Age-associated Bone Research Articles

Comparison of Outcomes of the Third Generation Minimally Invasive Chevron-Akin Procedure Between Elderly and Younger Patients with Hallux Valgus

Category: Bunion; Other Introduction/Purpose: Hallux valgus is known to be more prevalent and present with greater severity in the elderly population compared to the younger age group. However, surgical treatment of hallux valgus in the elderly carries a risk of suboptimal results owing to porotic bone quality and multiple comorbidities typical of this age group. Although the third-generation minimally invasive Chevron and Akin osteotomy (MICA) technique, which features minimal soft tissue disruption and rigid internal fixation, may be an attractive option for surgical treatment of hallux valgus in the elderly, its outcomes have not been thoroughly investigated. This study sought to compare the outcomes of third-generation MICA procedures in patients aged 65 and older with those in the cohort under 65 years of age. Methods: In this retrospective cohort study, 76 patients (79 feet) who underwent the third generation MICA procedure for hallux valgus and were followed up for at least 1 year were stratified into 2 groups: ≥65 years old (Group 1: 36 feet) and < 65 years old (Group 2: 40 feet). Radiographic assessments included HV angle (HVA), intermetatarsal angle (IMA) and distal metatarsal articular angle (DMAA). Clinical assessment included the American Orthopaedic Foot & Ankle Society (AOFAS) score, pain visual analog scale (VAS), and patient satisfaction rating. Postoperative complications, including recurrence of the deformity, reduction loss and reoperation, were assessed. Results: The average age at the time of surgery in group 1 was 70.9 ± 5.8 (range 65~90) years, and in group 2, 50.2 ± 2.8 (range 21~ 63), respectively. In the elderly group, Bone Mineral Density (BMD) was measured -2.9. The mean HVA, IMA and DMAA significantly improved at the final follow-up in both groups, respectively (all P < .001). Both preoperative and postoperative HVA were significantly greater in group 1 (both, P < .001), and the change of this parameter after surgery was also lower in group 1 than in group 2 (P < .001). There were significantly higher rates of reduction loss (16% vs 2.5%) and recurrence (11.1% vs 0.5%). However, clinical scores and patient satisfaction rates did not differ significantly. Conclusion: The present study demonstrates that chronological age, specifically categorized as 65 years or older, exhibits no substantial influence on the clinical outcomes following third-generation MICA surgery. Nevertheless, it is imperative to adequately apprise patients of the potential for reduction loss and recurrence, possibly due to age-associated bone fragility.

CD11B + CD36 + cells are bone anabolic macrophages that limit age-associated bone loss.

Disruptions in the bone remodeling cycle that occur with increasing age lead to degeneration of the skeleton and increased risk of fragility fractures. Our understanding of how the bone remodeling process within cortical bone is controlled and altered with age in males and females is limited. Here, we generated bone marrow chimeric mice to understand the impacts of age and sex on the bone remodeling process. We demonstrate that transplantation of aged male or female bone marrow into young lethally irradiated male hosts unexpectedly enhances cortical bone mass without an impacting cancellous bone. Our single cell RNA-sequencing data show that mice reconstituted with aged bone marrow exhibited subsets of cells marked by CD11B/CD36 expression that demonstrate enhanced production of anabolic cytokines as compared to young counterparts, and that these myeloid subsets exist under conditions of normal physiology in aged mice. Importantly, CD11B + CD36 + cells do not differentiate into osteoclasts in vitro, and CD36 does not mark TRAP+ cells in vivo. Instead, CD36 + cells localize to resorption sites, including within cortical bone defects, suggesting their involvement in cortical bone remodeling and healing. CD11B + CD36 + cells also express elevated levels of bone anabolic WNT ligands, especially Wnt6. In functional assays, we demonstrate that soluble factors produced by CD11B + CD36 + cells enhance osteoblast progenitor commitment, mineralization, and activation of WNT signaling in vitro. Moreover, CD11B/CD36 exquisitely mark a subset of anabolic myeloid cells within human bone marrow. In conclusion, our studies identified a novel population of aged macrophages that limit cortical bone loss.

Glycemic Marker Correlation with Collagen Denaturation and Non-Enzymatic Collagen Cross-Linking in Age-Associated Bone Resistance

The primary clinical indicator of fracture risk among the elderly is low bone mass, yet it accounts for less than half of fractures in individuals over 50 years. Age is recognized to influence bone quality, affecting bone structure and properties. Previous research indicates that age diminishes tissue plasticity and toughness conferred by collagen, suggesting that age-related changes in the collagen environment may contribute to bone fragility. This study explores the relationship between age-related collagen impairment, specifically the accumulation of non-enzymatic collagen cross-linking and molecular collagen denaturation, and bone toughness in middle-aged and older patients (postmenopausal 50–70 years old and senile osteoporosis age > 70 years old). Additionally, it examines the influence of blood glucose and HbA1c levels, as well as body mass index (BMI), on these factors. Despite not finding any differences in fracture toughness between groups, we found a significant correlation between hemoglobin A1c and collagen integrity (collagen denaturation and non-enzymatic cross-linking).

Targeting mitochondrial dysfunction using methylene blue or mitoquinone to improve skeletal aging.

Methylene blue (MB) is a well-established antioxidant that has been shown to improve mitochondrial function in both in vitro and in vivo settings. Mitoquinone (MitoQ) is a selective antioxidant that specifically targets mitochondria and effectively reduces the accumulation of reactive oxygen species. To investigate the effect of long-term administration of MB on skeletal morphology, we administered MB to aged (18 months old) female C57BL/J6 mice, as well as to adult male and female mice with a genetically diverse background (UM-HET3). Additionally, we used MitoQ as an alternative approach to target mitochondrial oxidative stress during aging in adult female and male UM-HET3 mice. Although we observed some beneficial effects of MB and MitoQ in vitro, the administration of these compounds in vivo did not alter the progression of age-induced bone loss. Specifically, treating 18-month-old female mice with MB for 6 or 12 months did not have an effect on age-related bone loss. Similarly, long-term treatment with MB from 7 to 22 months or with MitoQ from 4 to 22 months of age did not affect the morphology of cortical bone at the mid-diaphysis of the femur, trabecular bone at the distal-metaphysis of the femur, or trabecular bone at the lumbar vertebra-5 in UM-HET3 mice. Based on our findings, it appears that long-term treatment with MB or MitoQ alone, as a means to reduce skeletal oxidative stress, is insufficient to inhibit age-associated bone loss. This supports the notion that interventions solely with antioxidants may not provide adequate protection against skeletal aging.

Integrative analyses of genetic characteristics associated with skeletal endothelial cells.

The osseous vascular endothelium encompasses a vast intricate framework that regulates bone remodeling. Osteoporosis, an age-associated systemic bone disease, is characterized by the degeneration of the vascular architecture. Nevertheless, the precise mechanisms underpinning the metamorphosis of endothelial cells (ECs) with advancing age remain predominantly enigmatic. In this study, we conducted a systematic analysis of differentially expressed genes (DEGs) and the associated pathways in juvenile and mature femoral ECs, utilizing data sourced from the Gene Expression Omnibus (GEO) repositories (GSE148804) and employing bioinformatics tools. Through this approach, we successfully discerned six pivotal genes, namely Adamts1, Adamts2, Adamts4, Adamts14, Col5a1, and Col5a2. Subsequently, we constructed a miRNA-mRNA network based on miRNAs displaying differential expression between CD31hiEMCNhi and CD31lowEMCNlow ECs, utilizing online repositories for prediction. The expression of miR-466i-3p and miR-466i-5p in bone marrow ECs exhibited an inverse correlation with age. Our in vivo experiments additionally unveiled miR-466i-5p as a pivotal regulator in osseous ECs and a promising therapeutic target for age-related osteoporosis.

Quantitative Analysis of Age-Associated Bone Mineral Density Variations via Automated Segmentation: Using CT Scans and Radon Transform to Accurately Examine and Assess the Vertebrae

Quantitative Analysis of Age-Associated Bone Mineral Density Variations via Automated Segmentation: Using CT Scans and Radon Transform to Accurately Examine and Assess the Vertebrae

Ineffective Erythropoiesis with Aging in Adults with Sickle Cell Disease

INTRODUCTION In the United States, life expectancy for adults with sickle cell disease (SCD) has improved over the last several decades and providers are now encountering increasing numbers of patients in the fourth, fifth and sixth decades. One notable feature of older SCD patients is lower ‘steady state’ hemoglobin (Hb) levels. Although it is well established that lower Hb levels are associated with increased adverse outcomes including pulmonary hypertension, stroke, cognitive decline, acute chest syndrome, frequency of vaso-occlusive crisis and shortened survival, a full understanding of the mechanisms accounting for the more prevalent and severe anemia in older SCD patients remains to be elucidated. Involved factors may include the long-term effect of chronic hemolysis, age-associated increased inflammation, progressive renal insufficiency and an age-related decline in nutritional adequacy. Despite lacking a uniform explanation for anemia applicable to individual SCD patients, it remains a management goal to optimize Hb level as possible within constraints associated with the development of secondary hemochromatosis. We postulate that the pathways to anemia in SCD patients may change with advancing age and that a better understanding of these pathways may result in a more directed intervention for the older sickle cell patient. METHODS Adult patients visiting Inova Adult Sickle Cell Center over a 6-month period (January through June 2023) with Hb levels below 9 g/dL were evaluated by a contemporary hematologic panel that included complete blood count with differential, reticulocyte count, comprehensive metabolic panel (included total bilirubin and creatinine), and lactate dehydrogenase (LDH). The reticulocyte proliferation index (RPI) was determined utilizing a readily accessible calculator (MedCalc) and a univariate regression analysis was performed for RPI against age. An RPI of < 2 was considered evidence for hypoproliferative anemia. RESULTS Of the 139 unique adult SCD patients visiting our clinic in this 6-month period, 60 had Hb levels equal to or below 9 g/dL. Of these, 8 patients had serum creatinine levels ≥ 1.5 mg/dL and were considered to have hypoproliferative anemia based on renal insufficiency. Among the remaining 52 patients, the mean age was 34.9 years, mean Hb was 7.49 g/dL and the mean RPI was 2.52. In patients with age ≤ 40 years, mean RPI was 2.74, compared to mean RPI of 1.61 in patients with age > 40 years. The mean Hb was 7.55 g/dL vs 7.21 g/dL mean total bilirubin was 3.08 mg/dL vs 2.15 mg/dL and mean LDH was 509 U/L vs 434 U/L in these two age groups (Fig 1). Regression analysis indicated that an increase in age corresponded to a reduction in RPI at 95% confidence level (Fig 2; regression coefficient: -0.04; 95% CI: -0.08 - -0.003; p-value: 0.03). CONCLUSION From the data we gathered, we conclude that despite chronic hemolysis in sickle cell disease, low RPI and ineffective erythropoiesis are evident to some extent in the majority of adult SCD patients even in the absence of renal failure and this significantly increases with age. Older patients with SCD are more likely to have ineffective erythropoiesis. We speculate that this may be evidence for age-associated bone marrow failure not unlike myelodysplasia (MDS) which occurs with increased frequency in the general population with advancing age. If future studies confirm disordered erythropoiesis, clonal hematopoiesis, or other markers of MDS, novel treatments known to be effective in patients with MDS may be examined in selected anemic SCD patients.

Nore1 inhibits age-associated myeloid lineage skewing and clonal hematopoiesis but facilitates termination of emergency (stress) granulopoiesis

Age-associated bone marrow changes include myeloid skewing and mutations that lead to clonal hematopoiesis. Molecular mechanisms for these events are ill defined, but decreased expression of Irf8/Icsbp (interferon regulatory factor 8/interferon consensus sequence binding protein) in aging hematopoietic stem cells may contribute. Irf8 functions as a leukemia suppressor for chronic myeloid leukemia, and young Irf8-/- mice have neutrophilia with progression to acute myeloid leukemia (AML) with aging. Irf8 is also required to terminate emergency granulopoiesis during the innate immune response, suggesting this may be the physiologic counterpart to leukemia suppression by this transcription factor. Identifying Irf8 effectors may define mediators of both events and thus contributors to age-related bone marrow disorders. In this study, we identified RASSF5 (encoding Nore1) as an Irf8 target gene and investigated the role of Nore1 in hematopoiesis. We found Irf8 activates RASSF5 transcription and increases Nore1a expression during emergency granulopoiesis. Similar to Irf8-/- mice, we found that young Rassf5-/- mice had increased neutrophils and progressed to AML with aging. We identified enhanced DNA damage, excess clonal hematopoiesis, and a distinct mutation profile in hematopoietic stem cells from aging Rassf5-/- mice compared with wildtype. We found sustained emergency granulopoiesis in Rassf5-/- mice, with repeated episodes accelerating AML, also similar to Irf8-/- mice. Identifying Nore1a downstream from Irf8 defines a pathway involved in leukemia suppression and the innate immune response and suggests a novel molecular mechanism contributing to age-related clonal myeloid disorders.

Piezo1 opposes age-associated cortical bone loss.

As we age, our bones undergo a process of loss, often accompanied by muscle weakness and reduced physical activity. This is exacerbated by decreased responsiveness to mechanical stimulation in aged skeleton, leading to the hypothesis that decreased mechanical stimulation plays an important role in age-related bone loss. Piezo1, a mechanosensitive ion channel, is critical for bone homeostasis and mechanotransduction. Here, we observed a decrease in Piezo1 expression with age in both murine and human cortical bone. Furthermore, loss of Piezo1 in osteoblasts and osteocytes resulted in an increase in age-associated cortical bone loss compared to control mice. The loss of cortical bone was due to an expansion of the endosteal perimeter resulting from increased endocortical resorption. In addition, expression of Tnfrsf11b, encoding anti-osteoclastogenic protein OPG, decreases with Piezo1 in vitro and in vivo in bone cells, suggesting that Piezo1 suppresses osteoclast formation by promoting Tnfrsf11b expression. Our results highlight the importance of Piezo1-mediated mechanical signaling in protecting against age-associated cortical bone loss by inhibiting bone resorption in mice.

Age-associated declining of the regeneration potential of skeletal stem/progenitor cells.

Bone fractures represent a significant health burden worldwide, mainly because of the rising number of elderly people. As people become older, the risk and the frequency of bone fractures increase drastically. Such increase arises from loss of skeletal integrity and is also associated to a reduction of the bone regeneration potential. Central to loss of skeletal integrity and reduction of regeneration potential are the skeletal stem/progenitor cells (SSPCs), as they are responsible for the growth, regeneration, and repair of the bone tissue. However, the exact identity of the SSPCs has not yet been determined. Consequently, their functions, and especially dysfunctions, during aging have never been fully characterized. In this review, with the final goal of describing SSPCs dysfunctions associated to aging, we first discuss some of the most recent findings about their identification. Then, we focus on how SSPCs participate in the normal bone regeneration process and how aging can modify their regeneration potential, ultimately leading to age-associated bone fractures and lack of repair. Novel perspectives based on our experience are also provided.

Glucagon-Like Peptide-2 Ameliorates Age-Associated Bone Loss and Gut Barrier Dysfunction in Senescence-Accelerated Mouse Prone 6 Mice

Introduction: Senile osteoporosis is one of the most common age-related diseases worldwide. Glucagon like peptide-2 (GLP-2), a naturally occurring gastrointestinal peptide, possesses therapeutic effects on bone loss in postmenopausal women and ovariectomized rats. However, the role of GLP-2 in senile osteoporosis and underlying mechanisms has not been explored. Methods: GLP-2 was subcutaneously injected into the 6-month-old male senile osteoporosis model of senescence-accelerated mouse prone 6 (SAMP6) mice for 6 weeks. SAMP6 subjected to normal saline and senescence-accelerated mouse resistant 1 served as control groups. Micro-computed tomography was performed to evaluate the bone mass and microarchitecture of the mice. Osteoblastic and osteoclastic activities were determined by biochemical, quantitative real-time PCR, histological, and histomorphometric analyses combined with hematoxylin-eosin, toluidine blue, and tartrate-resistant acid phosphatase staining. We also examined the proteins and structure of intestinal tight junction using immunohistochemical assay as well as a transmission electron microscope. Serum inflammation marker levels were measured using ELISA. Additionally, anti-oxidative enzymes GPX-4 and SOD-2 and receptors of GLP-2 and vitamin D expression in the ileum and colon were detected under immunofluorescence staining. Results: Six-week GLP-2 treatment attenuated bone loss in SAMP6 mice, as evidenced by increased bone mineral density, improved microarchitecture in femora, and enhanced osteogenic activities. In contrast, the activity of osteoclastic activity was not obviously inhibited. Moreover, GLP-2 ameliorated tight junction structure and protein expression in the intestinal barrier, which was accompanied by the reduction of TNF-α level. The expression of receptors of intestinal GLP-2 and vitamin D in the ileum was elevated. Furthermore, the oxidative stress in the intestines was improved by increasing the GPX-4 and SOD-2 signaling. Conclusion: Our findings suggest that GLP-2 could ameliorate age-associated bone loss, tight junction structure, and improved antioxidant enzyme activity in the gut in SAMP6 mice. Amelioration of gut barrier dysfunction may potentially contribute to improving bone formation and provide evidence for targeting the entero-bone axis in the treatment of senile osteoporosis.

A multimodal 3D imaging approach of pore networks in the human femur to assess age-associated vascular expansion and Lacuno-Canalicular reduction.

Cellular communication in the mechanosensory osteocyte Lacuno-Canalicular Network (LCN) regulates bone tissue remodeling throughout life. Age-associated declines in LCN size and connectivity dysregulate mechanosensitivity to localized remodeling needs of aging or damaged tissue, compromising bone quality. Synchrotron radiation-based micro-Computed Tomography (SRμCT) and Confocal Laser Scanning Microscopy (CLSM) were employed to visualize LCN and vascular canal morphometry in an age series of the anterior femur (males n=14, females n=11, age range=19-101, mean age=55). Age-associated increases in vascular porosity were driven by pore coalescence, including a significant expansion in pore diameter and a significant decline in pore density. In contrast, the LCN showed significant age-associated reductions in lacunar volume fraction, mean diameter, and density, and in canalicular volume fraction and connectivity density. Lacunar density was significantly lower in females across the lifespan, exacerbating their age-associated decline. Canalicular connectivity density was also significantly lower in females but approached comparable declining male values in older age. Our data illuminate the trajectory and potential morphometric sources of age-associated bone loss. Increased vascular porosity contributes to bone fragility with aging, while an increasingly reduced and disconnected LCN undermines the mechanosensitivity required to repair and reinforce bone. Understanding why and how this degradation occurs is essential for improving the diagnosis and treatment of age-related changes in bone quality and fragility.

Moderate-Intensity Exercise Preserves Bone Mineral Density and Improves Femoral Trabecular Bone Microarchitecture in Middle-Aged Mice.

BackgroundAging leads to significant bone loss and elevated osteoporosis risk. Exercise slows age-related bone loss; however, the effects of various moderate-intensity exercise training volumes on bone metabolism remain unclear. This study aimed to determine the degree to which different volumes of moderate-intensity aerobic exercise training influence bone mineral density (BMD), bone mineral content (BMC), femoral trabecular bone microarchitecture, and cortical bone in middle-aged mice.MethodsTwenty middle-aged male C57BL/6 mice were randomly assigned 8 weeks of either (1) non-exercise (CON); (2) moderate-intensity with high-volume exercise (EX_MHV); or (3) moderate-intensity with low-volume exercise (EX_MLV) (N=6–7, respectively). Femoral BMD and BMC were evaluated using dual energy X-ray absorptiometry, and trabecular and cortical bone were measured using micro-computed tomography.ResultsFemoral BMD in EX_MHV but not EX_MLV was significantly higher (P<0.05) than in CON. The distal femoral fractional trabecular bone volume/tissue volume (BV/TV, %) was significantly higher (P<0.05) in both EX_MHV and EX_MLV than in CON mice. Increased BV/TV was induced by significantly increased trabecular thickness (mm) and tended to be higher (P<0.10) in BV (mm3) and lower in trabecular separation (mm) in EX_MHV and EX_MLV than in CON. The femoral mid-diaphysis cortical bone was stronger in EX_MLV than EX_MHV.ConclusionsLong-term moderate-intensity aerobic exercise with low to high volumes can be thought to have a positive effect on hindlimb BMD and attenuate age-associated trabecular bone loss in the femur. Moderate-intensity aerobic exercise may be an effective and applicable exercise regimen to prevent age-related loss of BMD and BV.

Targeting Sclerostin and Dkk1 at Optimized Proportions of Low-Dose Antibody Achieves Similar Skeletal Benefits to Higher-Dose Sclerostin Targeting in the Mature Adult and Aged Skeleton.

Age-associated low bone mass disease is a growing problem in the US. Development of osteoanabolic therapies for treating skeletal fragility has lagged behind anti-catabolic therapies, but several bone-building molecules are clinically available. We reported previously that antibody-based neutralization of the Lrp5/Lrp6 inhibitor Dkk1 has minimal effects on bone gain, but can potentiate the already potent osteoanabolic effects of sclerostin inhibition (another Lrp5/Lrp6 inhibitor highly expressed by osteocytes). In this communication, we test whether an optimized ratio of sclerostin and Dkk1 antibodies (Scl-mAb and Dkk1-mAb, respectively), administered at low doses, can maintain the same bone-building effects as higher dose Scl-mAb, in adult (6 months of age) and aged (20 months of age) wild-type mice. A 3:1 dose of Scl-mAb:Dkk1-mAb at 12.5 mg/kg was equally efficacious as 25 mg/kg of Scl-mAb in both age groups, using radiographic (DXA, µCT), biomechanical, (3-point bending tests), and histological (fluorochrome-based bone formation parameters) outcome measures. For some bone properties, including trabecular thickness and bone mineral density in the spine, and endocortical bone formation rates in the femur, the 3:1 treatment was associated with significantly improved skeletal properties compared to twice the dose of Scl-mAb. Cortical porosity in aged mice was also reduced by both Scl-mAb and low-dose 3:1 treatment. Overall, both treatments were efficacious in the mature adult (6 mo.) and aged (20 mo.) skeletons, suggesting Wnt targeting is a viable strategy for improving skeletal fragility in the very old. Further, the data suggest that low dose of combination therapy can be at least equally efficacious as higher doses of Scl-mAb monotherapy.

Adolescent female handball players present greater bone mass content than soccer players: A cross-sectional study.

Osteoporosis is a systemic disease affecting half of women over the age of 50years. Considering that almost 90% of peak of bone mass is achieved until the second decade of life, ensuring a maximal bone mineral content acquisition may compensate for age-associated bone loss. Among several other factors, physical activity has been recommended to improve bone mass acquisition. However, it is unknown whether athletes involved with sports with different impact loading characteristics differ in regards to bone mass measurements. To compare the bone mass content, bone mass density and lean mass of young female soccer players (odd-impact loading exercise), handball players (high-impact loading exercises) and non-athletes. A total of 115 female handball players (15.5±1.3years, 165.2±5.6cm and 61.9±9.3kg) and 142 soccer players (15.5±1.5years, 163.7±6.6cm and 56.5±7.7kg) were evaluated for body composition using a dual-emission X-ray absorptiometry system, and 136 female non-athletes (data from NHANES) (15.1±1.32years, 163.5±5.8cm and 67.2±19.4kg) were considered as the control. Handball players presented higher bone mass content values than soccer players for upper limbs (294.8±40.2g and 270.7±45.7g, p<0.001), lower limbs (1011.6±145.5g and 967.7±144.3g, p=0.035), trunk (911.1±182.5g and 841.6±163.7g, p=0.001), ribs (312.4±69.9g and 272.9±58.0g, p<0.001), spine (245.1±46.8g and 222.0±45.1g, p<0.001) and total bone mass (2708.7±384.1g and 2534.8±386.0g, p<0.001). Moreover, non-athletes presented lower bone mass content for lower limbs (740.6±132.3g, p<0.001), trunk (539.7±98.6g, p<0.001), ribs (138.2±29.9g, p<0.001), pelvis (238.9±54.6g, p<0.001), spine (152.8±26.4g, p<0.001) and total bone mass (1987.5±311.3g, p<0.001) than both handball and soccer players. Handball players also presented higher bone mass density values than soccer players for trunk, ribs and spine (p<0.05) and handball and soccer players presented higher bone mass density than non-athletes for all measurements (p<0.005). Finally, the non-athletes' lower limb lean mass was lower than soccer and handball players values (p<0.05). Adolescent females engaged in handball training for at least one year present higher bone mass contents than those who are engaged in soccer training, which, in turn, present higher bone mass contents than non-athletes. These results might be used by physicians and healthcare providers to justify the choice of a particular sport to enhance bone mass gain in female adolescents.

Neutralization of oxidized phospholipids attenuates age-associated bone loss in mice.

Oxidized phospholipids (OxPLs) are pro‐inflammatory molecules that affect bone remodeling under physiological conditions. Transgenic expression of a single‐chain variable fragment (scFv) of the antigen‐binding domain of E06, an IgM natural antibody that recognizes the phosphocholine (PC) moiety of OxPLs, increases trabecular and cortical bone in adult male and female mice by increasing bone formation. OxPLs increase with age, while natural antibodies decrease. Age‐related bone loss is associated with increased oxidative stress and lipid peroxidation and is characterized by a decline in osteoblast number and bone formation, raising the possibility that increased OxPLs, together with the decline of natural antibodies, contribute to age‐related bone loss. We show here that transgenic expression of E06‐scFv attenuated the age‐associated loss of spinal, femoral, and total bone mineral density in both female and male mice aged up to 22 and 24 months, respectively. E06‐scFv attenuated the age‐associated decline in trabecular bone, but not cortical bone, and this effect was associated with an increase in osteoblasts and a decrease in osteoclasts. Furthermore, RNA‐seq analysis showed that E06‐scFv increased Wnt10b expression in vertebral bone in aged mice, indicating that blocking OxPLs increases Wnt signaling. Unlike age‐related bone loss, E06‐scFv did not attenuate the bone loss caused by estrogen deficiency or unloading in adult mice. These results demonstrate that OxPLs contribute to age‐associated bone loss. Neutralization of OxPLs, therefore, is a promising therapeutic target for senile osteoporosis, as well as atherosclerosis and non‐alcoholic steatohepatitis (NASH), two other conditions shown to be attenuated by E06‐scFv in mice.

Age-related expansion and increased osteoclastogenic potential of myeloid-derived suppressor cells

Aging is associated with excessive bone loss that is not counteracted with the development of new bone. However, the mechanisms underlying age-related bone loss are not completely clear. Myeloid-derived suppressor cells (MDSCs) are a population of heterogenous immature myeloid cells with immunosuppressive functions that are known to stimulate tumor-induced bone lysis. In this study, we investigated the association of MDSCs and age-related bone loss in mice. Our results shown that aging increased the accumulation of MDSCs in the bone marrow and spleen, while in the meantime potentiated the osteoclastogenic activity of the CD11b+Ly6ChiLy6G+ monocytic subpopulation of MDSCs. In addition, CD11b+Ly6ChiLy6G+ MDSCs from old mice exhibited increased expression of c-fms compared to young mice, and were more sensitive to RANKL-induced osteoclast gene expression. On the other hand, old mice showed elevated production of IL-6 and receptor activator of nuclear factor kappa-B ligand (RANKL) in the circulation. Furthermore, IL-6 and RANKL were able to induce the proliferation of CD11b+Ly6ChiLy6G+ MDSCs and up-regulate c-fms expression. Moreover, CD11b+Ly6ChiLy6G+ MDSCs obtained from old mice showed increased antigen-specific T cell suppressive function, pStat3 expression, and cytokine production in response to inflammatory stimulation, compared to those cells obtained from young mice. Our findings suggest that CD11b+Ly6ChiLy6G+ MDSCs are a source of osteoclast precursors that together with the presence of persistent, low-grade inflammation, contribute to age-associated bone loss in mice.

Blocking Oxidized Phospholipids Attenuates the Age-Associated, but Not the Ovariectomy- or Unloading- Induced, Bone Loss in Mice

Oxidized phospholipids (OxPL), such as oxidized phosphatidylcholine, are generated by oxidative stress (OS)-induced lipid peroxidation. E06 IgM is a natural antibody that recognizes the phosphocholine (PC) moiety of OxPLs, but not native PLs. Generation of transgenic mice expressing a single chain (scFv) form of its antigen-binding domain, “E06-scFv” mice, protects against atherosclerosis, hepatic steatosis and high fat diet-induced loss of bone mass. In addition, E06-scFv increases cancellous and cortical bone mass in both male and female adult mice fed chow diet, by increasing bone formation. Age-related bone loss is associated with increased OS and lipid peroxidation, and is characterized by a reduction in osteoblast number and bone formation. Oxidative stress is involved also in the bone loss caused by sex-steroid deficiency and elevated OS markers are found in unloading-induced bone loss, raising the possibility that an increase of OxPLs induced by OS might be contributing to the pathogenesis of these conditions as well. We aged homozygous E06-scFv transgenic female and male mice and their wild-type littermates up to 22 and 24 months respectively. Serial DXA BMD every 3 months showed that overexpression of E06-scFv attenuated the age-associated bone loss in both sexes. In addition, male and female E06-scFv transgenic mice also accumulated less fat mass than WT littermates during aging. Micro-CT analysis revealed that E06-scFv attenuated the age-associated decline in cancellous, but not cortical, bone mass. The histological analysis of the vertebrae indicated that the aged E06-scFv transgenic mice had increased osteoblasts and decreased osteoclasts compared to the WT mice. To investigate whether the beneficial effect of the E06-scFv could be seen after ovariectomy, 4.5 month old E06-scFv homozygous females and WT controls were ovariectomized (OVX). DXA and micro-CT measurements 6 weeks post- surgery indicated that, unlike aging, E06-scFv did not protect against OVX-induced bone loss in either the cancellous or the cortical compartment. Lastly, we tail-suspended 5.5 month old male mice and sacrificed them 21 days later. E06-scFv transgenic mice had similar cortical bone loss compared to WT mice. In conclusion, the E06-scFV transgene attenuates the age-associated cancellous bone loss in both female and male mice, but has no effect on the OVX- or unloading-induced bone loss. These results fully support our hypothesis that an increase in PC-OxPLs with age, caused at least in part by a decrease in natural anti-PC antibodies, contributes to the age-associated bone loss. This evidence provides proof of concept that blocking PC-OxPLs represents a therapeutic approach to countering the increase of PC-OxPLs with age and their adverse effects on age-related bone loss as well as atherosclerosis and NASH. It also confirms that the mechanisms of cancellous and cortical bone loss are distinct.

Allyl sulfide promotes osteoblast differentiation and bone density via reducing mitochondrial DNA release mediated Kdm6b/H3K27me3 epigenetic mechanism

Age-associated bone loss or osteoporosis is a common clinical manifestation during aging (AG). The mechanism underlying age-associated osteoblast dysfunction induced by oxidative damage in the mitochondria and loss of bone density remains elusive. Here, we demonstrated the effect of allyl sulfide (AS), a natural organosulfur compound, on mitochondrial (mt) function in bone marrow-derived mesenchymal stem cells (BMMSCs) and bone density in AG mice. The data demonstrate that AS treatment in AG mice promotes BMMSCs differentiation and mineralization via inhibition of mitochondrial oxidative damage. The data also indicate that AG related mito-damage was associated with reduced mitochondrial biogenesis and oxidative phosphorylation, and release of a greater concentration of mtDNA. Furthermore, the data showed that mtDNA caused histone H3K27 demethylase inhibition, KDM6B, and subsequent inflammation by unbalancing mitochondrial redox homeostasis. KDM6B overexpression in AG BMMSCs or AS administration in AG mice restores osteogenesis and bone density in vitro and in vivo. Mechanistically, AS or the mitochondrial-specific antioxidant Mito-TEMPO increased KDM6B expression and upregulated the expression of Runx2 in BMMSCs, probably via epigenetic inhibition of H3K27me3 methylation at the promoter. These data uncover the previously undefined role of AS mediated prevention of mtDNA release, promoting osteogenesis and bone density via an epigenetic mechanism. Therefore, AS could be a potential drug target for the treatment of aging-associated osteoporosis.

Osteoclast fusion and bone loss are restricted by interferon inducible guanylate binding proteins

Chronic inflammation during many diseases is associated with bone loss. While interferons (IFNs) are often inhibitory to osteoclast formation, the complex role that IFN and interferon-stimulated genes (ISGs) play in osteoimmunology during inflammatory diseases is still poorly understood. We show that mice deficient in IFN signaling components including IFN alpha and beta receptor 1 (IFNAR1), interferon regulatory factor 1 (IRF1), IRF9, and STAT1 each have reduced bone density and increased osteoclastogenesis compared to wild type mice. The IFN-inducible guanylate-binding proteins (GBPs) on mouse chromosome 3 (GBP1, GBP2, GBP3, GBP5, GBP7) are required to negatively regulate age-associated bone loss and osteoclastogenesis. Mechanistically, GBP2 and GBP5 both negatively regulate in vitro osteoclast differentiation, and loss of GBP5, but not GBP2, results in greater age-associated bone loss in mice. Moreover, mice deficient in GBP5 or chromosome 3 GBPs have greater LPS-mediated inflammatory bone loss compared to wild type mice. Overall, we find that GBP5 contributes to restricting age-associated and inflammation-induced bone loss by negatively regulating osteoclastogenesis.