body less the head; lumbar spine), serum calcium, or bone turnover markers. Fracture incidence was very low in both groups (0.93% compared with 1.32%). The null findings of these subtrials indicate that vitamin D supplementation alone does not reduce fractures or improve BMC or serum remodeling markers in schoolchildren despite vitamin D deficiency or insufficiency. 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