Tibial Bone Marrow Research Articles

Lovastatin Combination Therapy Increases the Survival and Proliferation of Rat Bone Marrow-Derived Mesenchymal Stem Cells Against the Inflammatory Activity of Lipopolysaccharide.

Oxidative stress hurts the survival of transplanted mesenchymal stem cells (MSCs). Lipopolysaccharide (LPS) preconditioning inhibits apoptotic death in MSCs. Also, Lovastatin's protective effect was reported on MSCs. Here, we investigated the potential of LPS and Lovastatin combination therapy on the survival and proliferation of MSCs. MSCs harvested from adult rats (240-260 g) femur and tibia bone marrow. Third passage MSCs were divided into 6 groups control group, LPS, LPS + Lovastatin (10 and 15 µM), and Lovastatin (10 and 15 µM). Cell survival and proliferation were assessed using an MTT assay 24 h after LPS, Lovastatin, or LPS + Lovastatin treatment. Also, Malondialdehyde (MDA) as a lipid peroxidation marker and antioxidant enzymes such as Glutathione peroxidase (GPX) and Superoxide dismutase (SOD) activity levels evaluated. Finally, the expression level of tumor protein P53 (P53) and octamer-binding transcription factor 4 (OCT4) genes were measured by qRT-PCR test. Lovastatin 10 μM potentiated proliferation and survival of MSCs. It can increase the activity of GPX and SOD. 10 µM Lovastatin could not affect MDA amounts but decreased the expression levels of P53 and Oct4 significantly. Nevertheless, treatment with LPS reduced the survival and proliferation of MSCs, along with a significant reduction in GPX activity. LPS + Lovastatin could increase SOD activity, however, GPX enzyme activity and MSCs proliferation did not change so, and it was not effective. We propose Lovastatin at the dose of 10 µM as a suitable combination agent to increase the survival and proliferation of MSCs in oxidative stress conditions.

Differences in running biomechanics and tibiotalar cartilage T2 relaxation time between runners with and without asymptomatic tibial sesamoid bone marrow edema – clinical case series

ABSTRACT The impact of asymptomatic bone marrow edema (BME) in sesamoids on running biomechanics remains largely unknown, but exploring this relationship could aid early detection of forefoot-related injuries with cost-effective modalities. This study aimed to compare the running biomechanics of runners with asymptomatic BME in the tibial sesamoids with that of healthy controls. Four runners with asymptomatic BME in tibial sesamoids and four healthy runners participated. Lower extremity joint kinetics and kinematics were assessed along with plantar pressure while running at a self-selected speed. The T2 relaxation time of the tibiotalar cartilage was measured using MRI-derived T2 maps. Compared to the non-injured group, the injured group exhibited significantly lower plantar pressure under the hallux (p = 0.001), increased peak ankle rotation angles (p = 0.025), reduced ankle power generation (p = 0.049), and increased knee extension torque (p = 0.015). No significant differences in T2 values of the tibiotalar cartilage were observed. It appears that runners with asymptomatic BME adapted their running strategies by modifying their ankle biomechanics and reducing plantar pressure, even in the absence of pain. While the precise implications of these biomechanical alterations warrant further investigation, this study provides valuable insights into the relationship between asymptomatic BME and running biomechanics.

Abstract 4207: Zoledronic acid reduces quiescent bone-disseminated human ER+ breast cancer tumor cell burden and estrogen-responsiveness in a pre-clinical model

Abstract Unique to early-stage ER+ breast cancer, ER+ tumor cells already bone disseminated at time of diagnosis can remain dormant at this site until an initiating event (e.g., activating ESR1 mutation acquisition) allows for osteolytic bone metastases (BMET) progression even decades later. Adjuvant use of anti-resorptive bisphosphonates, but not denosumab, reduces BMET risk in post-menopausal women with early stage breast cancer, suggesting protective effects may be unrelated to changes in bone turnover. Pre-clinical modeling of dormant, bone-disseminated ER+ breast cancer cells may enhance our understanding and thus our ability to better target these inherently treatment-resistant cells. In studies reported here, a model of bone-disseminated ER+ dormancy was characterized and used to query effects of zoledronic acid (ZA) on dormant cells. When female nude mice were inoculated (intracardiac) with human ER+ MCF-7 cells labelled with DiD, a membrane dye that dissipates with each cell division, DiD-positive tumor cell number in harvested tibial bone marrow remained unchanged from 24 hours to 28 days post-tumor cell inoculation. When 17ß-estradiol (E2) treatment (0.72 mg 60-day pellets) was initiated on D28 following tumor cell inoculation, detectable DiD+ tumor cell number in harvested tibial bone marrow increased 1.8-fold within one week (D35 vs. D28, p < 0.0001), consistent with initiation of E2-stimulated proliferation, and osteolytic, histology-confirmed BMET appeared subsequently with 60% incidence by 6-weeks post-start of E2 treatment. When mice were instead zoledronic acid-treated during the 28d dormancy period (70 ug/kg subcutaneously on D7 and on D21 post-inoculation), DiD-positive cell number in marrow harvested on D28 was reduced by more than half vs. vehicle-treated controls (p< 0.05). Moreover, DiD-positive cell numbers on D35, one week after start of E2-treatment, remained unchanged in tibial marrow of ZA-treated mice and were thus substantially (3.8-fold) lower than controls (p< 0.001). When followed for 6-weeks after initiation of E2 treatment, no osteolytic BMET were detected in ZA-treated mice. In toto, these results recapitulate clinical findings, thus validating the methodologic approach, and suggest that in addition to reducing dormant ER+ tumor cell burden in bone, bisphosphonates may also alter their estrogen responsiveness. Acknowledgements: Special thanks to undergraduates Alyssa Magee and Geethika Ameneni for assistance with data acquisition, and the METAvivor Foundation and NIH-NCI (NCI R03 CA181893, R01 CA174926, P30 CA023074, T32 CA009213) for financial support. Citation Format: Julia N. Cheng, Jennifer B. Frye, Susan A. Whitman, Janet L. Funk. Zoledronic acid reduces quiescent bone-disseminated human ER+ breast cancer tumor cell burden and estrogen-responsiveness in a pre-clinical model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4207.

Porcine Mandibular Bone Marrow-Derived Mesenchymal Stem Cell (BMSC)-Derived Extracellular Vesicles Can Promote the Osteogenic Differentiation Capacity of Porcine Tibial-Derived BMSCs.

Existing research suggests that bone marrow-derived mesenchymal stem cells (BMSCs) may promote endogenous bone repair. This may be through the secretion of factors that stimulate repair processes or directly through differentiation into osteoblast-progenitor cells. However, the osteogenic potential of BMSCs varies among different tissue sources (e.g., mandibular versus long BMSCs). The main aim of this study was to investigate the difference in osteogenic differentiation capacity between mandibular BMSCs (mBMSCs) and tibial BMSCs (tBMSCs). Bioinformatics analysis of the GSE81430 dataset taken from the Gene Expression Omnibus (GEO) database was performed using GEO2R. BMSCs were isolated from mandibular and tibial bone marrow tissue samples. Healthy pigs (n = 3) (registered at the State Office for Nature, Environment, and Consumer Protection, North Rhine-Westphalia (LANUV) 81-02.04.2020.A215) were used for this purpose. Cell morphology and osteogenic differentiation were evaluated in mBMSCs and tBMSCs. The expression levels of toll-like receptor 4 (TLR4) and nuclear transcription factor κB (NF-κB) were analyzed using quantitative polymerase chain reaction (qPCR) and Western blot (WB), respectively. In addition, mBMSC-derived extracellular vesicles (mBMSC-EVs) were gained and used as osteogenic stimuli for tBMSCs. Cell morphology and osteogenic differentiation capacity were assessed after mBMSC-EV stimulation. Bioinformatic analysis indicated that the difference in the activation of the TLR4/NF-κB pathway was more pronounced compared to all other examined genes. Specifically, this demonstrated significant downregulation, whereas only 5-7 upregulated genes displayed significant variances. The mBMSC group showed stronger osteogenic differentiation capacity compared to the tBMSC group, confirmed via ALP, ARS, and von Kossa staining. Furthermore, qPCR and WB analysis revealed a significant decrease in the expression of the TLR4/NF-κB pathway in the mBMSC group compared to the tBMSC group (TLR4 fold changes: mBMSCs vs. tBMSCs p < 0.05; NF-κB fold changes: mBMSCs vs. tBMSCs p < 0.05). The osteogenic differentiation capacity was enhanced, and qPCR and WB analysis revealed a significant decrease in the expression of TLR4 and NF-κB in the tBMSC group with mBMSC-EVs added compared to tBMSCs alone (TLR4 fold changes: p < 0.05; NF-κB fold changes: p < 0.05). Our results indicate that mBMSC-EVs can promote the osteogenic differentiation of tBMSCs in vitro. The results also provide insights into the osteogenic mechanism of mBMSCs via TLR4/NF-κB signaling pathway activation. This discovery promises a fresh perspective on the treatment of bone fractures or malunions, potentially offering a novel therapeutic method.

Prostate cancer therapy using immune checkpoint molecules to target recombinant dendritic cells.

We developed immune checkpoint molecules to target recombinant dendritic cells (DCs) and verified their anti-tumor efficacy and immune response against prostate cancer. DCs were generated from mononuclear cells in the tibia and femur bone marrow of mice. We knocked down the programmed death ligand 1 (PD-L1) on monocyte-derived DCs through siRNA PD-L1. Cell surface antigens were immune fluorescently stained through flow cytometry to analyze cultured cell phenotypes. Furthermore, we evaluated the efficacy of monocyte-derived DCs and recombinant DCs in a prostate cancer mouse model with subcutaneous TRAMP-C1 cells. Lastly, DC-induced mixed lymphocyte and lymphocyte-only proliferations were compared to determine cultured DCs' function. Compared to the control group, siRNA PD-L1 therapeutic DC-treated mice exhibited significantly inhibited tumor volume and increased tumor cell apoptosis. Remarkably, this treatment substantially augmented interferon-gamma and interleukin-2 production by stimulating T-cells in an allogeneic mixed lymphocyte reaction. Moreover, we demonstrated that PD-L1 gene silencing improved cell proliferation and cytokine production. We developed monocyte-derived DCs transfected with PD-L1 siRNA from mouse bone marrow. Our study highlights that PD-L1 inhibition in DCs increases antigen-specific immune responses, corroborating previous immunotherapy methodology findings regarding castration-resistant prostate cancer.

Investigating Risk Factors and Magnetic Resonance Imaging (MRI)-Based Grading of Subchondral Incomplete Fracture (SIF) of Medial Femoral Condyle.

Subchondral insufficiency fractures (SIF) of the knee joint are prevalent in osteoporosis patients over the age of 55. Early diagnosis of SIF fracture of the medial femoral condyle is crucial for delaying disease progression, early therapy, and potential disease reversal. Magnetic resonance imaging (MRI) is useful in detecting SIF, which is often undetectable on initial radiographs. This study aimed at developing a grading system for subchondral insufficiency fractures (SIF) based on MRI to predict outcomes and evaluate risk factors. In this study, MRI was used to examine SIF risk variables in the medial condyle of the femur to help clinicians diagnose, treat, and delay the condition. A total of 386 patients with SIF from 2019 to 2021 were retrospectively analyzed and divided into 106 patients in the disease group and 280 patients in the control group according to whether they had SIF. The lesion site, meniscus, ligament, and other parameters were evaluated and compared. At the same time, a grading system was introduced to stratify and statistically analyze the size of the lesion area, the degree of bone marrow edema (BME), meniscus tears, and other parameters in the patients. Most SIF were low-grade (LG) fractures, and the predictors of LG and high-grade (HG) fractures included heel tear (P =0.031), degree of medial malleolus degeneration (P < 0.001), advanced age (P < 0.001), and lesion size (P < 0.001). The prognostic factors that showed significant differences between the two groups included age (P =0.027), gender (P =0.005), side (P =0.005), medial tibial plateau injury (P < 0.0001), femoral medullary bone marrow edema (P < 0.0001), medial tibial plateau bone marrow edema (P < 0.0001), meniscus body partial injury (P =0.016), heel tear (P =0.001), anterior cruciate ligament injury (P =0.002), and medial collateral ligament injury (P < 0.0001). This current study proposed an MRI-based grading system for inferior condylar fractures of the femur, in which HG inferior condylar fractures are associated with severe medial malleolus degeneration, advanced age, lesion size (correlation), and meniscus heel tears.

Larger Pre-operative Tibial Bone Marrow Lesion Volume Correlates with Better Clinical Outcomes after Medial Opening High Tibial Osteotomy

Larger Pre-operative Tibial Bone Marrow Lesion Volume Correlates with Better Clinical Outcomes after Medial Opening High Tibial Osteotomy

386 Stereotactic Implantation of Autologous Mesenchymal Stem Cells Improves Neurological Outcomes in a Rat Model of Chronic Middle Cerebral Artery Stroke

INTRODUCTION: Limited treatment options exist for patients with chronic infarcts. Multipotent adult mesenchymal stem cells (MSC) show promise as a treatment for chronic stroke. Allogeneic MSCs have been implanted successfully in chronic stroke1,2. However, no literature evaluates the use of autologous MSCs delivered via stereotactic implantation. Autologous MSCs provide safety and accessibility benefits. METHODS: Male Sprague-Dawley rats underwent transient right MCAO for 120 minutes. Brain MRI and sensorimotor behavior were used to evaluate stroke. Sixteen days post stroke, tibial bone marrow was aspirated and MSCs were grown in a closed bioreactor. At 28 days post stroke, 3 trajectories of MSCs were implanted in the peri-infarct zone using a stereotactic robot. The treatments consisted of group 1 (5 x 106 cell dose, N = 6), group 2 (1 x 106 cell dose, N = 6) and a control group (saline injection, N = 9). In a second cohort of animals, Q-dot labeled autologous MSCs were tracked in the brain for 7 or 30 days. RESULTS: Behavior as determined by the modified neurological severity score (0-16) significantly improved in both groups. Stroke volume did not decrease. Histological analysis confirmed increased astrocyte and microglia presence in the affected hemisphere. Quantum dot analysis of injections showed presence of MSCs along trajectory as well as targeting perilesional brain tissue at 7 and 30 days post-implantation. CONCLUSIONS: Stereotactic injection of autologous MSCs represent an effective treatment for chronic stroke. Cells were effective at the lowest dose studied, with no added benefit at higher cell dosages. Unlike allogeneic MSCs which disappear over days to weeks3, autologous MSCs persist long term in the brain, potentially providing continued benefit. This treatment warrants human trials to study its effectiveness.

Development of a novel regenerative therapy for malignant bone tumors using an autograft containing tumor inactivated by high hydrostatic pressurization (HHP).

Surgical resection of malignant bone tumors leads to significant defects in the normal surrounding tissues that should be reconstructed to avoid amputation. Our research aimed to inactivate osteosarcoma (OS)-affected bone to obtain autologous bone grafts for bone defect reconstruction using a novel therapy called high hydrostatic pressurization (HHP) therapy. The key points are complete tumor death and preservation of the non-denatured native extracellular matrix (ECM) and bone tissue by HHP. Previously, we found that HHP at 200MPa for 10min can completely inactivate cells in normal skin and skin tumors, including malignant melanoma and squamous cell carcinoma while maintaining their original biochemical properties and biological components. Based on our previous research, this study used HHP at 200MPa for 10min to eradicate OS. We prepared an OS cell line (LM8), pressurized it at 200MPa for 10min, and confirmed its inactivation through morphological observation, WST-8 assay, and live/dead assay. We then injected OS cells with or without HHP into the bone marrow of the murine tibia, after which we implanted tumor tissues with or without HHP into the anterior surface of the tibia. After HHP, OS cells did not proliferate and were assessed using a live/dead assay. The pressurized cells and tumors did not grow after implantation. The pressurized bone was well prepared as tumor-free autologous bone tissues, resulting in the complete eradication of OS. This straightforward and short-pressing treatment was proven to process the tumor-affected bone to make a transplantable and tumor-free autologous bone substitute.

Repeatability of Quantitative Magnetic Resonance Imaging Biomarkers in the Tibia Bone Marrow of a Murine Myelofibrosis Model.

Quantitative MRI biomarkers are sought to replace painful and invasive sequential bone-marrow biopsies routinely used for myelofibrosis (MF) cancer monitoring and treatment assessment. Repeatability of MRI-based quantitative imaging biomarker (QIB) measurements was investigated for apparent diffusion coefficient (ADC), proton density fat fraction (PDFF), and magnetization transfer ratio (MTR) in a JAK2 V617F hematopoietic transplant model of MF. Repeatability coefficients (RCs) were determined for three defined tibia bone-marrow sections (2-9 mm; 10-12 mm; and 12.5-13.5 mm from the knee joint) across 15 diseased mice from 20-37 test-retest pairs. Scans were performed on consecutive days every two weeks for a period of 10 weeks starting 3-4 weeks after transplant. The mean RC with (95% confidence interval (CI)) for these sections, respectively, were for ADC: 0.037 (0.031, 0.050), 0.087 (0.069, 0.116), and 0.030 (0.022, 0.044) μm2/ms; for PDFF: 1.6 (1.3, 2.0), 15.5 (12.5, 20.2), and 25.5 (12.0, 33.0)%; and for MTR: 0.16 (0.14, 0.19), 0.11 (0.09, 0.15), and 0.09 (0.08, 0.15). Change-trend analysis of these QIBs identified a dynamic section within the mid-tibial bone marrow in which confident changes (exceeding RC) could be observed after a four-week interval between scans across all measured MRI-based QIBs. Our results demonstrate the capability to derive quantitative imaging metrics from mouse tibia bone marrow for monitoring significant longitudinal MF changes.

Frequency of anterolateral ligament tears and ramp lesions in patients with anterior cruciate ligament tears and associated injuries indicative for these lesions—a retrospective MRI analysis

ObjectivesTo assess the frequency of anterolateral ligament (ALL) tears and ramp lesions (RL) detected with MRI in patients with anterior cruciate ligament (ACL) tears and to describe associated injuries indicative for these lesions.MethodsIn this retrospective study, 164 patients with surgically verified ACL tears were included. Preoperative MRI scans were reviewed for ALL tears and different types of RL. All coexisting meniscal tears, tears of the medial (MCL) and lateral collateral band (LCL), and posterior-medial tibial bone marrow edema (BME) were recorded. The frequency of ALL tears and RL was assessed and coexisting injuries were correlated using Pearson’s chi-square test. A p < 0.05 was defined as statistically significant. In cases of multiple testing, Bonferroni’s correction was applied.ResultsALL tears and RL combined were detected in 28 patients (17.1%), ALL tears in 48 patients (29.3%), and RL in 54 patients (32.9%) which were significantly associated to each other. ALL tears were significantly associated with tears of the posterior horn of the lateral meniscus (PHLM), BME, and with tears of the LCL and MCL. RL were significantly associated with tears of the posterior horn of the medial (PHMM) and PHLM, with BME, and with tears of the LCL.ConclusionsACL tears are associated with RL or ALL tears in about one-third of cases and with both lesions combined in about one-fifth of cases. ALL tears and RL are significantly associated with additional posttraumatic injuries, which can thus be indicative of these lesions.Key Points• ACL tears were associated with ramp lesions or ALL tears in about one-third of the cases.• Ramp lesions and ALL tears were significantly associated with each other, tear in the PHLM, tear in the LCL, and BME.• ALL tears were more frequently associated with instable classified ramp lesion type 4b and type 5.

GPR84 potently inhibits osteoclastogenesis and alleviates osteolysis in bone metastasis of colorectal cancer

The expression of GPR84 in bone marrow-derived monocytes/macrophages (BMMs) can inhibit osteoclast formation; however, its role in bone metastasis of colorectal cancer (CRC) is still unknown. To investigate the effects of GPR84 on bone metastasis of CRC, the murine CRC cell line MC-38 was injected into tibial bone marrow. We found that the expression of GPR84 in BMMs was gradually downregulated during bone metastasis of CRC, and the activation of GPR84 significantly prevented osteoclastogenesis in the tumor microenvironment. Mechanistically, the MAPK pathway mediated the effects of GPR84 on osteoclast formation. Moreover, we found that IL-11 at least partly inhibited the expression of GPR84 in the tumor microenvironment through the inactivation of STAT1. Additionally, activation of GPR84 could prevent osteolysis during bone metastasis of CRC. Our results suggest that CRC cells downregulate the expression of GPR84 in BMMs to promote osteoclastogenesis in an IL-11-dependent manner. Thus, GPR84 could be a potential therapeutic target to attenuate bone destruction induced by CRC metastasis.

Activation of HCN channels caused by elevated cAMP levels in periaqueductal gray promotes bone cancer pain

The periaqueductal gray (PAG) is an important relay center for the descending pathways that regulate nociceptive information transduction. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in the nerve injury-induced pain hypersensitivity. Previous studies have identified that HCN1 and HCN2 channel protein located in the ventral-lateral periaqueductal gray (vlPAG), a region important for pain regulation. However, it is not clear whether the HCN channel in vlPAG is involved in bone cancer pain (BCP). In this study, we assessed the role of HCN channels in BCP by measuring changes of HCN channel expression and activity in vlPAG neurons in bone cancer rats. In the present study, the BCP model was established by injecting SHZ-88 breast cancer cells into the right tibia bone marrow in rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured to evaluate pain behavior in rats. HCN1 and HCN2 channels expression in vlPAG were detected by using Western Blot and immunohistochemistry. In addition, the cAMP level in vlPAG neurons was detected by ELISA, and HCN channel current (Ih) of vlPAG neurons was recorded by whole cell patch-clamp to evaluate HCN channel activity. As a result, decreased MWT and TWL were observed in rats on 7d after SHZ-88 cell inoculation, and the allodynia was sustained until 21d after inoculation. At the same time, HCN1 and HCN2 channels expression and neuronal Ih in vlPAG were significantly increased in BCP rats. In addition, the level of cAMP in vlPAG also increased after SHZ-88 cell inoculation. Furthermore, intravlPAG injection of ZD7288 (HCN channels antagonist) could significantly reduce hyperalgesia and the elevation of cAMP in vlPAG in BCP rats. Our observations suggest that the elevation of cAMP may promote the activation of HCN channels in vlPAG in bone cancer rats, thereby promoting the development of bone cancer pain.

Larger bone marrow lesion volume before medial open-wedge high tibial osteotomy correlates with better improvement of clinical scores in patients with knee osteoarthritis.

This study evaluated the clinical outcomes of medial open-wedge high tibial osteotomy (MOWHTO) and bone marrow lesion (BML) scores and volumes. The hypotheses were that quantitative BML volume is more associated with clinical outcomes of MOWHTO than qualitative BML evaluations, and pre-operative BML volume is associated with the improvement of clinical outcomes. Patients who underwent MOWHTO were retrospectively enrolled. The Knee Injury and Osteoarthritis Outcome Score (KOOS) was recorded before the initial surgery and at plate removal surgery. Using pre-operative short-time inversion recovery magnetic resonance imaging, BMLs were evaluated using three qualitative scores, reflecting the maximum length, proportion, and intensity of BML. For quantification, BMLs of the femur and tibia were separately defined as lesions with a threshold greater than the mean signal intensity plus two standard deviations, using the corresponding lateral condyles as controls. The association between the KOOS scales and BML scores/volume was evaluated using Spearman's correlation coefficient. Multivariate linear regression analyses for post-operative KOOS scales were performed using the tibial BML volume as one of the four independent variables. The final analysis included 40 MOWHTO cases. Two qualitative BML scores correlated only with pre-operative KOOS sports. Femoral and tibial BML volumes were correlated with post-operative KOOS QOL (ρ = 0.40, p = 0.01) and sports (ρ = 0.36, p = 0.02), respectively. Tibial BML volume was significantly correlated with all five delta KOOS scales (ρ = 0.39-0.51, p = 0.01-0.001), however, femoral BML volume was only correlated with delta KOOS QOL (ρ = 0.41, p = 0.01). In multivariate analyses, tibial BML volume was a significant positive predictor for every post-operative KOOS scale, while post-operative % mechanical axis was also a positive significant variable, except post-operative KOOS pain. Tibial BML volume was positively correlated with one post-operative KOOS scale and all delta KOOS scales. A larger pre-operative tibial BML and appropriate alignment correction were associated with a better post-operative KOOS scales. Pre-operative large BML had no negative influence on post-operative clinical outcomes; hence, surgeons need not hesitate to perform MOWHTO in patients with large BMLs in the medial condyles. Retrospective case series, Level IV.

Magnetic resonance imaging of a pediatric case of arthritis associated with acute lymphoblastic leukemia: A case report

In the present case report, a 3-year-old girl presented with a 1-week history of spontaneously resolving right knee pain. After 1 month, the patient had trouble ambulating due to painful swelling of their ankle. Rheumatic disease, specifically juvenile idiopathic arthritis, was considered. Blood examination could not be conducted because their blood sample was coagulated. T1-weighted magnetic resonance imaging (MRI) revealed abnormally low signals in the femur, tibia, fibula and foot bone marrow. Contrast-enhanced T1-weighted MRI revealed synovial contrast enhancement and synovial fluid retention in the right ankle joint. Blood analysis revealed a white blood cell count of 40,000/µl (blasts, 66%). In addition, a monoclonal increase in the number of lymphoblasts was observed. The patient was subsequently diagnosed with B-cell precursor acute lymphoblastic leukemia. Reports on leukemic arthritis resembling synovitis on MRI remain limited. The findings of this report indicated that pediatricians should consider leukemia in children presenting with joint symptoms.

Long Bone Mineral Loss, Bone Microstructural Changes and Oxidative Stress After Eimeria Challenge in Broilers.

The objective of this study was to evaluate the impact of coccidiosis on bone quality and antioxidant status in the liver and bone marrow of broiler chickens. A total of 360 13-day old male broilers (Cobb 500) were randomly assigned to different groups (negative control, low, medium-low, medium-high, and highest dose groups) and orally gavaged with different concentrations of Eimeria oocysts solution. Broiler tibia and tibia bone marrow were collected at 6 days post-infection (6 dpi) for bone 3-D structural analyses and the gene expression related to osteogenesis, oxidative stress, and adipogenesis using micro-computed tomography (micro-CT) and real-time qPCR analysis, respectively. Metaphyseal bone mineral density and content were reduced in response to the increase of Eimeria challenge dose, and poor trabecular bone traits were observed in the high inoculation group. However, there were no significant structural changes in metaphyseal cortical bone. Medium-high Eimeria challenge dose significantly increased level of peroxisome proliferator-activated receptor gamma (PPARG, p < 0.05) and decreased levels of bone gamma-carboxyglutamate protein coding gene (BGLAP, p < 0.05) and fatty acid synthase coding gene (FASN, p < 0.05) in bone marrow. An increased mRNA level of superoxide dismutase type 1 (SOD1, p < 0.05) and heme oxygenase 1 (HMOX1, p < 0.05), and increased enzyme activity of superoxide dismutase (SOD, p < 0.05) were found in bone marrow of Eimeria challenged groups compared with that of non-infected control. Similarly, enzyme activity of SOD and the mRNA level of SOD1, HMOX1 and aflatoxin aldehyde reductase (AKE7A2) were increased in the liver of infected broilers (p < 0.05), whereas glutathione (GSH) content was lower in the medium-high challenge group (p < 0.05) compared with non-challenged control. Moreover, the mRNA expression of catalase (CAT) and nuclear factor kappa B1 (NFKB1) showed dose-depend response in the liver, where expression of CAT and NFKB1 was upregulated in the low challenge group but decreased with the higher Eimeria challenge dosage (p < 0.05). In conclusion, high challenge dose of Eimeria infection negatively affected the long bone development. The structural changes of tibia and decreased mineral content were mainly located at the trabecular bone of metaphyseal area. The change of redox and impaired antioxidant status following the Eimeria infection were observed in the liver and bone marrow of broilers.

Abstract 5985: Marrow immune landscape participates dormancy and activation of disseminated tumor cells in a breast cancer bone metastasis model

Abstract Breast cancer has the highest incidence rate among women and the second-highest mortality rate in cancer-related death in the world. Although the research on breast cancer treatment has made some progress, metastatic breast cancer is still incurable. Bone is the most common site for breast cancer metastasis. This work is dedicated to the use of mass cytometry and single cell analysis to explore the molecular alterations in the marrow immune microenvironment which participates the dormancy and activation of disseminated tumor cells (DTCs). Firstly, four Balb/c mouse breast cancer bone metastasis groups were established. Animals were divided into intracardiac injection group (n=10), subcutaneous injection group (n=10), tumor resection and suture group (n=10), and control group (n=10). In the intracardiac injection group, a breast cancer bone metastatic model was constructed by directly injecting mCherry-labeled 4T1 cells into the left ventricle. After detecting obvious bone metastasis by bioluminescence imaging (BLI), femur and tibia bone marrow were excised for flow cytometry cell sorting, mass cytometry and single cell analysis. Both the subcutaneous and the resection and suture groups were injected with mCherry-labeled 4T1 cells into the right-back of Balb/c mice. In the tumor resection and suture group, the tumor was removed at the time when the subcutaneous tumor was just palpable. In the subcutaneous injection group, the animals were sacrificed when tumor volume reached 1 cubic centimeter. The classification of bone marrow cells was determined by using fluorescent dye labeled CD45, CD4, CD8, CD19, NK1.1, CD11b, CD11c, IA/IE, Ly6C, Ly6G and other antibodies. Constructed as a local immune landscape in the marrow, certain numbers of B cells and T cells, in the bone marrow of mice with breast cancer bone metastasis were significantly less than those in control mice, indicating that the acquired immunity of the mice with bone metastasis is diminished. In addition, the proportions of CD11b+ myeloid cells and neutrophils in the marrow of mice with bone metastasis were significantly higher than that in the control group, whereas the proportion of macrophages and eosinophils were all significantly decreased indicating that the bone marrow immune environment has changed in the process of tumor occurrence and metastasis. Research is ongoing, including ex-marrow spleen cell analysis in those animals, to explore the mechanism of the interaction between the immune microenvironment and tumor cells that determines tumor heterogeneity and sensitivity to therapy. This work was supported by NSFC projects 81773146, 81972766, 81972420; Shenzhen Science and Technology Commission Project JCYJ20180302174235893; and College students' innovative entrepreneurial training program (S202114325028) Citation Format: Yingxuan Gong, Yinghao Wang, Xinyu Jiang, Xiuzhi Li, Yueming Li, Ming Chang, Yi Lu, Jian Zhang. Marrow immune landscape participates dormancy and activation of disseminated tumor cells in a breast cancer bone metastasis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5985.

Abdominal adipose tissue and liver fat imaging in very low birth weight adults born preterm: birth cohort with sibling-controls

Preterm birth at very low birth weight (VLBW, < 1500 g) is associated with an accumulation of cardiovascular and metabolic risk factors from childhood at least to middle age. Small-scale studies suggest that this could partly be explained by increased visceral or ectopic fat. We performed magnetic resonance imaging on 78 adults born preterm at VLBW in Finland between 1978 and 1990 and 72 term same-sex siblings as controls, with a mean age of 29 years. We collected T1-weighted images from the abdomen, and magnetic resonance spectra from the liver, subcutaneous abdominal adipose tissue, and tibia. The adipose tissue volumes of VLBW adults did not differ from their term siblings when adjusting for age, sex, and maternal and perinatal factors. The mean differences were as follows: subcutaneous − 0.48% (95% CI − 14.8%, 16.3%), visceral 7.96% (95% CI − 10.4%, 30.1%), and total abdominal fat quantity 1.05% (95% CI − 13.7%, 18.4%). Hepatic triglyceride content was also similar. VLBW individuals displayed less unsaturation in subcutaneous adipose tissue (− 4.74%, 95% CI − 9.2%, − 0.1%) but not in tibial bone marrow (1.68%, 95% CI − 1.86%, 5.35%). VLBW adults displayed similar adipose tissue volumes and hepatic triglyceride content as their term siblings. Previously reported differences could thus partly be due to genetic or environmental characteristics shared between siblings. The VLBW group displayed less unsaturation in subcutaneous abdominal adipose tissue, suggesting differences in its metabolic activity and energy storage.

Factor analysis of effective platelet-producing ability of fetal liver-derived cells

Objective: To study the different factors affecting platelet production post transplantation of hematopoietic stem cells (HSCs) isolated from different sources in order to explore novel options for treating platelet depletion following HSCs transplantation. Methods: HSCs and their downstream derivatives including myeloid and lymphoid cells (i.e., collective of mononuclear cells (MNCs)), were isolated from E14.5 fetal liver (FL) and bone marrow (BM) of 8-week-old mice by Ficoll separation technique. These cells were subsequently transplanted into the tibia bone marrow cavity of recipient mice post lethal myeloablative treatment in order to construct the FL-MNCs and BM-MNCs transplantation mouse model. Routine blood indices were examined in these recipient mice. The chimeric rate of donor cells in recipient peripheral blood cells were determined by flow cytometry. Different groups of cells involved in platelet reconstruction were analyzed. CD41+megakaryocytes were sorted from fetal liver or bone marrow using magnetic beads, which were then induced to differentiate into platelets in an in vitro assay. Quantitative RT-PCR was used to detect the expression of platelet-related genes in CD41+megakaryocytes from the two sources. Results: Both the FL-MNCs and the BM-MNCs transplantation groups resumed normal hematopoiesis at the 4th week after transplantation, and the blood cells of the recipient mice were largely replaced by the donor cells. Compared with the mice transplanted with BM-MNCs, the platelet level of mice transplanted with FL-MNCs recovered faster and were maintained at a higher level. At week 4, the PLT level of the FL-MNCs group was (1.45±0.37)×1012/L, and of the BM-MNCs group was (1.22±0.24)×1012/L, P<0.05. The FL-MNCs contain a higher proportion of hematopoietic stem cells (Lin-Sca-1+c-Kit+)(7.60%±1.40%) compared to the BM-MNCs (1.10%±0.46%), P<0.01; the proportion of the megakaryocyte progenitor cells (Lin-Sca-1-c-Kit+CD41+CD150+) and mature megakaryocyte cells (CD41+CD42b+), also differ significantly between the FL-MNCs (3.05%±0.22%, 1.60%±0.06%, respectively) and the BM-MNCs (0.15%±0.02%, 0.87%±0.11%, respectively) groups, both P<0.01. In vitro functional studies showed that FL-MNCs-CD41+megakaryocytes could produce proplatelet-like cells more quickly after induction, with proplatelet-like cells formation on day 3 and significant platelet-like particle formation on day 5, in contrast to bone marrow-derived BM-MNCs-CD41+megakaryocytes that failed to form proplatelet-like cell on day 5. In addition, FL-MNCs-CD41+cells expressed higher levels of platelet-related genes, Mpl (3.25-fold), Fog1 (3-fold), and Gata1 (1.5-fold) (P<0.05). Conclusion: Compared with the BM-MNCs group, the FL-MNCs transplantation group appears to have a more efficient platelet implantation effect in the HSCs transplantation recipient in vivo, as well as a higher platelet differentiation rate in vitro. This might be related to a higher proportion of megakaryocytes and higher expression levels of genes such as Mpl, Fog1, and Gata1 that could be important for platelet formation in FL-MNCs-CD41+cells. Further exploration of the specific functions of these genes and the characteristics of the different proportions of the donor cells will provide valuable clues for the future treatment of platelets reconstitution after HSCs transplantation clinically.

Effects of non-muscle myosin Ⅱ silenced bone marrow-derived mesenchymal stem cells transplantation on lung extracellular matrix in rats after endotoxin/lipopolysaccharide-induced acute lung injury

Effects of non-muscle myosin Ⅱ silenced bone marrow-derived mesenchymal stem cells transplantation on lung extracellular matrix in rats after endotoxin/lipopolysaccharide-induced acute lung injury