Young Bone Research Articles

Role for BLT1 in regulating inflammation within adipose tissue immune cells of aged mice

BackgroundAging is a complex biological process characterized by obesity and immunosenescence throughout the organism. Immunosenescence involves a decline in immune function and the increase in chronic-low grade inflammation, called inflammaging. Adipose tissue expansion, particularly that of visceral adipose tissue (VAT), is associated with an increase in pro-inflammatory macrophages that play an important role in modulating immune responses and producing inflammatory cytokines. The leukotriene B4 receptor 1 (BLT1) is a regulator of obesity-induced inflammation. Its ligand, LTB4, acts as a chemoattractant for immune cells and induces inflammation. Studies have shown that BLT1 is crucial for cytokine production during lipopolysaccharide (LPS) endotoxemia challenge in younger organisms. However, the expression patterns and function of BLT1 in older organisms remains unknown.ResultsIn this study, we investigated BLT1 expression in immune cell subsets within the VAT of aged male and female mice. Moreover, we examined how antagonizing BLT1 signaling could alter the inflammatory response to LPS in aged mice. Our results demonstrate that aged mice exhibit increased adiposity and inflammation, characterized by elevated frequencies of B and T cells, along with pro-inflammatory macrophages in VAT. BLT1 expression is the highest in VAT macrophages. LPS and LTB4 treatment result in increased BLT1 in young and aged bone marrow-derived macrophages (BMDMs). However, LTB4 treatment resulted in amplified Il6 from aged, but not young BMDMs. Treatment of aged mice with the BLT1 antagonist, U75302, followed by LPS-induced endotoxemia resulted in an increase in anti-inflammatory macrophages, reduced phosphorylated NFκB and reduced Il6.ConclusionsThis study provides valuable insights into the age- and sex- specific changes in BLT1 expression on immune cell subsets within VAT. This study offers support for the potential of BLT1 in modulating inflammation in aging.

Single-cell mitochondrial sequencing reveals low-frequency mitochondrial mutations in naturally aging mice.

Mitochondria play a crucial role in numerous biological processes; however, limited methods and research have focused on revealing mitochondrial heterogeneity at the single-cell level. In this study, we optimized the DNBelab C4 single-cell ATAC (assay for transposase-accessible chromatin) sequencing workflow for single-cell mitochondrial sequencing (C4_mtscATAC-seq). We validated the effectiveness of our C4_mtscATAC-seq protocol by sequencing the HEK-293T cell line with two biological replicates, successfully capturing both mitochondrial content (~68% of total sequencing data) and open chromatin status simultaneously. Subsequently, we applied C4_mtscATAC-seq to investigate two mouse tissues, spleen and bone marrow, obtained from two mice aged 2 months and two mice aged 23 months. Our findings revealed higher mitochondrial DNA (mtDNA) content in young tissues compared to more variable mitochondrial content in aged tissues, consistent with higher activity scores of nuclear genes associated with mitochondrial replication and transcription in young tissues. We detected a total of 22, 15, and 21 mtDNA mutations in the young spleen, aged spleen, and bone marrow, respectively, with most variant allele frequencies (VAF) below 1%. Moreover, we observed a higher number of mtDNA mutations with higher VAF in aged tissues compared to young tissues. Importantly, we identified three mtDNA variations (m.9821A>T, m.15219T>C, and m.15984C>T) with the highest VAF in both aged spleen and aged bone marrow. By comparing cells with and without these mtDNA variations, we analyzed differential open chromatin status to identify potential genes associated with these mtDNA variations, including transcription factors such as KLF15 and NRF1. Our study presents an alternative single-cell mitochondrial sequencing method and provides crude insights into age-related single-cell mitochondrial variations.

Three-dimensional chromatin reorganization regulates B cell development during ageing

The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes young and old bone marrow progenitor (pro-) B cells. These changes result in increased interactions at the compartment level and reduced interactions within topologically associated domains (TADs). The gene encoding Ebf1, a key B cell regulator, switches from compartment A to B with age. Genetically reducing Ebf1 recapitulates some features of old pro-B cells. TADs that are most reduced with age contain genes important for B cell development, including the immunoglobulin heavy chain (Igh) locus. Weaker intra-TAD interactions at Igh correlate with altered variable (V), diversity (D) and joining (J) gene recombination. Our observations implicate three-dimensional chromatin reorganization as a major driver of pro-B cell phenotypes that impair B lymphopoiesis with age.

Rejuvenation of peripheral immune cells attenuates Alzheimer's disease-like pathologies and behavioral deficits in a mouse model.

Immunosenescence contributes to systematic aging and plays a role in the pathogenesis of Alzheimer's disease (AD). Therefore, the objective of this study was to investigate the potential of immune rejuvenation as a therapeutic strategy for AD. To achieve this, the immune systems of aged APP/PS1 mice were rejuvenated through young bone marrow transplantation (BMT). Single-cell RNA sequencing revealed that young BMT restored the expression of aging- and AD-related genes in multiple cell types within blood immune cells. The level of circulating senescence-associated secretory phenotype proteins was decreased following young BMT. Notably, young BMT resulted in a significant reduction in cerebral Aβ plaque burden, neuronal degeneration, neuroinflammation, and improvement of behavioral deficits in aged APP/PS1 mice. The ameliorated cerebral amyloidosis was associated with an enhanced Aβ clearance of peripheral monocytes. In conclusion, our study provides evidence that immune system rejuvenation represents a promising therapeutic approach for AD.

Reference intervals for bone marrow cells in juvenile and young adult cats bone marrow cytology.

The current bone marrow (BM) reference intervals (RI) are based on a limited number of cats. Age-related changes in BM variables might be important,possibly affecting the interpretation of the results. Establish BM cytologic reference intervals (RIs) and association of age and sex on these findings, in healthy juvenile and young adult cats. BM aspirates of cats deemed healthy based on history and clinical, CBC, serum chemistry findings, and negative retrovirus serology were obtained and examined cytologically. The examination included a 1000-nucleated differential cell count and cell morphologic assessment. RIs were calculated using parametric, robust, and nonparametric methods. The cytologic findings were examined for associations with sex and age. The study included 40 cats (females, 22 [55%]; males, 18 [45%]) with a median age of 1.5 years (range 0.5-5). Most calculated RIs were similar to those previously reported. BM plasma cell and monocyte counts were weakly and positively correlated with age (rs, .312 and .373, respectively; P < .05). Metarubricytes were higher infemales (mean, 25.1%; SD, 6.0%) than males (mean, 21.2%; SD, 6.0%; P < .05). The BM differential cell counts determined in this study can serve as RIs for cats aged 0.5-5 years.

TREATMENT OF YATROGENIC POST-TRAUMATIC NEUROPATHY ASSOCIATED WITH ENDODONTIC THERAPY USING 3D TECHNOLOGIES

Post-traumatic neuropathy is one of the complications of endodontic treatment, represents a serious problem, manifested by sensory disturbances and has a significant negative impact on the quality of life of patients. Mechanical pressure on the filler causes swelling and hematoma and, due to the chemical action of the substance, neurotoxicity. The decision to treat a patient with trigeminal neuropathy requires complex treatment. Early surgical removal of excess endodontic material resulting from endodontic treatment provides the best prognosis for recovery. With the development of modern digital technologies, advances have been made in the endodontic treatment process. The presented clinical case describes original method for the treatment of endodontic inferior alveolar nerve injuries associated with endodontic therapy using surgical template obtained by three-dimensional software modeling. Using computed tomography software, a template was developed for drilling the bone in the projection of the localization of the material. The surgical template is printed with a 3D printer, after installation in the oral cavity, osteotomies are made under X-ray and excess material is removed, after which the patient is prescribed complex treatment. Results: After 2 weeks, the patient&amp;#39;s complaints disappeared. Sensory impairments were restored and had a positive impact on the patient’s quality of life. A control X-ray examination revealed regeneration of young bone tissue at the osteotomy site. Conclusion: The introduction of digital planning software and the use of 3D printed surgical guides in endodontic microsurgery have significantly improved treatment planning as well as operative and psychological comfort for patients.

In Vitro Osteogenesis Study of Shell Nacre Cement with Older and Young Donor Bone Marrow Mesenchymal Stem/Stromal Cells.

Bone void-filling cements are one of the preferred materials for managing irregular bone voids, particularly in the geriatric population who undergo many orthopedic surgeries. However, bone marrow mesenchymal stem/stromal cells (BM-MSCs) of older-age donors often exhibit reduced osteogenic capacity. Hence, it is crucial to evaluate candidate bone substitute materials with BM-MSCs from the geriatric population to determine the true osteogenic potential, thus simulating the clinical situation. With this concept, we investigated the osteogenic potential of shell nacre cement (SNC), a bone void-filling cement based on shell nacre powder and ladder-structured siloxane methacrylate, using older donor BM-MSCs (age > 55 years) and young donor BM-MSCs (age < 30 years). Direct and indirect cytotoxicity studies conducted with human BM-MSCs confirmed the non-cytotoxic nature of SNC. The standard colony-forming unit-fibroblast (CFU-F) assay and population doubling (PD) time assays revealed a significant reduction in the proliferation potential (p < 0.0001, p < 0.05) in older donor BM-MSCs compared to young donor BM-MSCs. Correspondingly, older donor BM-MSCs contained higher proportions of senescent, β-galactosidase (SA-β gal)-positive cells (nearly 2-fold, p < 0.001). In contrast, the proliferation capacity of older donor BM-MSCs, measured as the area density of CellTrackerTM green positive cells, was similar to that of young donor BM-MSCs following a 7-day culture on SNC. Furthermore, after 14 days of osteoinduction on SNC, scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS) showed that the amount of calcium and phosphorus deposited by young and older donor BM-MSCs on SNC was comparable. A similar trend was observed in the expression of the osteogenesis-related genes BMP2, RUNX2, ALP, COL1A1, OMD and SPARC. Overall, the results of this study indicated that SNC would be a promising candidate for managing bone voids in all age groups.

The osteocytic actions of glucocorticoids on bone mass, mechanical properties, or perilacunar remodeling outcomes are not rescued by PTH(1-34).

Glucocorticoids (GC) and parathyroid hormone (PTH) are widely used therapeutic endocrine hormones where their effects on bone and joint arise from actions on multiple skeletal cell types. In osteocytes, GC and PTH exert opposing effects on perilacunar canalicular remodeling (PLR). Suppressed PLR can impair bone quality and joint homeostasis, including in GC-induced osteonecrosis. However, combined effects of GC and PTH on PLR are unknown. Given the untapped potential to target osteocytes to improve skeletal health, this study sought to test the feasibility of therapeutically mitigating PLR suppression. Focusing on subchondral bone and joint homeostasis, we hypothesize that PTH(1-34), a PLR agonist, could rescue GC-suppressed PLR. The skeletal effects of GC and PTH(1-34), alone or combined, were examined in male and female mice by micro-computed tomography, mechanical testing, histology, and gene expression analysis. For each outcome, females were more responsive to GC and PTH(1-34) than males. GC and PTH(1-34) exerted regional differences, with GC increasing trabecular bone volume but reducing cortical bone thickness, stiffness, and ultimate force. Despite PTH(1-34)'s anabolic effects on trabecular bone, it did not rescue GC's catabolic effects on cortical bone. Likewise, cartilage integrity and subchondral bone apoptosis, tartrate-resistant acid phosphatase (TRAP) activity, and osteocyte lacunocanalicular networks showed no evidence that PTH(1-34) could offset GC-dependent effects. Rather, GC and PTH(1-34) each increased cortical bone gene expression implicated in bone resorption by osteoclasts and osteocytes, including Acp5, Mmp13, Atp6v0d2, Ctsk, differences maintained when GC and PTH(1-34) were combined. Since PTH(1-34) is insufficient to rescue GC's effects on young female mouse bone, future studies are needed to determine if osteocyte PLR suppression, due to GC, aging, or other factors, can be offset by a PLR agonist.

Primary Total Hip Arthroplasty in Juvenile Idiopathic Arthritis: Survivorship After a Median Follow-Up of 12 Years

BackgroundJuvenile idiopathic arthritis (JIA) is a chronic inflammatory condition of childhood that frequently affects the hip. Total hip arthroplasty (THA) in JIA can be challenging due to the patient’s young age, small proportion, complex anatomy, and bone loss. Outcome data are limited. MethodsWe reviewed prospectively collected data in 57 JIA patients (83 hips) who underwent THA between 1986 and 2020 by a single surgeon. The median patient age at surgery was 26 years (range, 14 to 62). Reoperation-free survival was assessed via the cumulative incidence function, accounting for the competing risk of death. Relationships between patient and implant factors and survivorship were evaluated by stratification of the cumulative incidence function and Gray’s tests. Wilcoxon signed rank tests were used to assess the preoperative to latest postoperative change in patient-reported outcome measures. ResultsAt a median (interquartile range) of 12 (4, 20) years of follow-up, 13 (16%) patients underwent reoperation, most commonly for polyethylene wear and osteolysis (7 hips). The estimated incidence of 10-year, 20-year, and 30-year revision (95% confidence interval) were 11.3% (4.5, 21.6%), 18.5% (8.9, 30.9%), and 40.6% (19.4, 60.9%), respectively. There were no differences in survival based on patient age, sex, implant fixation method, polyethylene type, or thickness. All patient-reported outcome measures improved from preoperative to latest follow-up. ConclusionsPrimary THA is a durable and effective treatment for JIA patients with severe hip involvement and results in major improvements in pain and function. We did not identify any factors predictive of failure.

Variación de las propiedades mecánicas del hueso inmaduro en las etapas más tempranas del desarrollo

IntroductionBone as a material varies its composition and mechanical properties throughout life. Although these variations are better understood in adulthood, there is little experimental information on the variation of these properties in early stages of development. The objective of this study is to analyze the mechanical behavior and chemical properties of cortical bone tissue from two animal species in these earliest stages. Material and methodologyTwenty specimens of cortical bone were manufactured from bovine and ovine species that were in different stages of development (feeding exclusively on breast milk, in the transition period to feed or pasture, and young animals but on a solid food diet). The specimens were subjected to tensile tests, recorded with a high-speed camera to obtain deformation maps. Measurements of the tensile force until the specimen broke were also carried out. A fractographic study was carried out with a scanning electron microscope to analyze the fracture surface and an analysis of the amount of calcium in each of the specimens using X-ray dispersion spectroscopy. ResultsA statistically significant and positive correlation was found between the elastic modulus of the specimens and their calcium content. A trend towards more rigid behavior with age was observed. ConclusionsYoung bone tissue tends to stiffen with age as the calcium content increases with an increase in elastic modulus.

Detection and Prevention of ADAR1p150-Induced Hematopoietic Stem and Progenitor Cell Aging

Introduction: Over the last decade, ADAR1p150 has been linked by our research team and others to malignant progenitor reprogramming and therapeutic resistance in a broad array of malignancies. However, ADAR1p150's role in accelerated hematopoietic stem and progenitor cell (HSPC) aging in response to inflammatory cytokines had not been clearly elucidated. Here, we investigate ADAR1 splice isoform switching and the ADAR1p150 inhibitory effects of Rebecsinib on human HSPC function in normal humanized aged normal bone marrow (aNBM) HSPC mouse models. Methods: Whole transcriptome RNA sequencing (RNA-seq) was utilized with splice isoform and editome analysis to detect ADAR1 splice isoform switching and adenosine to inosine RNA editing. Rebecsinib (17S-FD-895), a pre-IND drug candidate, has shown promising potential as an inhibitor of ADAR1p150-mediated RNA editing ( Crews, Ma...Jamieson. Cell Stem Cell 2023), which is an important therapeutic target in patients with myelofibrosis or acute myeloid leukemia that overexpress ADAR1p150 compared with ADAR1p110. Human CD34 + cells, isolated from aged normal bone marrow (aNBM) from patients undergoing elective hip replacement surgery, were transduced with a lentiviral ADAR1-nano-luciferase reporter and transplanted into NSG-SGM3 adult mice that secrete human SCF, IL-3, and GM-CSF cytokines that support normal hematopoiesis. After engraftment confirmation peripheral blood human CD45 flow cytometric assessment and IVIS 200 system imaging of ADAR1-nano-luc-GFP reporter expressing HSPC engraftment, the mice were randomly divided into vehicle-treated and Rebecsinib-treated groups (15mg/kg, twice a week for two weeks). Peripheral blood, bone marrow, and spleen were collected upon treatment completion for analysis. Results: By HSPC RNA-seq, we detected an imbalance between ADAR1p150 and ADAR1p110 expression together with increased RNA editing and splicing alterations using whole transcriptome RNA sequencing (RNA-seq) of aged compared with young bone marrow hematopoietic stem and progenitor cells (HSPCs). However, the functional relevance of ADAR1 splice isoform switching had not been evaluated in human-aged normal bone marrow HSPC xenograft (PDX) mouse models nor had this cytokine-induced HSPC aging been reversed. Flow cytometric analysis and immunohistochemistry staining revealed that Rebecsinib treatment at a dose of 10mg/kg, twice a week for two weeks, effectively spared normal human immune cells (CD3+, CD14+, and CD19+ cells). Remarkably, Rebecsinib treatment not only preserved Hematopoietic Stem (CD34+CD38-) cells and Progenitor (CD34+CD38+) cells in peripheral blood, bone marrow, and spleen but also promoted their expansion. These findings demonstrate the potential of Rebecsinib to enhance the retention of HSPC populations in the bone marrow niche. Conclusions: Our pre-clinical experiments utilizing RNA-seq, a novel ADAR1-nano-luciferase-GFP reporter, and aNBM PDX mouse models provide compelling evidence supporting the potential of Rebecsinib to restore the balance of ADAR1p150:p110 ratios and engraftment in humanized aNBM HSPC mouse models. These findings highlight Rebecsinib as a promising therapeutic candidate for targeting leukemia and myelofibrosis while preserving essential HSPC populations.

Oncostatin M (OSM) Regulates an Inflammatory Cytokine Network in Dnmt3a-Mutant Hematopoietic Stem Cells

During aging, somatic mutations are known to accumulate in the hematopoietic stem and progenitor cell (HSPC) compartment. While most of these somatic mutations are considered ‘neutral’, a subset can confer a selective growth advantage to HSPCs, leading to a condition termed age-associated clonal hematopoiesis (CH). While CH is not a disease per se, it is associated with increased risk of age-associated pathologies such as blood cancers and ischemic stroke. Understanding the cellular and molecular mechanisms underlying the selective advantage of HSPCs harboring somatic mutations, and how and in whom this is a risk factor for age-associated disease, will further inform our understanding of CH-associated conditions. Using an inducible Dnmt3a-mutant mouse model of CH ( Dnmt3a R878H/+), we previously reported that transplant of Dnmt3a-mutant HSCs into 14-month-old recipient mice promotes their selective growth advantage compared to transplant into 2-month-old recipient mice (SanMiguel et al., Cancer Discovery 2022). This led us to hypothesize that differences in soluble factors in the middle-aged versus young bone marrow context contribute to Dnmt3a-mutant HSC expansion. We began with re-analysis of our HSC RNA-seq data from that study, identifying a hallmark inflammatory response signature in Dnmt3a-mutant versus control HSCs in middle-aged but not in young recipient mice. Examining this gene signature, we found that the top gene encoding a soluble factor and increased in expression in Dnmt3a-mutant HSCs was Oncostatin M ( Osm), anIL-6 family cytokine that activates JAK/STAT signaling and is involved in the immunopathogenesis of myeloma. Given that activation of OSM signaling is associated with expanded Dnmt3a-mutant hematopoiesis in the middle-aged BM microenvironment, we hypothesized that OSM as a single stimulus would be sufficient to promote the selective advantage of young Dnmt3a-mutant HSPCs. We found that recombinant OSM did not result in altered proliferation, apoptosis, hematopoietic engraftment, or myeloid differentiation of young Dnmt3a-mutant HSCs. Despite this, we found that young Dnmt3a-mutant HSCs do transcriptionally upregulate an inflammatory cytokine network in response to acute OSM stimulation as evidenced by significant upregulation of the genes encoding IL-6, IL-1β and TNFα. OSM-stimulated Dnmt3a-mutant HSCs also demonstrate upregulation of the anti-inflammatory genes Socs3, Atf3 and Nr4a1 which creates a negative feedback loop that limits sustained activation of the inflammatory network. In the context of an aged bone marrow context with chronically elevated levels of OSM, Dnmt3a-mutant HSCs upregulate pro-inflammatory genes but not the anti-inflammatory genes Socs3, Atf3 and Nr4a1. The results from our studies suggest that chronic inflammation with aging exhausts the regulatory mechanisms that HSCs in young mice use to resolve inflammatory states and that OSM is a master regulator of an inflammatory network that contributes to age-associated CH.

In vitro biomechanical analysis of a locking self-compression screw model applied to Pauwels III and comminuted femoral neck fractures

Femoral neck fractures (FNFs) affect the young adult population and are intimately related to high-energy trauma. Despite innovations in osteosynthesis materials, the rate of complications remains at 10%–59% in Pauwels type III (PIII) fractures. The authors thus propose a fixation model with a novel self-compression screw, comparing it to a sliding hip screw plate associated with a derotation screw in the fixation of a PIII fracture with posterior inferior comminution. The finite element method (FEM) was used in this comparison along with a virtual femur model with structural characteristics similar to those of a healthy young human bone. We formed 4 groups: Group 1 (GC), intact bone; Group 2 (SHS+S), sliding hip screw plate with derotation screw; Group 3 (XS), X-pin + SS (self-compression model with superior positioning screw); Group 4 (XI), X-pin + IS (self-compression model with inferior positioning screw). A 700 N monotonic load was applied to the apex of the femur head towards the ground so that stress was mainly focused on the fracture site and osteosynthesis. Analyses included total displacement and maximum principal stress and were performed for all groups. Fracture displacement, rotation, and von Mises were assessed only in groups that underwent osteosynthesis. Total displacement values in groups with self-compression screws (XS and XI) were closer to those for healthy femurs, with a 38.5% reduction when comparing the XS group with the SHS+S group. Fracture displacement and rotation values presented reductions of over 60% when comparing the XS and XI groups with the SHS+S group. Equivalent Von Mises stress values were similar between XS and XI and presented a reduction of approximately 5.25% when compared with the SHS+S group. Our FEM analyses demonstrated that the self-compression screw model has potential biomechanical advantages over the SHS+S model.

Adenovirus-Based Gene Therapy for Bone Regeneration: A Comparative Analysis of In Vivo and Ex Vivo BMP2 Gene Delivery.

Adenovirus-mediated gene therapy is a promising tool in bone regenerative medicine. In this work, gene-activated matrices (GAMs) composed of (1) polylactide granules (PLA), which serve as a depot for genetic constructs or matrices for cell attachment, (2) a PRP-based fibrin clot, which is a source of growth factors and a binding gel, and (3) a BMP2 gene providing osteoinductive properties were studied. The study aims to compare the effectiveness of in vivo and ex vivo gene therapy based on adenoviral constructs with the BMP2 gene, PLA particles, and a fibrin clot for bone defect healing. GAMs with Ad-BMP2 and MSC(Ad-BMP2) show osteoinductive properties both in vitro and in vivo. However, MSCs incubated with GAMs containing transduced cells showed a more significant increase in osteopontin gene expression, protein production, Alpl activity, and matrix mineralization. Implantation of the studied matrices into critical-size calvarial defects after 56 days promotes the formation of young bone. The efficiency of neoosteogenesis and the volume fraction of newly formed bone tissue are higher with PLA/PRP-MSC(Ad-BMP2) implantation (33%) than PLA/PRP-Ad-BMP2 (28%). Thus, ex vivo adenoviral gene therapy with the BMP2 gene has proven to be a more effective approach than the in vivo delivery of gene constructs for bone regeneration.

Young Sca-1+ bone marrow stem cell-derived exosomes preserve visual function via the miR-150-5p/MEKK3/JNK/c-Jun pathway to reduce M1 microglial polarization

BackgroundPolarization of microglia, the resident retinal immune cells, plays important roles in mediating both injury and repair responses post-retinal ischemia–reperfusion (I/R) injury, which is one of the main pathological mechanisms behind ganglion cell apoptosis. Aging could perturb microglial balances, resulting in lowered post-I/R retinal repair. Young bone marrow (BM) stem cell antigen 1-positive (Sca-1+) cells have been demonstrated to have higher reparative capabilities post-I/R retinal injury when transplanted into old mice, where they were able to home and differentiate into retinal microglia.MethodsExosomes were enriched from young Sca-1+ or Sca-1− cells, and injected into the vitreous humor of old mice post-retinal I/R. Bioinformatics analyses, including miRNA sequencing, was used to analyze exosome contents, which was confirmed by RT-qPCR. Western blot was then performed to examine expression levels of inflammatory factors and underlying signaling pathway proteins, while immunofluorescence staining was used to examine the extent of pro-inflammatory M1 microglial polarization. Fluoro-Gold labelling was then utilized to identify viable ganglion cells, while H&E staining was used to examine retinal morphology post-I/R and exosome treatment.ResultsSca-1+ exosome-injected mice yielded better visual functional preservation and lowered inflammatory factors, compared to Sca-1−, at days 1, 3, and 7 days post-I/R. miRNA sequencing found that Sca-1+ exosomes had higher miR-150-5p levels, compared to Sca-1− exosomes, which was confirmed by RT-qPCR. Mechanistic analysis found that miR-150-5p from Sca-1+ exosomes repressed the mitogen-activated protein kinase kinase kinase 3 (MEKK3)/JNK/c-Jun axis, leading to IL-6 and TNF-α downregulation, and subsequently reduced microglial polarization, all of which contributes to reduced ganglion cell apoptosis and preservation of proper retinal morphology.ConclusionThis study elucidates a potential new therapeutic approach for neuroprotection against I/R injury, via delivering miR-150-5p-enriched Sca-1+ exosomes, which targets the miR-150-5p/MEKK3/JNK/c-Jun axis, thereby serving as a cell-free remedy for treating retinal I/R injury and preserving visual functioning.

Comparative analysis of Tet2 catalytic-deficient and knockout bone marrow over time

TET2 is a member of the Ten-eleven translocation (Tet) family of DNA dioxygenases that regulate gene expression by promoting DNA demethylation (enzymatic activity) and partnering with chromatin regulatory complexes (nonenzymatic functions). TET2 is highly expressed in the hematopoietic lineage, where its molecular functions are the subject of continuous investigations because of the prevalence of TET2 mutations in hematologic malignancies. Previously, we have implicated Tet2 catalytic and noncatalytic functions in the regulation of myeloid and lymphoid lineages, respectively. However, the impact of these functions of Tet2 on hematopoiesis as the bone marrow ages remains unclear. Here, we conducted comparative transplantations and transcriptomic analyses of 3-, 6-, 9-, and 12-month-old Tet2 catalytic mutant (Mut) and knockout (KO) bone marrow. Tet2 Mut bone marrow of all ages exclusively caused hematopoietic disorders of the myeloid lineage. In contrast, young Tet2 KO bone marrow developed both lymphoid and myeloid diseases, whereas older Tet2 KO bone marrow predominantly elicited myeloid disorders with shorter latency than age-matched Tet2 Mut bone marrow. We identified robust gene dysregulation in Tet2 KO Lin- cells at 6 months that involved lymphoma and myelodysplastic syndrome and/or leukemia-causing genes, many of which were hypermethylated early in life. There was a shift from lymphoid to myeloid gene deregulation in Tet2 KO Lin- cells with age, underpinning the higher incidence of myeloid diseases. These findings expand on the dynamic regulation of bone marrow by Tet2 and show that its catalytic-dependent and -independent roles have distinct impacts on myeloid and lymphoid lineages with age.

Elastic Modulus and Its Relation to Apparent Mineral Density in Juvenile Equine Bones of the Lower Limb.

Density-modulus relationships are necessary to develop finite element models of bones that may be used to evaluate local tissue response to different physical activities. It is unknown if juvenile equine trabecular bone may be described by the same density-modulus as adult equine bone, and how the density-modulus relationship varies with anatomical location and loading direction. To answer these questions, trabecular bone cores from the third metacarpal (MC3) and proximal phalanx (P1) bones of juvenile horses (age <1 yr) were machined in the longitudinal (n = 134) and transverse (n = 90) directions and mechanically tested in compression. Elastic modulus was related to apparent computed tomography density of each sample using power law regressions. We found that density-modulus relationships for juvenile equine trabecular bone were significantly different for each anatomical location (MC3 versus P1) and orientation (longitudinal versus transverse). Use of the incorrect density-modulus relationship resulted in increased root mean squared percent error of the modulus prediction by 8-17%. When our juvenile density-modulus relationship was compared to one of an equivalent location in adult horses, the adult relationship resulted in an approximately 80% increase in error of the modulus prediction. Moving forward, more accurate models of young bone can be developed and used to evaluate potential exercise regimens designed to encourage bone adaptation.

Pro-Osteogenic and Anti-Inflammatory Synergistic Effect of Orthosilicic Acid, Vitamin K2, Curcumin, Polydatin and Quercetin Combination in Young and Senescent Bone Marrow-Derived Mesenchymal Stromal Cells.

During aging, bone marrow mesenchymal stromal cells (MSCs)-the precursors of osteoblasts-undergo cellular senescence, losing their osteogenic potential and acquiring a pro-inflammatory secretory phenotype. These dysfunctions cause bone loss and lead to osteoporosis. Prevention and intervention at an early stage of bone loss are important, and naturally active compounds could represent a valid help in addition to diet. Here, we tested the hypothesis that the combination of two pro-osteogenic factors, namely orthosilicic acid (OA) and vitamin K2 (VK2), and three other anti-inflammatory compounds, namely curcumin (CUR), polydatin (PD) and quercetin (QCT)-that mirror the nutraceutical BlastiMin Complex® (Mivell, Italy)-would be effective in promoting MSC osteogenesis, even of replicative senescent cells (sMSCs), and inhibiting their pro-inflammatory phenotype in vitro. Results showed that when used at non-cytotoxic doses, (i) the association of OA and VK2 promoted MSC differentiation into osteoblasts, even when cultured without other pro-differentiating factors; and (ii) CUR, PD and QCT exerted an anti-inflammatory effect on sMSCs, and also synergized with OA and VK2 in promoting the expression of the pivotal osteogenic marker ALP in these cells. Overall, these data suggest a potential role of using a combination of all of these natural compounds as a supplement to prevent or control the progression of age-related osteoporosis.

Osteocyte apoptosis and cellular micropetrosis signify skeletal aging in type 1 diabetes.

Bone fragility is a profound complication of type 1 diabetes mellitus (T1DM), increasing patient morbidity. Within the mineralized bone matrix, osteocytes build a mechanosensitive network that orchestrates bone remodeling; thus, osteocyte viability is crucial for maintaining bone homeostasis. In human cortical bone specimens from individuals with T1DM, we found signs of accelerated osteocyte apoptosis and local mineralization of osteocyte lacunae (micropetrosis) compared with samples from age-matched controls. Such morphological changes were seen in the relatively young osteonal bone matrix on the periosteal side, and micropetrosis coincided with microdamage accumulation, implying that T1DM drives local skeletal aging and thereby impairs the biomechanical competence of the bone tissue. The consequent dysfunction of the osteocyte network hampers bone remodeling and decreases bone repair mechanisms, potentially contributing to the enhanced fracture risk seen in individuals with T1DM. STATEMENT OF SIGNIFICANCE: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that causes hyperglycemia. Increased bone fragility is one of the complications associated with T1DM. Our latest study on T1DM-affected human cortical bone identified the viability of osteocytes, the primary bone cells, as a potentially critical factor in T1DM-bone disease. We linked T1DM with increased osteocyte apoptosis and local accumulation of mineralized lacunar spaces and microdamage. Such structural changes in bone tissue suggest that T1DM speeds up the adverse effects of aging, leading to the premature death of osteocytes and potentially contributing to diabetes-related bone fragility.

Bone marrow endosteal stem cells dictate active osteogenesis and aggressive tumorigenesis

The bone marrow contains various populations of skeletal stem cells (SSCs) in the stromal compartment, which are important regulators of bone formation. It is well-described that leptin receptor (LepR)+ perivascular stromal cells provide a major source of bone-forming osteoblasts in adult and aged bone marrow. However, the identity of SSCs in young bone marrow and how they coordinate active bone formation remains unclear. Here we show that bone marrow endosteal SSCs are defined by fibroblast growth factor receptor 3 (Fgfr3) and osteoblast-chondrocyte transitional (OCT) identities with some characteristics of bone osteoblasts and chondrocytes. These Fgfr3-creER-marked endosteal stromal cells contribute to a stem cell fraction in young stages, which is later replaced by Lepr-cre-marked stromal cells in adult stages. Further, Fgfr3+ endosteal stromal cells give rise to aggressive osteosarcoma-like lesions upon loss of p53 tumor suppressor through unregulated self-renewal and aberrant osteogenic fates. Therefore, Fgfr3+ endosteal SSCs are abundant in young bone marrow and provide a robust source of osteoblasts, contributing to both normal and aberrant osteogenesis.