Musculoskeletal basic science continues to advance our understanding of pathologies encountered in clinical practice and to drive the development of new therapeutics by identifying suitable targets. Over the past year, substantial contributions were made to our understanding of the chronification of pain, stem cell biology, bone innervation, bone and cartilage remodeling, mechanosensing, skeletal development, and aging. Also, highly debated clinical topics related to routine vitamin D supplementation or the effects of cognition on bone health were addressed with high-quality studies. Because of the large number of pertinent publications and the limited size of this review, only certain groundbreaking studies were selected. Recognizing the inherent subjectivity of article selection, I prioritized articles in which the authors reported original disruptive findings or clarified persistent problems with high-level evidence. No preference was given to the specific musculoskeletal areas covered by the articles beyond their quality and impact on orthopaedic science. Of 20 seminal musculoskeletal science articles highlighted in this update, the vast majority were published in multidisciplinary journals with very high impact factors that customarily disseminate breakthrough discoveries in natural science. Pain Resolution Compared with Pain Chronification Pain inherently accompanies active inflammation; however, it is unclear how pain resolves or becomes chronic when inflammation ceases. Van der Vlist et al.1 mechanistically studied the role of macrophages in the resolution of inflammatory pain. In carrageenan-induced hyperalgesia as a transient pain model in mice, monocyte-derived M2-polarized macrophages infiltrated the corresponding afferent dorsal root ganglia (DRGs) during active pain and left after the pain had resolved. A diphtheria-toxin-regulated selective depletion of monocytes and macrophages failed to achieve timely resolution of pain independently of inflammation, whereas immunosuppressive dexamethasone relieved pain only when administered before but not after inducing inflammation. Intrathecal delivery of monocytes or M2-polarized macrophages to the depleted mice reestablished pain resolution, but the transfer of M1 macrophages did not. The M2 macrophages secreted vesicles laden with mitochondria, which were absorbed by the DRG neurons. Exogenous delivery of vesicles with mitochondria rapidly resolved pain in macrophage-depleted mice. The transfer of mitochondria from macrophages to the DRG neurons is mediated via the CD200 receptor on macrophages and the iSec1/Gm609 ligand on the neurons. This pioneering study identifies a vesicle-mediated mitochondrial transfer between macrophages and the DRG sensory neurons during timely resolution of inflammatory pain, thereby offering a novel approach to chronic pain therapy. Autonomic Innervation of Bone In postganglionic synapses, the parasympathetic innervation is cholinergic and the sympathetic innervation is primarily adrenergic; however, postnatally, some sympathetic neurons switch their neurotransmitter from noradrenaline to acetylcholine. Gadomski et al.2 identified the factors driving this cholinergic switch and its implications for bone development and growth in prenatal and postnatal murine models. Immunofluorescence mapping in ChAT-IRES-cre knock-in mice identified cholinergic nerves in periosteal and perivascular regions of the femoral cortex and marked non-neuronal lining cells near the growth plate. Cholinergic-nerve-deficient Gfrα2−/− knockouts demonstrated a reduction of cholinergic innervation in the femoral cortex, but not in the non-neuronal lining cells. Timed ablation with 6-hydroxydopamine revealed that the cholinergic nerves were, indeed, sympathetic and the cholinergic switch was regulated by interleukin-6 (IL-6). The authors established a neurotrophic dependency between sympathetic cholinergic fibers and osteocytes. Knocking out the cholinergic nerves in Gfrα2−/− mice rendered an osteoporotic phenotype more pronounced in females. Moderate exercise mitigated these changes by an IL-6-dependent expansion of sympathetic cholinergic nerves. The study revealed unique sympathetic cholinergic neuronal interfaces at bone lining cells and osteocytes to regulate bone development, growth, and adaptative remodeling. Traumatic Brain Injury and Bone Healing Traumatic brain injury has been associated with heterotopic ossification and exuberant fracture healing. Previous studies implicated a disruption of the blood-brain barrier and leakage of neuronal or glial cells and/or molecules that create an osteogenic environment but failed to delineate their identity. By following traumatic brain injury clinically in human patients and experimentally in rats, Xia et al.3 demonstrated that extracellular small vesicles enriched with miRNA appear in the circulation. Upon traumatic brain injury, injured neurons, primarily in the hippocampus, release these vesicles, which specifically target osteoprogenitors to stimulate bone formation. Plasma profiling identified miR-328a-3p and miR-150-5p in the vesicles; osteogenesis was promoted by miR-328a-3p downregulating Foxo4 and miR-150-5p downregulating Cbl. Exogenous delivery of vesicles with miR-328a-3p cargo stimulated healing of drill-hole defects in the tibiae of rats without traumatic brain injury that was comparable with animals after traumatic brain injury. Inhibitors of vesicle secretion or of miR-328a-3p or miR-150-5p suppressed the osteogenic differentiation of bone progenitors. This work unveils interactions of the injured hippocampal neurons with bone formation and identifies new mediators in the crosstalk between brain and bone as potential targets for bone-healing enhancement. Skeletal Stem Cell Biology and Mechanosensing The lineage commitment and differentiation of mesenchymal stromal cells (MSCs) is controlled by signaling pathways and transcription factors. Kim et al.4 demonstrated that microtubule-associated serine/threonine-protein kinase 4 (Mast4) serves as an essential mediator of TGF-β and Wnt signal transduction during chondrogenic and osteogenic MSC differentiation. In chondrogenesis, Mast4 regulates Sox9 stability, and its suppression by TGF-β1 leads to increased Sox9 phosphorylation and subsequent degradation, ultimately augmenting chondrogenesis. Enhancing Mast4 by Wnt-mediated inhibition of glycogen synthase kinase-3 beta (GSK3β) promotes β-catenin nuclear localization and Runx2 transcriptional activity, resulting in osteogenesis. Complete Mast4 deficiency in Mast4−/− knockouts produces excessive cartilage and an osteoporotic phenotype, whereas a partial Mast4 depletion in MSCs facilitates cartilage formation and regeneration. These findings highlight the essential roles of Mast4 in determining whether MSC becomes cartilage or bone. Hematopoietic stem cells (HSCs) reside in a perivascular niche of bone marrow in which LepR+ stromal cells and endothelial cells provide them trophic factors. Using lineage-tracing mice, Shen et al.5 demonstrated that the expression of osteolectin (Oln) in LepR+ marrow stroma localizes the Oln+ cells in the periarteriolar and the Oln− cells in the perisinusoidal areas of bone marrow. Unlike the LepR+Oln− cells prone to adipogenesis, LepR+Oln+ cells comprise rapidly dividing osteogenic progenitors that are activated upon fracture and are depleted upon aging. These cells colocalize and functionally link with lymphoid progenitors. The periarteriolar LepR+Oln+ cell niche promotes lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Mechanical stimulation appears essential for maintaining the periarteriolar niche. Hindlimb suspension decreases, whereas running increases, the periarteriolar LepR+Oln+ cells concomitantly with common lymphoid progenitors; this process relies on the mechanosensitive ion channel Piezo1. Conditional deletion of Piezo1 simultaneously depletes LepR+Oln+ cells and common lymphoid progenitors, produces an osteoporotic phenotype, and reduces bacterial clearance after acute infection. These results showed that the periarteriolar niche in adult bone marrow cooperatively regulates osteogenesis and lymphopoiesis and depends on mechanical stimulation. Peng et al.6 described reticulocalbin-2 (RCN2) as a lipolytic factor released upon bone loading during exercise. RCN2 activates lipolysis in bone marrow adipose to supply free fatty acids as a major substrate to generate energy for osteogenesis and lymphopoiesis. Lipid scarcity or impaired utilization suppresses osteogenesis and immune function. Exercise stimulates bone marrow macrophages to increase expression of RCN2, and macrophage mechanosensing is mediated by Piezo1. Deleting the Piezo1 gene in macrophages leads to a reduction of RCN2 expression. Ablation of Rcn2 leads to concomitant impairment of osteogenesis and lymphopoiesis, which cannot be rescued by exercise, whereas exogenous administration of recombinant RCN2 alleviates bone loss and improves immunity despite limb unloading. Furthermore, the expression of RCN2 significantly declines in older animals, and the administration of recombinant RCN2 arrests age and sedentariness-induced changes. Dzamukova et al.7 addressed the mechanisms of cessation and control of bone growth after reaching skeletal maturity. Active bone growth requires highly angiogenic type-H vessels rather than quiescent type-L vessels; type-H vessels are present exclusively at active bone growth sites, such as the ossification front and periosteum. Utilizing laser microdissection of a single capillary with surrounding cells in the ossification front of juvenile and adult mice, the authors established that dentin matrix protein 1 (DMP1) secreted by osteoblasts transforms type-H vessels into type-L vessels to limit bone growth and enhance its mineralization. The process is initiated by an increase in mechanical loading due to body mass accrual after adolescence. The activation of Piezo1 in osteoblasts causes upregulation of Fam20C, which phosphorylates DMP1. A burst secretion of DMP1 from osteoblasts suppresses vascular endothelial growth factor (VEGF) signaling by averting phosphorylation of VEGF receptor 2. These findings are truly relevant for bone growth abnormities associated with aberrant angiogenesis. Skeletal Aging, Senescence, and Senolytics Skeletal aging may be inherent to stem cells and/or involve local or systemic factors of the host. To establish the intrinsic factors compared with extrinsic factors in skeletal stem cells (SSCs) during aging, Ambrosi et al.8 tested bone and cartilage-forming stem cells of young mice (2 months old) and old mice (24 months old). SSCs were characterized as a self-renewing population, which gives rise to bone, cartilage, and stroma, but not fat. SSCs isolated from uninjured and fractured bones of young and old hosts demonstrated reduced proliferative and differentiation potential and a shift toward pro-myeloid lineages. Heterotopic transplantation of young SSCs compared with old SSCs into a young host revealed a robust bone induction by the former and much smaller, less mature bone by the latter, whereas old SSCs exposed to young circulation using heterochronic parabiosis did not rejuvenate within their endogenous microenvironment. These results indicate that skeletal aging primarily involves changes inherent to SSCs rather than the systemic circulatory environment. Also, old SSCs expressed high levels of colony stimulating factor 1 (CSF1), which promotes osteoclastogenesis. CSF1 delivery to a femoral fracture in young mice impaired healing, whereas CSF1 inhibition in old mice enhanced bone morphogenetic protein-2 (BMP-2)-stimulated bone healing. This study provided compelling evidence that skeletal aging is inherently rooted in skeletal stem cells, not in the systemic circulatory factors of the host. Skeletal aging shifts bone remodeling toward resorption as bone-forming cells undergo senescence. Senescent cells release cytokines and mediators called the senescence-associated secretory phenotype (SASP), which creates a microenvironment further promoting senescence. Li et al.9 established that senescent neutrophils and macrophages in bone marrow secrete grancalcin (GCA). GCA, expressed preferentially in neutrophils and marrow M1 macrophages, progressively increases with age and proinflammatory stimulation. Delivery of recombinant GCA induces pronounced skeletal aging in young mice and causes adipose changes in bone marrow with suppression of osteoblasts and increased bone resorption. The deletion of the Gca gene in these cells considerably delays age-related bone loss; similarly, delivery of antibodies neutralizing GCA suppresses skeletal aging. Although cellular senescence is widely attributable to aging, some senescent cells may exhibit desirable regulatory function. Saul et al.10 established that cell senescence and SASP increase during fracture healing in young mice. The expression of Cdkn1aCip1 increased twentyfold and Cdkn2aInk4a increased a hundredfold in fractures at 14 days. Senescence markers senescence-associated distension of satellites (SADS) and telomere-associated foci (TAF) were also high in the callus. The complete deletion of Cdkn2aInk4a resulted in greater callus formation, although the overall fracture-healing time was unaltered. SASP components were highly heterogenous, and the most expressed factors included Igfbp3, Igfbp4, VegfA, Mmp13, and Foxo4. The weekly administration of dasatinib plus quercetin (D+Q) senolytics demonstrated a significant clearance of senescent cells and SASP in the callus, leading to improved fracture healing. The study indicated that senescent cells and SASP accumulate transiently in fracture callus in young mice and their clearance with D+Q senolytics improves fracture healing. The merits of D+Q senolytics for age-dependent intervertebral disc degeneration were assessed by Novais et al.11, who treated C57BL/6 mice beginning at 6, 14, and 18 months of age and analyzed them as old at 23 months. Interestingly, 6-month and 14-month D+Q-treated mice had a lower incidence of disc degeneration, and treatment resulted in a significant decrease of senescence markers p16INK4a and p19ARF and of SASP molecules IL-6 and Mmp13. D+Q treatment also preserved disc cell viability, phenotype, and matrix content. Although transcriptomic analysis demonstrated disc compartment-specific effects of the treatment, cell death and cytokine response pathways were commonly modulated, suggesting that senolytics may offer an effective option to mitigate age-dependent intervertebral disc degeneration. Bone and Cartilage Remodeling Osteoclasts, formed by the cell-to-cell fusion of monocytes and macrophages of hematopoietic linages, were presumed to undergo apoptosis after their bone resorptive function stops. By tracking the fate of osteoclasts using real-time imaging of cellular dynamics in the tibiae of living mice, McDonald et al.12 established that osteoclasts have an alternative fate, which involves splitting into daughter cells, osteomorphs. This osteoclast fission is distinct from apoptosis and is regulated by the receptor activator of nuclear factor kappa-B ligand (RANKL). When suppressed, RANKL causes osteomorphs to accumulate as a recycling pool and, when reactivated, causes them to reassemble into functional osteoclasts. These findings explain clinically observed accelerated bone loss following discontinuation of denosumab, an inhibitor RANKL. Single-cell RNA sequencing (scRNAseq) showed that osteomorphs differ from osteoclasts and macrophages, expressing genes linked to maintaining the structural and functional bone phenotype. An analysis of human orthologs of osteomorph-upregulated genes revealed a strong correlation with rare monogenic skeletal dysplasia and association with polygenetic bone mineral pathologies. The osteoclast recycling discovered in this study is a new and exciting addition to our understanding of skeletal diseases associated with dysregulated bone remodeling. Despite available treatment options, effectively arresting excessive bone resorption poses problems. Arandjelovic et al.13 established that ELMO1 is an important signaling node in molecular complexes activating osteoclasts. These authors tested 4 mouse models of osteoporosis or joint destruction: osteoprotegerin (Opg) knockouts, ovariectomy, and collagen or K/BxN-induced inflammatory arthritides. Although Opg−/− knockouts reliably rendered an osteoporotic phenotype, Elmo1−/−Opg−/− double knockouts showed significant and lasting improvement of bone mineral density and microarchitecture unaffected by aging. Similar results were observed in the ovariectomized Elmo1−/− knockouts. In a collagen-induced arthritis model, bone erosions developed with overexpression of osteoclast genes; analogous bone and joint destruction ensued in a K/BxN-induced arthritis model. In both arthritides, Elmo1−/− knockouts exhibited a marked reduction of osteoclastic markers and apparent bone and joint preservation. Transcriptomic studies established that ELMO1 regulates the osteoclast release of cathepsin G and myeloperoxidase required for bone degradation and enhances the osteoclast resorptive sealing zone. Thus, inhibiting ELMO1 may be highly relevant to treating osteoporosis and arthritis. The identity of a cell responsible for cartilage resorption remains controversial. In endochondral osteogenesis, the removal of cartilage matrix and hypertrophic chondrocytes is essential for initiating angiogenesis and subsequent ossification. Previous studies proposed an elusive mononucleated cell—the septoclast, a cell rich in the cysteine proteinase cathepsin B and fatty acid-binding protein 5 (FABP5). Sivaraj et al.14 corroborated that cartilage matrix degradation and chondrocyte phagocytosis are functions of FABP+ septoclasts. Using high-resolution imaging and transcriptomic studies, these investigators assessed cartilage degradation during developmental and regenerative osteogenesis and established that FABP5+ septoclasts are of mesenchymal origin and are present at sites of endochondral bone healing and near the growth plate, representing a dynamic population at the chondro-osseous interface. Furthermore, septoclasts contribute to developmental bone growth by interacting with endothelial cells via the Notch signaling pathway. At the termination of skeletal growth, septoclasts disappear, but they reemerge during endochondral bone healing. The study substantiated the major involvement of FABP5+ septoclasts in cartilage degradation and offered opportunities to modulate the process. Pathological Bone Formation The surgical removal of heterotopic ossification can be invasive and hazardous, particularly when the ossification forms adjacent to vital neurovascular structures. Jin et al.15 designed a cellular system to biologically remove heterotopic ossification. The system consists of in vitro-engineered osteoclasts with a covalently bound tetracycline layer to harness their osteolytic properties preferentially to heterotopic ossification. Native osteoclasts do not effectively target heterotopic ossification, whereas the tetracycline-bound osteoclasts (TC-OCs) exhibit much greater capacity to migrate and adhere to heterotopic ossification, driven by the high affinity of tetracycline for calcium. The system was validated in vitro and in vivo. In a culture setting, TC-OCs resorbed bone ossicles at twice the efficiency of native osteoclasts, yielding 3 times greater removal of calcium from the bone matrix. For in vivo validation, 3 heterotopic ossification animal models were used: rat Achilles trauma-tenotomy, intramuscular BMP-2 injection, and murine transgenic Mkx−/− knockouts. In the tenotomy model, TC-OC injection into the site of heterotopic ossification progressively reduced heterotopic ossification volume, whereas, in the BMP-2 and Mkx−/− models, abolition of heterotopic ossification by TC-OCs was even more apparent. Because tetracycline exhibits inherent fluorescent properties, this cell-based heterotopic ossification removal system enables detection and monitoring of heterotopic ossification targeting. Pathological bone formation was also investigated by Shao et al.16 using a transgenic CD4-Cre;Ptpn11f/f mouse model, which spontaneously develops features of ankylosing spondylitis. The skeletal phenotype of these mice exhibits progressive abnormal bone formation, leading to massive osteophyte formation and ankylosis at the hip, knee, sacroiliac joints, and spine at 12 months of age. The study demonstrated that aberrant chondrocytes in these mice induced joint ankylosis and osteophyte formation through endochondral osteogenesis mediated by the BMP6/pSmad1/5 and Hedgehog signaling pathways. Administration of sonidegib, which inhibits smoothened (Smo), and thereby the BMP-Hedgehog pathways, abolished aberrant chondrogenesis and significantly ameliorated the ankylosing spondylitis-like phenotype. These findings indicate that targeting dysfunctional chondrocytes with an Smo inhibitor may present a promising strategy for arresting aberrant bone formation in ankylosing spondylitis. Odomzo (sonidegib; Sun Pharma Global) is a drug currently used to treat basal cell carcinoma; thus, its use may be expeditiously extended to treating ankylosing spondylitis following clinical trials. Cognitive Decline and Bone Health Dementia and osteoporosis frequently accompany each other; however, it is unclear whether their relationship is causative or just due to similar elderly onset. A high prevalence of osteoporosis and fragility fractures in patients with cognitive decline has been reported, yet longitudinal studies to delineate this relationship independently of aging have been lacking. Bliuc et al.17 addressed that void by examining the associations between cognitive decline and bone loss and between clinically important cognitive loss (≥3 points on the Mini Mental State Examination) and fracture. A cohort of 2,361 elderly patients (1,741 women and 620 men, ≥65 years of age) were randomly recruited from participants of the Canadian Multicentre Osteoporosis Study and were followed for 10 years. Cognitive decline was associated with bone loss in women (6.5% for each percentage drop in the Mini Mental State Examination) independent of age, education, comorbidities, and lifestyle factors, but not in men. In women, fracture risk increased significantly; for men, the sample size was too small. A clinically important cognitive decline over 5 years was associated with an increased incidence of hip, vertebral, and other fractures over 10 years. The study demonstrates the clinically important relationship of cognitive decline with bone loss and fracture risk in women. A mechanistic study to elucidate a relationship between the most common dementia, Alzheimer disease, and reduced bone mass was conducted by Guo et al.18. Tg2576 mice were used as an Alzheimer disease model, which ubiquitously expresses Swedish mutant amyloid precursor protein (APPswe), and their bone phenotype was characterized. APPswe mutation in humans is associated with early-onset Alzheimer disease. Tg2572 mice develop early osteoporotic changes long before any pathology is detectable in the brain. In transgenic osteocalcin-cre TgAPPswe mice, which conditionally express APPswe only in cells of the osteoblast lineage, osteoporotic changes were consistent with those in the Tg2576 mice, indicating that APPswe plays a cell-autonomous role in the suppression of bone formation. Further, the study identified hepcidin, a hepatic factor regulating iron homeostasis, as downstream APPswe. Hepcidin was highly expressed in Tg2576 mice in the serum and in the liver, muscle, and osteoblast-lineage cells. Increased hepcidin expression is caused by proinflammatory cytokines, iron overload, BMP-6, and endoplasmic reticulum stress, factors implicated in the pathogenesis of both Alzheimer disease and osteoporosis. Vitamin D Supplementation and Bone Health Vitamin D3 supplements are widely recommended for bone health, although evidence supporting their benefits is lacking. LeBoff et al.19 conducted an ancillary study of the Vitamin D and Omega-3 Trial (VITAL) and demonstrated that vitamin D supplementation did not significantly lower fractures in middle-aged and older adults (age of ≥55 years in women and ≥50 years in men). VITAL was a 2 × 2-factorial randomized controlled trial to investigate the ability of daily vitamin D3 (2,000 IU) and/or omega-3 fatty acids (1 g) to prevent cancer and cardiovascular disease; thus, the participants were not recruited on the basis of the vitamin D level, bone mass, or osteoporosis. VITAL has been the largest, most extensive, and most definitive trial to date, with 25,871 participants followed for 5.3 years. Comparing the VITAL groups treated with vitamin D alone and with placebo found no significant effect on overall, nonvertebral, or hip fractures, even in subjects with low 25-hydroxyvitamin D3 levels. Subgroup analyses based on sex, age, race or ethnicity, and body mass index also did not demonstrate an effect. As the largest randomized controlled trial offering the highest evidence, these findings explicitly demonstrated that routine daily vitamin D3 supplementation does not prevent fractures in healthy middle-aged and older adults. In a double-blinded, randomized controlled trial, Gaffney-Stomberg et al.20 also challenged the skeletal benefits of routine daily supplementation with vitamin D3 (1,000 IU) and calcium (1 g) throughout an 8-week course of basic combat training in young (21 ± 3.5 years) and healthy military recruits. Although administering vitamin D3 with calcium prevented the increase of blood markers of bone resorption (CTX, TRAP) that was seen in the placebo group, the adverse effects on tibial mineral density and microarchitecture of the recruits during intense military basic combat training were not significantly less than in the placebo group. This study extends the lack of benefits of routine vitamin D supplementation to include young and healthy individuals. Upcoming Meetings and Events Related to Orthopaedic Basic Science The 2023 Annual Meeting of the Orthopaedic Research Society (ORS) will be held on February 10 to 14, 2023, in Dallas, Texas, United States. The 2023 OARSI (Osteoarthritis Research Society International) World Congress on Osteoarthritis will be held on March 17 to 20, 2023, in Denver, Colorado, United States. The Gordon Research Conference, Cartilage Biology, Structure and Function: From Development to Regeneration, will be held on March 19 to 24, 2023, in Barga, Italy. The International Society for Stem Cell Research (ISSCR) 2023 Annual Meeting will be held on June 14 to 17, 2023, in Boston, Massachusetts, United States. The 17th World Congress of the International Cartilage Society will be held on September 9 to 12, 2023, in Sitges-Barcelona, Spain. The 31st Annual Meeting of the European Orthopaedic Research Society will be held on October 4 to 6, 2023, in Porto, Portugal. The American Society for Bone and Mineral Research (ASBMR) 2023 Annual Meeting will be held on October 13 to 16, 2023, in Vancouver, British Columbia, Canada.
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