Search

Purpose of Review: The purpose of this review is to highlight the evidence of microvascular dysfunction in bone and marrow and its relation to poor skeletal outcomes in diabetes mellitus., Recent Findings: Diabetes mellitus is characterized by chronic hyperglycemia, which may lead to microangiopathy and macroangiopathy. Micro- and macroangiopathy have been diagnosed in Type 1 and Type 2 diabetes, coinciding with osteopenia, osteoporosis, enhanced fracture risk and delayed fracture healing. Microangiopathy has been reported in the skeleton, correlating with reduced blood flow and perfusion, vasomotor dysfunction, microvascular rarefaction, reduced angiogenic capabilities, and augmented vascular permeability. Microangiopathy within the skeleton may be detrimental to bone and manifest as, among other clinical abnormalities, reduced mass, enhanced fracture risk, and delayed fracture healing. More investigations are required to elucidate the various mechanisms by which diabetic microvascular dysfunction impacts the skeleton., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The mechanical stimuli generated by body exercise can be transmitted from cortical bone into the deep bone marrow (mechanopropagation). Excitingly, a mechanosensitive perivascular stem cell niche is recently identified within the bone marrow for osteogenesis and lymphopoiesis. Although it is long known that they are maintained by exercise-induced mechanical stimulation, the mechanopropagation from compact bone to deep bone marrow vasculature remains elusive of this fundamental mechanobiology field. No experimental system is available yet to directly understand such exercise-induced mechanopropagation at the bone-vessel interface. To this end, taking advantage of the revolutionary in vivo 3D deep bone imaging, an integrated computational biomechanics framework to quantitatively evaluate the mechanopropagation capabilities for bone marrow arterioles, arteries, and sinusoids is devised. As a highlight, the 3D geometries of blood vessels are smoothly reconstructed in the presence of vessel wall thickness and intravascular pulse pressure. By implementing the 5-parameter Mooney-Rivlin model that simulates the hyperelastic vessel properties, finite element analysis to thoroughly investigate the mechanical effects of exercise-induced intravascular vibratory stretching on bone marrow vasculature is performed. In addition, the blood pressure and cortical bone bending effects on vascular mechanoproperties are examined. For the first time, movement-induced mechanopropagation from the hard cortical bone to the soft vasculature in the bone marrow is numerically simulated. It is concluded that arterioles and arteries are much more efficient in propagating mechanical force than sinusoids due to their stiffness. In the future, this in-silico approach can be combined with other clinical imaging modalities for subject/patient-specific vascular reconstruction and biomechanical analysis, providing large-scale phenotypic data for personalized mechanobiology discovery., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Multiple myeloma (MM) is an incurable B cell malignancy characterized by the accumulation of monoclonal abnormal plasma cells in the bone marrow (BM). It has been a significant challenge to study the spatiotemporal interactions of MM cancer cells with the embedded microenvironments of BM. Here we report a microfluidic device which was designed to mimic several physiological features of the BM niche: (1) sinusoidal circulation, (2) sinusoidal endothelium, and (3) stroma. The endothelial and stromal compartments were constructed and used to demonstrate the device's utility by spatiotemporally characterizing the CXCL12-mediated egression of MM cells from the BM stroma and its effects on the barrier function of endothelial cells (ECs). We found that the egression of MM cells resulted in less organized and loosely connected ECs, the widening of EC junction pores, and increased permeability through ECs, but without significantly affecting the number density of viable ECs. The results suggest that the device can be used to study the physical and secreted factors determining the trafficking of cancer cells through BM. The sinusoidal flow feature of the device provides an integral element for further creating systemic models of cancers that reside or metastasize to the BM niche., (© 2022. The Author(s).)

The fate of hematopoietic stem cells (HSCs) can be directed by microenvironmental factors including extracellular calcium ion concentration ([Ca 2+ ] e ), but the local [Ca 2+ ] e around individual HSCs in vivo remains unknown. Here we develop intravital ratiometric analyses to quantify the absolute pH and [Ca 2+ ] e in the mouse calvarial bone marrow, taking into account the pH sensitivity of the calcium probe and the wavelength-dependent optical loss through bone. Unexpectedly, the mean [Ca 2+ ] e in the bone marrow (1.0 ± 0.54 mM) is not significantly different from the blood serum, but the HSCs are found in locations with elevated local [Ca 2+ ] e (1.5 ± 0.57 mM). With aging, a significant increase in [Ca 2+ ] e is found in M-type cavities that exclusively support clonal expansion of activated HSCs. This work thus establishes a tool to investigate [Ca 2+ ] e and pH in the HSC niche with high spatial resolution and can be broadly applied to other tissue types., (© 2022. The Author(s).)

Endothelial cells play a critical role in supporting postnatal hematopoiesis in the bone marrow. Unique endothelial cells, together with various perivascular cells, form different types of vascular structures, constructing a vast microvascular delivery and trafficking network for blood cells, oxygen, and nutrition. These blood vessels build distinct hematopoietic stem and progenitor cell niches, which offer not only sites of residence for blood cells but also indispensable signals directing HSPC homing, self-renewal, and multilineage differentiation. Deep insight into the structure and function of the BM vasculature niche and its participation in hematopoiesis is necessary to develop advanced strategies for the reconstitution of hematopoiesis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bone marrow (BM) angiogenesis significantly influences disease progression in multiple myeloma (MM) patients and correlates with adverse prognosis. The present study shows a statistically significant correlation of the AP-1 family member JunB with VEGF, VEGFB, and IGF1 expression levels in MM. In contrast to the angiogenic master regulator Hif-1α, JunB protein levels were independent of hypoxia. Results in tumor-cell models that allow the induction of JunB knockdown or JunB activation, respectively, corroborated the functional role of JunB in the production and secretion of these angiogenic factors (AFs). Consequently, conditioned media derived from MM cells after JunB knockdown or JunB activation either inhibited or stimulated in vitro angiogenesis. The impact of JunB on MM BM angiogenesis was finally confirmed in a dynamic 3D model of the BM microenvironment, a xenograft mouse model as well as in patient-derived BM sections. In summary, in continuation of our previous study (Fan et al., 2017), the present report reveals for the first time that JunB is not only a mediator of MM cell survival, proliferation, and drug resistance, but also a promoter of AF transcription and consequently of MM BM angiogenesis. Our results thereby underscore worldwide efforts to target AP-1 transcription factors such as JunB as a promising strategy in MM therapy., (© 2021. The Author(s).)

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized β-catenin in BM-SEC (Ctnnb1 OE-SEC ) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII-PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1 OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1 OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease., (© 2021. The Author(s).)

Neural differentiation of mesenchymal stromal cells has been widely studied. However, a comparative characterization of ultrastructural changes during neural differentiation has not been performed. In this study, we conducted scanning electron microscopy and transmission electron microscopy analysis to show the morphological changes in mesenchymal stromal cells upon induction of neural differentiation. In addition, transmission electron microscopy results demonstrated ultrastructural differences between human cranial bone marrow mesenchymal stromal cells and iliac crest bone marrow mesenchymal stromal cells. We propose that enriched microvesicles in cranial bone marrow mesenchymal stromal cells may be responsible for the increased efficiency of neural differentiation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: During acute myeloid leukemia (AML) growth, the bone marrow (BM) niche acquires significant vascular changes that can be offset by therapeutic blast cytoreduction. The molecular mechanisms of this vascular plasticity remain to be fully elucidated. Herein, we report on the changes that occur in the vascular compartment of the FLT3-ITD+ AML BM niche pre and post treatment and their impact on leukemic stem cells (LSCs)., Methods: BM vasculature was evaluated in FLT3-ITD+ AML models (Mll PTD/WT /Flt3 ITD/ITD mouse and patient-derived xenograft) by 3D confocal imaging of long bones, calvarium vascular permeability assays, and flow cytometry analysis. Cytokine levels were measured by Luminex assay and miR-126 levels evaluated by Q-RT-PCR and miRNA staining. Wild-type (wt) and Mll PTD/WT /Flt3 ITD/ITD mice with endothelial cell (EC) miR-126 knockout or overexpression served as controls. The impact of treatment-induced BM vascular changes on LSC activity was evaluated by secondary transplantation of BM cells after administration of tyrosine kinase inhibitors (TKIs) to Mll PTD/WT /Flt3 ITD/ITD mice with/without either EC miR-126 KO or co-treatment with tumor necrosis factor alpha (TNFα) or anti-miR-126 miRisten., Results: In the normal BM niche, CD31 + Sca-1 high ECs lining arterioles have miR-126 levels higher than CD31 + Sca-1 low ECs lining sinusoids. We noted that during FLT3-ITD+ AML growth, the BM niche lost arterioles and gained sinusoids. These changes were mediated by TNFα, a cytokine produced by AML blasts, which induced EC miR-126 downregulation and caused depletion of CD31 + Sca-1 high ECs and gain in CD31 + Sca-1 low ECs. Loss of miR-126 high ECs led to a decreased EC miR-126 supply to LSCs, which then entered the cell cycle and promoted leukemia growth. Accordingly, antileukemic treatment with TKI decreased the BM blast-produced TNFα and increased miR-126 high ECs and the EC miR-126 supply to LSCs. High miR-126 levels safeguarded LSCs, as shown by more severe disease in secondary transplanted mice. Conversely, EC miR-126 deprivation via genetic or pharmacological EC miR-126 knock-down prevented treatment-induced BM miR-126 high EC expansion and in turn LSC protection., Conclusions: Treatment-induced CD31 + Sca-1 high EC re-vascularization of the leukemic BM niche may represent a LSC extrinsic mechanism of treatment resistance that can be overcome with therapeutic EC miR-126 deprivation., (© 2021. The Author(s).)

Background: Iron deficiency anemia (IDA)-induced reactive thrombocytosis can occur in children and adults. The underlying mechanism for this phenomenon is indeterminate. Traditional cytokines such as thrombopoietin (TPO), interleukin-6 (IL-6), and IL-11 involved in megakaryopoiesis have not been shown to be the cause. Recent studies suggest that growth factors and signaling molecules involved with angiogenesis influence the proliferation and differentiation of megakaryocytes., Methods: We investigated the possible association between angiogenic cytokines with reactive thrombocytosis due to IDA in an iron-deficient (ID) rat model. Complete blood count, iron panels, and TPO levels were measured at baseline and 5 weeks later in both control (C) and ID rats. Angiogenic cytokines were evaluated in the bone marrow in all rats., Results: We successfully induced IDA in our rats by phlebotomy and reduced iron diet. We did not find an increase of TPO in ID rats. A review of the bone marrow showed an increase in the number of megakaryocytes, vascular structures, as well as increased intensity of stain for vascular endothelial growth factor (VEGF), and CXC chemokine receptor 4 (CXCR4) in rats with IDA compared to controls., Conclusions: Our results of histological bone marrow data suggest an important role for angiogenesis in the development of IDA-induced thrombocytosis., Impact: Thrombocytosis is common with IDA in both children and adults, but the mechanism is unclear. We confirmed that TPO is not the major driver of iron deficiency-associated thrombocytosis. We confirmed the increase in the number of megakaryocytes in the bone marrow despite stable TPO levels. We provided evidence supporting an important role of angiogenesis in megakaryocytopoiesis/thrombopoiesis with increased vascular structures and angiogenic cytokines in the bone marrow of iron-deficient rats. The demonstration that angiogenesis may play an important role in secondary thrombocytosis could lead to a new approach in treating symptomatic reactive thrombocytosis by targeting angiogenesis., (© 2021. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

This translational study aimed at gaining insight into the effects of lenalidomide in acute myeloid leukemia (AML). Forty-one AML patients aged 66 or older of the Swiss cohort of the HOVON-103 AML/SAKK30/10 study were included. After randomization, they received standard induction chemotherapy with or without lenalidomide. Bone marrow biopsies at diagnosis and before the 2nd induction cycle were obtained to assess the therapeutic impact on leukemic blasts and microenvironment. Increased bone marrow angiogenesis, as assessed by microvessel density (MVD), was found at AML diagnosis and differed significantly between the WHO categories. Morphological analysis revealed a higher initial MVD in AML with myelodysplasia-related changes (AML-MRC) and a more substantial decrease of microvascularization after lenalidomide exposure. A slight increase of T-bet-positive TH1-equivalents was identifiable under lenalidomide. In the subgroup of patients with AML-MRC, the progression-free survival differed between the two treatment regimens, showing a potential but not significant benefit of lenalidomide. We found no correlation between the cereblon genotype (the target of lenalidomide) and treatment response or prognosis. In conclusion, addition of lenalidomide may be beneficial to elderly patients suffering from AML-MRC, where it leads to a reduction of microvascularization and, probably, to an intensified specific T cell-driven anti-leukemic response.

Objective: The vascularization of subchondral bone plays a significant role in the progression of knee osteoarthritis (OA). Treatment with platelet-rich plasma (PRP) has positive effects on cartilage lesions. However, PRP's efficacy for subchondral bone marrow lesions and the relationship of these lesions to cartilage are still undiscovered. Therefore, our aims were first to longitudinally investigate the change in subchondral flow by dynamic contrast enhanced MRI and degeneration of cartilage by MRI T2 ∗ in an anterior cruciate transection rodent (ACLT) model, and second to examine changes in parameters after intra-articular PRP injection., Design: A 32-week investigation in 18 rats allocated to sham-control, ACLT with normal saline injection (ACLT + NS), and ACLT with PRP injection groups ended with histological evaluation. Another rat was used as a donor of allogenic PRP., Results: Compared to the sham-control group, the ACLT + NS group had higher subchondral blood volume A (0.051, 95% confidence interval: 0.009, 0.092) and lower venous washout k el (-0.030: -0.055, -0.005) from week 4; lower permeability k ep from week 18 (-0.954: -1.339, -0.569); higher cartilage T2 ∗ values (1.803: 1.504, 2.102) reflecting collagen loss beginning at week 10. For the PRP treatment group, subchondral bone marrow A and cartilage T2 ∗ decreased from week 10. Histological results confirmed and were correlated with the MRI findings., Conclusion: Subchondral hyper-perfusion plays a vital role in the pathogenesis of OA and was associated with cartilage degeneration. The efficacy of PRP can be observed from reduced perfusion and MRI T2 ∗ values., (Copyright © 2021 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The bone marrow is a complex ecosystem in which hematopoietic and non-hematopoietic cells reside. In this review, we discuss the bone marrow niches in mice that facilitate the survival, maintenance, and differentiation of cells of hematopoietic origin based on the recent literature. Our review places a special focus on the hematopoietic multipotent progenitors and on plasma cells, corresponding to the last stage of the B-cell lineage, that play a key role in the humoral memory response. We highlight the similarities between the microenvironments necessary for the establishment and the maintenance of these two immune cell subsets, and how the chemokine CXCL12/CXCR4 signaling axis contributes to these processes. Finally, we bring elements to address the following question: are multipotent progenitors and plasma cells neighbors or roommates within the bone marrow?, Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bonaud, Lemos, Espéli and Balabanian.)

The bone marrow is the major hematopoietic organ, consisting of distinct microenvironmental niches for the production of hematopoietic cells. Advanced visualizing methods are required to define and better understand the interactions between stromal and hematopoietic cells. In this chapter, we describe an ex vivo whole-mount imaging technique of the bone marrow, which allows for a fast, high-quality, and three-dimensional visualization of different bone marrow components. We provide a guide for conducting adoptive transfer experiments of fluorescently labeled leukocytes and visualizing their location in the bone marrow with respect to the bone marrow vasculature. This method presents a quick, easy, and inexpensive approach to image the bone marrow in three dimensions.

Purpose: Although vascular alterations in solid tumor malignancies are known to decrease therapeutic delivery, the effects of leukemia-induced bone marrow vasculature (BMV) alterations on therapeutic delivery are not well known. Additionally, functional quantitative measurements of the leukemic BMV during chemotherapy and radiation therapy are limited, largely due to a lack of high-resolution imaging techniques available preclinically. This study develops a murine model using compartmental modeling for quantitative multiphoton microscopy (QMPM) to characterize the malignant BMV before and during treatment., Methods and Materials: Using QMPM, live time-lapsed images of dextran leakage from the local BMV to the surrounding bone marrow of mice bearing acute lymphoblastic leukemia (ALL) were taken and fit to a 2-compartment model to measure the transfer rate (K trans ), fractional extracellular extravascular space (ν ec ), and vascular permeability parameters, as well as functional single-vessel characteristics. In response to leukemia-induced BMV alterations, the effects of 2 to 4 Gy low-dose radiation therapy (LDRT) on the BMV, drug delivery, and mouse survival were assessed post-treatment to determine whether neoadjuvant LDRT before chemotherapy improves treatment outcome., Results: Mice bearing ALL had significantly altered K trans , increased ν ec , and increased permeability compared with healthy mice. Angiogenesis, decreased single-vessel perfusion, and decreased vessel diameter were observed. BMV alterations resulted in disease-dependent reductions in cellular uptake of Hoechst dye. LDRT to mice bearing ALL dilated BMV, increased single-vessel perfusion, and increased daunorubicin uptake by ALL cells. Consequently, LDRT administered to mice before receiving nilotinib significantly increased survival compared with mice receiving LDRT after nilotinib, demonstrating the importance of LDRT conditioning before therapeutic administration., Conclusion: The developed QMPM enables single-platform assessments of the pharmacokinetics of fluorescent agents and characterization of the BMV. Initial results suggest BMV alterations after neoadjuvant LDRT may contribute to enhanced drug delivery and increased treatment efficacy for ALL. The developed QMPM enables observations of the BMV for use in ALL treatment optimization., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Multiple Myeloma (MM) induces bone destruction, decreases bone formation, and increases marrow angiogenesis in patients. We reported that osteocytes (Ocys) directly interact with MM cells to increase tumor growth and expression of Ocy-derived factors that promote bone resorption and suppress bone formation. However, the contribution of Ocys to enhanced marrow vascularization in MM is unclear. Since the MM microenvironment is hypoxic, we assessed if hypoxia and/or interactions with MM cells increases pro-angiogenic signaling in Ocys. Hypoxia and/or co-culture with MM cells significantly increased Vegf-a expression in MLOA5-Ocys, and conditioned media (CM) from MLOA5s or MM-MLOA5 co-cultured in hypoxia, significantly increased endothelial tube length compared to normoxic CM. Further, Vegf-a knockdown in MLOA5s or primary Ocys co-cultured with MM cells or neutralizing Vegf-a in MM-Ocy co-culture CM completely blocked the increased endothelial activity. Importantly, Vegf-a-expressing Ocy numbers were significantly increased in MM-injected mouse bones, positively correlating with tumor vessel area. Finally, we demonstrate that direct contact with MM cells increases Ocy Fgf23, which enhanced Vegf-a expression in Ocys. Fgf23 deletion in Ocys blocked these changes. These results suggest hypoxia and MM cells induce a pro-angiogenic phenotype in Ocys via Fgf23 and Vegf-a signaling, which can promote MM-induced marrow vascularization.

Purpose: To describe bone perfusion and adiposity beyond the necrotic zone with quantitative MRI techniques in osteonecrosis of the femoral head (ONFH)., Method: In this cross-sectional multicentre study, we recruited patients suffering from late-stage ONFH or hip osteoarthritis. Hip MRI included quantitative MRI sequences: chemical-shift imaging and dynamic contrast-enhanced MRI. We drew regions of interest inside the necrotic zone (inner necrosis and its border) and outside (femoral head, neck and greater trochanter) in ONFH. In the control group, regions of interest were drawn in the femoral head, femoral neck and the greater trochanter. For each region of interest, we measured fat fraction, and calculated semi-quantitative (area under the curve, initial slope) and pharmacokinetic perfusion parameters (K trans and K ep )., Results: Thirty-two male adults (mean age 58 ± 9 years, range 38-74 years) were included. Sixteen patients formed the ONFH group and fifteen the control group; one was excluded. In the normal-appearing non-necrotic part of the femoral head, fat fraction was not significantly different in comparison with controls (p = 1), but Ktrans was significantly lower than in controls (0.012 ± 0.018 vs. 0.027 ± 0.045; p = 0.05). This perfusion parameter reflects exchanges between blood microvessels and bone marrow., Conclusions: Our results question the concept of adipose toxicity on the macroscopic scale, and bring up the concept of regional ischemic penumbra that goes beyond the visible necrotic zone. Further studies are required to test these hypotheses in larger populations and earlier disease states., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal and multidirectional differentiation capabilities. Dimethyloxalyl glycine (DMOG) mobilizes MSCs, and the hypoxia inducible factor-1 (HIF-1) signaling pathway plays an important role in MSC mobilization. We aimed to investigate the effect of DMOG on the HIF-1 pathway in MSCs. Rats were treated with DMOG, and the numbers of peripheral blood MSCs (PB-MSCs) and bone marrow MSCs (BM-MSCs) were detected by the Colony-forming unit fibroblastic (CFU-F) method. The growth curve, cell cycle and migration ability of PB-MSCs and BM-MSCs were detected by CCK-8, Flow cytometry and Transwell assays. Western blotting and real-time qPCR were used to detect the expression of the HIF-1 pathway. The number of bone marrow microvessels was detected by immunohistochemistry. DMOG significantly increased the numbers of PB-MSCs and BM-MSCs (P < 0.05). Further, the MSCs in peripheral blood and bone marrow still had the ability to proliferate and migrate after mobilization by DMOG. The expression levels of HIF-1α, stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) in MSCs were significantly regulated by DMOG (P < 0.05). The number of bone marrow microvessels decreased after the VEGF/VEGFR signaling pathway was blocked by SU5416 (P < 0.05). Therefore, these findings demonstrated that DMOG regulates the HIF-1α signaling pathway and promotes biological effects in MSCs.

Blood vessels serve as a versatile transport system and play crucial roles in organ development, regeneration, and stem cell behavior. In the skeletal system, certain capillaries support perivascular stem cells or osteoprogenitor cells and thereby regulate bone formation. Recent studies reported that a specialized capillary subtype, termed type H vessels, is shown to couple angiogenesis and osteogenesis in rodents and humans. They can be distinguished by specific cell surface markers, as the endothelial cells in the metaphysis and endosteum highly express the junctional protein CD31 and the sialoglycoprotein endomucin. Here, we provide an overview of the role of type H vessels in bone homeostasis and summarize their linkage with various cytokines to control bone cell behavior and bone formation. We also discuss the potential clinical application for bone disorders by targeting these specific vessels according to their physiological and pathobiological settings., (© 2020 New York Academy of Sciences.)

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81 -/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.

The skeleton is highly vascularized due to the various roles blood vessels play in the homeostasis of bone and marrow. For example, blood vessels provide nutrients, remove metabolic by-products, deliver systemic hormones, and circulate precursor cells to bone and marrow. In addition to these roles, bone blood vessels participate in a variety of other functions. This article provides an overview of the afferent, exchange and efferent vessels in bone and marrow and presents the morphological layout of these blood vessels regarding blood flow dynamics. In addition, this article discusses how bone blood vessels participate in bone development, maintenance, and repair. Further, mechanical loading-induced bone adaptation is presented regarding interstitial fluid flow and pressure, as regulated by the vascular system. The role of the sympathetic nervous system is discussed in relation to blood vessels and bone. Finally, vascular participation in bone accrual with intermittent parathyroid hormone administration, a medication prescribed to combat age-related bone loss, is described and age- and disease-related impairments in blood vessels are discussed in relation to bone and marrow dysfunction. © 2020 American Physiological Society. Compr Physiol 10:1009-1046, 2020., (Copyright © 2020 American Physiological Society. All rights reserved.)

Objective: To investigate the association between pain and perfusion in bone marrow lesions with and without cysts assessed dynamic contrast-enhanced (DCE)-MRI in patients with knee osteoarthritis., Subjects and Methods: In a cross-sectional setting, perfusion in bone marrow lesions was assessed using 3 Tesla MRI and correlated (Spearman's rank correlation) to pain using the knee injury and osteoarthritis outcome score (KOOS). Bone marrow lesions were assessed across the whole knee with DCE-MRI using heuristic variable and non-contrast-enhanced-MRI using MRI osteoarthritis knee score., Results: Data were available from 107 participants. The participants had a mean age of 60.8 years, mean BMI of 34.5 kg/m 2 , mean KOOS-pain of 63.7 (0-100 scale), and mean bone marrow lesion sum score of 6.5 (0-45 scale). The bivariate association between KOOS-pain and the heuristic perfusion variable time to peak in bone marrow lesions containing subchondral cysts showed a statistically significant correlation (r = 0.40; p = 0.002). The perfusion variables were not correlated with KOOS-pain in bone marrow lesions without cysts., Conclusion: In this cross-sectional study, the rate of perfusion (TTP) in bone marrow lesions containing subchondral cysts was associated with pain in patients with knee OA. DCE-MRI has a potential to be used for separating subtypes of OA.

Physiological regulation of blood flow in bone marrow is important to maintain oxygen and glucose supplies but also the physiological hypoxic state of the hematopoietic stem cell (HSC) niche. However, regulatory mechanisms underlying microcirculation in the bone marrow (BM) niche remain unclear. Here, we identify vessels functioning in control of blood flow in bone marrow and assess their contractility. To evaluate contractile potential of Alexa Fluor 633 (AF633; an arterial marker)-positive vessels, we performed immunohistochemistry for α-smooth muscle actin (α-SMA) and found it expressed around AF633 + vessels in the femoral and calvarial marrow. To validate AF633 + vessel contractility, we developed a simple system to locally administer vasoactive agents that penetrate BM through transcalvarial vessels. After exposure of the calvarial surface to FITC-dextran (70 kDa), FITC intensity in calvarial bone marrow gradually increased. When we evaluated the effect of transcalvarial administration (TCA) of norepinephrine (NE) on vascular tone of AF633 + arteries and behavior of transplanted blood cells, NE administration decreased artery diameter and transendothelial migration of transplanted cells, suggesting that adrenergic signaling regulates the HSC niche microcirculation and blood cell migration into the BM via effects on BMarteries. We conclude that TCA is a useful tool for bone marrow research.

Bone perfusion is an essential physiological measure reflecting vasculature status and tissue viability of the skeletal system. Arterial spin labeling (ASL), as a non-invasive and non-contrast enhanced perfusion imaging method, is an attractive approach for human research studies. To evaluate the feasibility of ASL perfusion imaging of knee bone marrow in the distal femoral condyle at a 3 T MRI scanner, a study was performed with eight healthy volunteers (three males and five females, 26 ± 2 years old) and two patients (male, 15 and 11 years old) with diagnosed stage II juvenile osteochondritis dissecans (JOCD). ASL imaging utilized a flow-sensitive alternating inversion recovery method for labeling and a single-shot fast spin echo sequence for image readout. In addition to quantitative knee bone marrow ASL imaging, studies were also performed to evaluate the effects of prolonged post-bolus delay and varied labeling size. ASL imaging was successfully performed with all volunteers. Despite the benefits of hyper-intensive signal suppression within bone marrow, the use of a prolonged post-bolus delay caused excessive perfusion signal decay, resulting in low perfusion signal-to-noise ratio (SNR) and poor image quality. Bone marrow perfusion signal changed with the labeling size, suggesting that the measured bone marrow perfusion signal is flow-associated. The means and standard deviations of bone marrow blood flow, spatial SNR, and temporal SNR from the quantitative perfusion study were 38.3 ± 5.2 mL/100 g/min, 3.31 ± 0.48, and 1.33 ± 0.31, respectively. The imaging results from JOCD patients demonstrated the potential of ASL imaging to detect disease-associated bone marrow perfusion changes. This study demonstrates that it is feasible to perform ASL imaging of knee bone marrow in the distal femoral condyle at 3 T.

Stem cells are nurtured and regulated by a specialized microenvironment known as stem cell niche. While the functions of the niches are well defined, their structure and location remain unclear. We have identified, in rat bone marrow, the seat of hematopoietic stem cells-extensively vascularized node-like compartments that fit the requirements for stem cell niche and that we called hemmules. Hemmules are round or oval structures of about one millimeter in diameter that are surrounded by a fine capsule, have afferent and efferent vessels, are filled with the extracellular matrix and mesenchymal, hematopoietic, endothelial stem cells, and contain cells of the megakaryocyte family, which are known for homeostatic quiescence and contribution to the bone marrow environment. We propose that hemmules are the long sought hematopoietic stem cell niches and that they are prototypical of stem cell niches in other organs.

Perfusion of the pelvic bone marrow is reduced in the postmenopausal group and with age. Quantitative dynamic contrast-enhanced MRI could reflect the blood supply characteristics and hemodynamic changes of the pelvic bone marrow. These results contribute to the description of osteoporosis in the postmenopausal females and the elderly., Introduction: To investigate the effect of menstrual status and age on the perfusion of pelvic bone marrow in adult females using quantitative dynamic contrast-enhanced MRI (DCE-MRI)., Methods: In total, 96 adult females who underwent DCE-MRI between September 2017 and December 2017 were included. All the subjects' quantitative DCE-MRI parameters of pelvic bone marrow were measured and retrospectively analyzed, including K trans (volume transfer constant), K ep (efflux rate constant), and V e (interstitial volume). According to their menstrual status, the subjects were divided into a premenopausal group (n = 39) and a postmenopausal group (n = 57), and the two groups were then divided into four subgroups according to age. The intraobserver reliability was assessed by the intraclass correlation coefficient (ICC). The parameters were compared between different menstrual status groups and age subgroups by Mann-Whitney test, and Spearman correlation analysis was used to evaluate the correlation between the age and the quantitative parameters., Results: The ICCs of the K trans , K ep , and V e values were 0.989, 0.974, and 0.920, respectively. K trans , K ep , and V e of the premenopausal group were significantly higher than those of the postmenopausal group (P < 0.05). The overall age was negatively correlated with K trans , K ep , and V e (r = - 0.590, - 0.357, and - 0.381, respectively, P < 0.05). In the premenopausal group, K trans and V e were significantly higher in subgroup 1 (≤ 40 years) compared with subgroup 2 (> 40 years) (P < 0.05), and age showed a negative correlation with K trans and V e (r = - 0.344 and - 0.334, respectively, P < 0.05). In the postmenopausal group, K trans and K ep were significantly higher in subgroup 3 (≤ 60 years) compared with subgroup 4 (> 60 years) (P < 0.05), and age showed a negative correlation with K trans and K ep (r = - 0.460 and - 0.303, respectively, P < 0.05)., Conclusion: Menstrual status and age have significant effects on the perfusion of the pelvic bone marrow microenvironment in adult females and that the microenvironment of the pelvic bone marrow displays different changes at different age stages. Quantitative DCE-MRI has contributed to the interpretation of the pelvic bone marrow perfusion status.

Objective: To characterize ossified bone marrow blood vessels and confirm the presence of ossified particles (OSP) in humans and rodents., Methods: Human bone marrow blood vessels were processed for scanning and transmission electron microscopy. Whole blood samples were collected from younger (26-39 years; n = 6) and older (55-63 years; n = 6) volunteers and male Fischer-344 rats (1 month, n = 7; 6 months, n = 7; 12 months, n = 7; 18-months, n = 6; 24 months, n = 8). OSP in the whole blood samples were sorted and imaged with microscopy to determine diameter, circularity, and solidity. Additionally, the chemical composition of OSP was determined via elemental analysis., Results: SEM revealed two types of ossified bone marrow blood vessels: that is, "transitioning" and "ossified." OSP were adhered to the surface of transitioning vessels and theoretically gain access to and circulate within the blood. The majority of OSP were ≤15 μm in diameter, but many were of sufficient size to serve as emboli (ie, >15 μm).OSP were predominately oblong in shape and several had jagged tips and edges., Conclusions: We introduce a novel, bone-like blood particle that may be diagnostic of bone marrow blood vessel ossification. Further, OSP may associate with several disease states (eg, atherosclerosis)., (© 2019 John Wiley & Sons Ltd.)

Zoledronic acid (ZOL) is an antiresorptive drug used to prevent bone loss in a variety of conditions, acting mainly through suppression of osteoclast activity. There is growing evidence that ZOL can also affect cells of the mesenchymal lineage in bone. We present novel data revealing significant changes in the abundance of perivascular mesenchymal stromal cells (MSCs)/osteoprogenitors and osteoblasts following the injection of ZOL, in vivo . In young mice with high bone turnover and an abundance of perivascular osteoprogenitors, ZOL significantly ( P < 0.0001) increased new bone formation. This was accompanied by a decline in osterix-positive osteoprogenitors and a corresponding increase in osteoblasts. However, these effects were not observed in mature mice with low bone turnover. Interestingly, the ZOL-induced changes in cells of the mesenchymal lineage occurred independently of effects on the osteogenic vasculature. Thus, we demonstrate that a single, clinically relevant dose of ZOL can induce new bone formation in microenvironments enriched for perivascular MSC/osteoprogenitors and high osteogenic potential. This arises from the differentiation of perivascular osterix-positive MSC/osteoprogenitors into osteoblasts at sites that are innately osteogenic. Collectively, our data demonstrate that ZOL affects multiple cell types in bone and has differential effects depending on the level of bone turnover.-Hughes, R., Chen, X., Hunter, K. D., Hobbs, J. K., Holen, I., Brown, N. J. Bone marrow osteoprogenitors are depleted whereas osteoblasts are expanded independent of the osteogenic vasculature in response to zoledronic acid.

Objective: Assess the link between bone marrow blood vessel ossification, Tb. and cortical bone, and hematological parameters across the lifespan in rats., Methods: Right femora and whole blood samples were taken from male Fischer-344 rats at 1, 6, 12, 18 and 24 months. Femora were scanned by micro-computed tomography (MicroCT) to determine bone marrow blood vessel ossification (ie, ossified vessel volume [OsVV], ossified vessel thickness (OsV.Th), ossified vessel density (OsV density), and structural model index [SMI]). Bone microarchitecture (ie, bone volume [BV/TV], trabecular thickness, trabecular number, and trabecular separation), density and SMI, and cortical bone parameters (ie, cortical shell thickness, porosity, and density) were also determined by MicroCT. Complete blood counts with differentials were conducted., Results: Ossified vessel volume increased throughout the lifespan, coinciding with reduced trabecular BV/TV and cortical shell thickness at 24 months. Many of the hematological parameters were unchanged (ie, white blood cells, lymphocyte number) or increased (monocyte number, percent monocyte, granulocyte number, percent granulocyte, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, red blood cell distribution width, platelet, mean platelet volume) with advancing age; however, red blood cells (RBC) and percent lymphocytes (LY%) were reduced at 24 months. In addition, OsV density was similar to trabecular bone density., Conclusions: Declines in trabecular BV/TV, cortical shell thickness, RBC, and LY% with advanced age coincided with augmented ossification of bone marrow blood vessels., (© 2019 John Wiley & Sons Ltd.)

Bone provides supportive microenvironments for hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) and is a frequent site of metastasis. While incidences of bone metastases increase with age, the properties of the bone marrow microenvironment that regulate dormancy and reactivation of disseminated tumor cells (DTCs) remain poorly understood. Here, we elucidate the age-associated changes in the bone secretome that trigger proliferation of HSCs, MSCs, and DTCs in the aging bone marrow microenvironment. Remarkably, a bone-specific mechanism involving expansion of pericytes and induction of quiescence-promoting secretome rendered this proliferative microenvironment resistant to radiation and chemotherapy. This bone-specific expansion of pericytes was triggered by an increase in PDGF signaling via remodeling of specialized type H blood vessels in response to therapy. The decline in bone marrow pericytes upon aging provides an explanation for loss of quiescence and expansion of cancer cells in the aged bone marrow microenvironment. Manipulation of blood flow - specifically, reduced blood flow - inhibited pericyte expansion, regulated endothelial PDGF-B expression, and rendered bone metastatic cancer cells susceptible to radiation and chemotherapy. Thus, our study provides a framework to recognize bone marrow vascular niches in age-associated increases in metastasis and to target angiocrine signals in therapeutic strategies to manage bone metastasis.

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy-derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated ( p < 0.001) in the MTX-alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 ( p < 0.001) in bone. In MTX-treated rats, genistein suppressed MTX-induced apoptosis of BM SECs ( p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF ( p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs., (© 2018 Wiley Periodicals, Inc.)

We evaluated pazopanib (800 mg orally QD) in patients not eligible for intensive treatment with relapsed/refractory AML or at initial diagnosis. Patients receiving pazopanib for > 14 days were analyzed for safety, tolerability, and efficacy. Co-primary endpoints were cumulative response rate and reduction of bone marrow microvessel density. Twenty patients (median age 76 years, range 52-86) were treated. Fifteen had relapsed/refractory and five had newly diagnosed AML. Median ECOG performance status was 1 (range 1-3). Four patients had adverse, 15 intermediate, and 1 patient favorable cytogenetic/molecular risk (ELN 2010 criteria). The safety profile of pazopanib was as reported. The most common adverse events of any grade were gastrointestinal. Two patients achieved PR (blast reduction > 50%), 14 stable disease (SD), and 4 progressive disease. Median PFS was 65 days (95% CI 29-105). After the end of the study, 1 CRi and 1 CRp occurred on demethylating agents, and 1 CR upon alloSCT. In these patients, SD and improved general condition on pazopanib allowed therapy escalation. Median OS for the overall study population was 191 days (95% CI 87-435) and 1-year survival was 35%. There was no significant change in microvessel density. Clinical trial information: NCT01361334.

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.

The BM serves as a blood-forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T-cell migration to and localization inside the BM. As BM accumulates many CXCR3-expressing memory CD8 + T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8 + T cells in BM, is critically important for homing of all CD8 + T-cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8 + T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM-1+VCAM-1+BP-1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL-7 and IL-15. We therefore conclude that CXCR4 is not only crucial for entry of CD8 + T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mounting evidence from animal studies suggests a role of the nervous system in bone physiology. However, little is known about the nerve fiber localization to human bone compartments and bone surface events. This study reveals the density and distribution of nerves in human bone and the association of nerve profiles to bone remodeling events and vascular structures in iliac crest biopsies isolated from patients diagnosed with primary hyperparathyroidism (PHPT). Bone sections were sequentially double-immunostained for tyrosine hydroxylase (TH), a marker for sympathetic nerves, followed by protein gene product 9.5 (PGP9.5), a pan-neuronal marker, or double-immunostained for either PGP9.5 or TH in combination with CD34, an endothelial marker. In the bone marrow, the nerve profile density was significantly higher above remodeling surfaces as compared to quiescent bone surfaces. Ninety-five percentages of all nerve profiles were associated with vascular structures with the highest association to capillaries and arterioles. Moreover, vasculature with innervation was denser above bone remodeling surfaces. Finally, the nerve profiles density was 5-fold higher in the intracortical pores compared to bone marrow and periosteum. In conclusion, the study shows an anatomical link between innervation and bone remodeling in human bone.

Closed circulatory systems (CCS) underlie the function of vertebrate organs, but in long bones their structure is unclear, although they constitute the exit route for bone marrow (BM) leukocytes. To understand neutrophil emigration from BM, we studied the vascular system of murine long bones. Here we show that hundreds of capillaries originate in BM, cross murine cortical bone perpendicularly along the shaft and connect to the periosteal circulation. Structures similar to these trans-cortical-vessels (TCVs) also exist in human limb bones. TCVs express arterial or venous markers and transport neutrophils. Furthermore, over 80% arterial and 59% venous blood passes through TCVs. Genetic and drug-mediated modulation of osteoclast count and activity leads to substantial changes in TCV numbers. In a murine model of chronic arthritic bone inflammation, new TCVs develop within weeks. Our data indicate that TCVs are a central component of the CCS in long bones and may represent an important route for immune cell export from the BM., Competing Interests: Competing Interests The authors declare no competing interests.

Decreasing bone marrow (BM) microvessel density and circulating angiogenic cytokine levels are promising strategies for the treatment of relapsed and resistant acute myeloid leukemia (AML). Previous studies have reported that wogonoside could inhibit the progression of AML and suppress angiogenesis in a solid tumor, but the correlation of these two effects was ignored. In this research, we determined whether wogonoside could inhibit angiogenesis in this hematologic malignancy. We found that wogonoside could inhibit tumor growth and progression, and prolong the survival of nude mice inoculated with U937/MDR. Besides, reducing BM angiogenesis might cause therapeutic effect against resistant AML. Therefore, coculture between AML cells and BM stromal cells was established to imitate their crosstalk. Then, the effect of wogonoside on BM angiogenesis was tested in vitro and in vivo. We found that wogonoside could suppress microvessel formation in the chicken chorioallantoic membrane assay model and matrigel plug assay. The mechanism research revealed that wogonoside could block the JAK2-STAT3 pathway in AML cells and stromal cells to break their positive feedback. We detected several cytokines related to AML or angiogenesis and found that secreted interleukin-8 was a significant angiogenic cytokine to induce BM angiogenesis. These findings supported that new diagnostics and promising treatment strategies could be developed in relapsed and resistant AML patients., (© 2018 Wiley Periodicals, Inc.)

Two-photon intravital microscopy (2P-IVM) is an advanced imaging technique that allows the visualization of dynamic cellular behavior deeply inside tissues and organs of living animals. Due to the deep tissue penetration, imaging of highly light-scattering tissue as the bone becomes feasible at subcellular resolution.To better understand the influence of blood flow on hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM) microvasculature of the calvarial bone, we analyzed blood flow dynamics and the influence of flow on the early homing behavior of HSPCs during their passage through BM microvessels. Here, we describe a 2P-IVM approach for direct measurements of red blood cell (RBC) velocities in the BM microvasculature using repetitive centerline scans at the level of individual arterial vessels and sinusoidal capillaries to obtain a detailed flow profile map. Furthermore, we explain the isolation and enrichment of HSPCs from long bones and the transplantation of these cells to study the early homing behavior of HSPCs in BM sinusoids at cellular resolution. This is achieved by high-resolution spatiotemporal imaging through a chronic cranial window using transgenic reporter mice.

Objectives: Hypovascularisation is thought to play an important role in the pathogenesis of osteoradionecrosis. The objective of this study was to assess the microvascular system in the irradiated mandibular bone marrow., Materials and Methods: Mandibular bone biopsies were taken from 20 irradiated patients and 24 controls. Blood vessels were visualized using CD34 antibody stain to detect endothelial cells. The vascular density (VD) and vascular area fraction (VAF) were measured. Mean vessel lumen area, perimeter and diameter of the vessels were calculated for each vessel. A distinction was made between large and small vessels (cut-off point <400 µm 2 )., Results: Vascular density and vascular area fraction were lower in the irradiated group. The mean vascular perimeter and mean vascular diameter were higher in samples with a local radiation dose of ≥50 Gy, whereas the percentage of small vessels was lower. Larger vessel perimeter is associated with higher radiation dose. A longer interval between biopsy and radiotherapy is associated with a larger mean vessel perimeter and a lower percentage of small vessels., Conclusions: Radiation dosages higher than 50 Gy mainly affect the smaller vessels. With increased time after irradiation, the share of smaller vessels in the mandibular bone marrow seems to decrease. In search of the exact mechanisms of irradiation damage and osteoradionecrosis of the mandible, the role of the microvascular system in the mandibular bone marrow should be further explored., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Retinoic acid receptor (RAR) signaling regulates bone structure and hematopoiesis through intrinsic and extrinsic mechanisms. This study aimed to establish how early in the osteoblast lineage loss of RARγ (Rarg) disrupts the bone marrow microenvironment. Bone structure was analyzed by micro-computed tomography (μCT) in Rarg -/- mice and mice with Rarg conditional deletion in Osterix-Cre-targeted osteoblast progenitors or Prrx1-Cre-targeted mesenchymal stem cells. Rarg -/- tibias exhibited less trabecular and cortical bone and impaired longitudinal and radial growth. The trabecular bone and longitudinal, but not radial, growth defects were recapitulated in Prrx1:Rarg Δ/Δ mice but not Osx1:Rarg Δ/Δ mice. Although both male and female Prrx1:Rarg Δ/Δ mice had low trabecular bone mass, males exhibited increased numbers of trabecular osteoclasts and Prrx1:Rarg Δ/Δ females had impaired mineral deposition. Both male and female Prrx1:Rarg Δ/Δ growth plates were narrower than controls and their epiphyses contained hypertrophic chondrocyte islands. Flow cytometry revealed that male Prrx1:Rarg Δ/Δ bone marrow exhibited elevated pro-B and pre-B lymphocyte numbers, accompanied by increased Cxcl12 expression in bone marrow cells. Prrx1:Rarg Δ/Δ bone marrow also had elevated megakaryocyte-derived Vegfa expression accompanied by smaller sinusoidal vessels. Thus, RARγ expression by Prrx1-Cre-targeted cells directly regulates endochondral bone formation and indirectly regulates tibial vascularization. Furthermore, RARγ expression by Prrx1-Cre-targeted cells extrinsically regulates osteoclastogenesis and B lymphopoiesis in male mice. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

In the bone marrow, the interaction of progenitor cells with the vasculature is fundamental for the release of blood cells into circulation. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising protein biomaterial for bone marrow tissue engineering, because of its tunable architecture and mechanical properties, the capacity to incorporate labile compounds without loss of bioactivity and the demonstrated ability to support blood cell formation without premature activation. In this study, we fabricated a custom perfusion chamber to contain a multi-channel lyophilized silk sponge mimicking the vascular network in the bone marrow niche. The perfusion system consisted in an inlet and an outlet and 2 splitters that allowed funneling flow in each single channel of the silk sponge. Computational Fluid Dynamic analysis demonstrated that this design permitted confined flow inside the vascular channels. The silk channeled sponge supported efficient platelet release from megakaryocytes (Mks). After seeding, the Mks localized along SDF-1α functionalized vascular channels in the sponge. Perfusion of the channels allowed the recovery of functional platelets as demonstrated by increased PAC-1 binding upon thrombin stimulation. Further, increasing the number of channels in the silk sponge resulted in a proportional increase in the numbers of platelets recovered, suggesting applicability to scale-up for platelet production. In conclusion, we have developed a scalable system consisting of a multi-channeled silk sponge incorporated in a perfusion chamber that can provide useful technology for functional platelet production ex vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Intravital multiphoton microscopy (iMPM) in mice provides access to cellular and molecular mechanisms of metastatic progression of cancers and the underlying interactions with the tumor stroma. Whereas iMPM of malignant disease has been performed for soft tissues, noninvasive iMPM of solid tumor in the bone is lacking. We combined miniaturized tissue-engineered bone constructs in nude mice with a skin window to noninvasively and repetitively monitor prostate cancer lesions by three-dimensional iMPM. In vivo ossicles developed large central cavities containing mature bone marrow surrounded by a thin cortex and enabled tumor implantation and longitudinal iMPM over weeks. Tumors grew inside the bone cavity and along the cortical bone interface and induced niches of osteoclast activation (focal osteolysis). Interventional bisphosphonate therapy reduced osteoclast kinetics and osteolysis without perturbing tumor growth, indicating dissociation of the tumor-stroma axis. The ossicle window, with its high cavity-to-cortex ratio and long-term functionality, thus allows for the mechanistic dissection of reciprocal epithelial tumor-bone interactions and therapy response., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Endothelial cells (ECs) contribute to haematopoietic stem cell (HSC) maintenance in bone marrow, but the differential contributions of EC subtypes remain unknown, owing to the lack of methods to separate with high purity arterial endothelial cells (AECs) from sinusoidal endothelial cells (SECs). Here we show that the combination of podoplanin (PDPN) and Sca-1 expression distinguishes AECs (CD45 - Ter119 - Sca-1 bright PDPN - ) from SECs (CD45 - Ter119 - Sca-1 dim PDPN + ). PDPN can be substituted for antibodies against the adhesion molecules ICAM1 or E-selectin. Unexpectedly, prospective isolation reveals that AECs secrete nearly all detectable EC-derived stem cell factors (SCF). Genetic deletion of Scf in AECs, but not SECs, significantly reduced functional HSCs. Lineage-tracing analyses suggest that AECs and SECs self-regenerate independently after severe genotoxic insults, indicating the persistence of, and recovery from, radio-resistant pre-specified EC precursors. AEC-derived SCF also promotes HSC recovery after myeloablation. These results thus uncover heterogeneity in the contribution of ECs in stem cell niches.

Inflammation coincides with diminished marrow function, vasodilation of blood vessels, and bone mass. Intermittent parathyroid hormone (PTH) administration independently improves marrow and vascular function, potentially impacting bone accrual. Currently, the influence of marrow and intermittent PTH administration on aged bone blood vessels has not been examined. Vasodilation of the femoral principal nutrient artery (PNA) was assessed in the presence and absence of marrow. Furthermore, we determined the influence of PTH 1-34 on 1) endothelium-dependent vasodilation and signaling pathways [i.e., nitric oxide (NO) and prostacyclin (PGI 2 )], 2) endothelium-independent vasodilation, 3) cytokine production by marrow cells, and 4) bone microarchitecture and bone static and dynamic properties. Young (4-6 mo) and old (22-24 mo) male Fischer-344 rats were treated with PTH 1-34 or a vehicle for 2 wk. In the absence and presence of marrow, femoral PNAs were given cumulative doses of acetylcholine, with and without the NO and PGI 2 blockers, and diethylamine NONOate. Marrow-derived cytokines and bone parameters in the distal femur were assessed. Exposure to marrow diminished endothelium-dependent vasodilation in young rats. Reduced bone volume and NO-mediated vasodilation occurred with old age and were partially reversed with PTH. Additionally, PTH treatment in old rats restored endothelium-dependent vasodilation in the presence of marrow and augmented IL-10, an anti-inflammatory cytokine. Endothelium-independent vasodilation was unaltered, and PTH treatment reduced osteoid surfaces in old rats. In conclusion, the marrow microenvironment reduced vascular function in young rats, and PTH treatment improved the marrow microenvironment and vasodilation with age. NEW & NOTEWORTHY This study investigated the influence of the marrow microenvironment on bone vascular function in young and old rats. An inflamed marrow microenvironment may reduce vasodilator capacity of bone blood vessels, diminishing delivery of blood flow to the skeleton. In young rats, the presence of the marrow reduced vasodilation in the femoral principal nutrient artery (PNA). However, intermittent parathyroid hormone administration (i.e., a treatment for osteoporosis) improved the marrow microenvironment and vasodilator capacity in old PNAs.

The bone marrow microenvironment is the preferred location of multiple myeloma, supporting tumor growth and development. It is composed of a collection of interacting subniches, including the endosteal and perivascular niche. Current in vitro models mimic either of these subniches. By developing a model combining both niches, this study aims to further enhance the ability to culture primary myeloma cells in vitro. Also, the dependency of myeloma cells on each niche was studied. A 3D bone marrow model containing two subniches was created using 3D bioprinting technology. We used a bioprintable pasty calcium phosphate cement (CPC) scaffold with seeded osteogenic multipotent mesenchymal stromal cells (O-MSCs) to model the endosteal niche, and Matrigel containing both endothelial progenitor cells (EPCs) and MSCs to model the perivascular niche. Within the model containing one or both of the niches, primary CD138 + myeloma cells were cultured and analyzed for both survival and proliferation. The 3D bone marrow model with combined subniches significantly increasing the proliferation of CD138 + myeloma cells compared to both environments separately. The developed model showed an essential role of the perivascular niche over the endosteal niche in supporting myeloma cells. The developed model can be used to study the expansion of primary myeloma cells and their interactions with varying bone marrow subniches.

Although the function of Runt-related (RUNX) transcription factors has been well characterized in leukemogenesis and regarded as an ideal target in antileukemia strategies, the effect of RUNX-inhibition therapy on bone marrow niche cells andr its impact on the engraftment of acute myeloid leukemia (AML) cells have largely been unknown. Here, we provide evidence suggesting the possible involvement of RUNX transcription factors in the transactivation of E-selectin , a member of selectin family of cell adhesion molecules, on the vascular endothelial cells of the mice bone marrow niche. In our experiments, gene switch-mediated silencing of RUNX downregulated E-selectin expression in the vascular niche and negatively controlled the engraftment of AML cells in the bone marrow, extending the overall survival of leukemic mice. Our work identified the novel role of RUNX family genes in the vascular niche and showed that the vascular niche, a home for AML cells, could be strategically targeted with RUNX-silencing antileukemia therapies. Considering the excellent efficacy of RUNX-inhibition therapy on AML cells themselves as we have previously reported, this strategy potentially targets AML cells both directly and indirectly, thus providing a better chance of cure for poor-prognostic AML patients., (© 2018 by The American Society of Hematology.)

In this study, we investigated microvascular perfusion status, changes to fat content and fatty acid composition in the bone marrow of rat femurs after total-body irradiation by quantitative permeability parameters of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and ex vivo high-resolution magic angle spinning (HRMAS) 1 H nuclear magnetic resonance spectroscopy (NMRS). Thirty-six Sprague-Dawley rats were randomly assigned to either an irradiated or nonirradiated control group. Permeability imaging using DCE-MRI and HRMAS 1 H NMRS was performed before irradiation, as well as at days 4 and 7 postirradiation. The volume transfer constant (K trans ) values increased to 2.219 ± 0.418/min ( P < 0.01) at day 4 and to 2.760 ± 0.217/min at day 7 ( P < 0.01) postirradiation. The plasma fraction (v p ) values gradually decreased. The proportion of (n-6) polyunsaturated fatty acids (PUFA) gradually reached a peak at day 7, the proportion of (n-3) PUFA gradually decreased and the proportion of saturated fatty acids gradually increased. After irradiation, K trans at different times showed significant negative correlation with (n-3) PUFA ( r = -0.6393, P < 0.01) and significant positive correlation with (n-6) PUFA ( r = 0.6841, P < 0.05). These findings indicate that bone marrow microcirculation perfusion and vascular permeability correlated with fat content at an early time point after irradiation. A pathophysiological mechanism may exist based on fat-vascular permeability in the case of injury to bone marrow microcirculation.