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Sample records for primary mouse bone

  1. Cerium oxide nanoparticles protect primary mouse bone marrow stromal cells from apoptosis induced by oxidative stress

    NASA Astrophysics Data System (ADS)

    Zhang, Qun; Ge, Kun; Duan, Jianlei; Chen, Shizhu; Zhang, Ran; Zhang, Cuimiao; Wang, Shuxiang; Zhang, Jinchao

    2014-11-01

    Cerium oxide nanoparticles (nanoceria) have been widely used in industries and biomedical fields due to its unique properties. Previous biodistribution studies of nanoceria in vivo have shown that they are accumulated in the bone of mice after intravenous administration, about 20 % of the total intake, however, the potential effect and the mechanism of nanoceria on bone metabolism are not well-understood. Our results showed that both 25 and 50 nm nanceria decreased the damage of cell viability induced by H2O2 in a dose-dependent manner. The apoptosis ratio of pre-incubated group with nanoceria was lower than the H2O2 group. The cellular uptake studies indicated that there was a dose-dependent accumulation of both two size nanoparticles in bone marrow stromal cells. Nanoceria could be uptaken by cells due to the synergistic effect of multiple endocytosis mechanisms, and then evenly distributed in the cytoplasm without entering the nucleus. Our results suggest that nanoceria could reduce intracellular ROS level induced by H2O2 in a dose-dependent manner, moreover, maintain the normal function of mitochondria, suggesting nanoceria may have potent applications for preventing or treating osteoporosis.

  2. Targeted drug delivery of near IR fluorescent doxorubicin-conjugated poly(ethylene glycol) bisphosphonate nanoparticles for diagnosis and therapy of primary and metastatic bone cancer in a mouse model.

    PubMed

    Rudnick-Glick, S; Corem-Salkmon, E; Grinberg, I; Margel, S

    2016-12-05

    Most primary and metastatic bone tumors demonstrate increased osteoclast activity and bone resorption. Current treatment is based on a combination of surgery, radiotherapy and chemotherapy. Severe side effects are associated with chemotherapy due to use of high dosage and nonspecific uptake. Bisphosphonates have a strong affinity to Ca(2+) ions and are widely used in the treatment of bone disorders. We have engineered a unique biodegradable bisphosphonate nanoparticle (NPs) bearing two functional surface groups: (1) primary amine groups for covalent attachment of a dye/drug (e.g. NIR dye Cy 7 or doxorubicin); (2) bisphosphonate groups for targeting and chelation to bone hydroxyapatite. In addition, these engineered NPs contain high polyethyleneglycol (PEG) concentration in order to increase their blood half life time. In vitro experiments on Saos-2 human osteosarcoma cell line, demonstrated that at a tenth of the concentration, doxorubicin-conjugated bisphosphonate NPs achieved a similar uptake to free doxorubicin. In vivo targeting experiments using the NIR fluorescence bisphosphonate NPs on both Soas-2 human osteosarcoma xenograft mouse model and orthotopic bone metastases mCherry-labeled 4T1 breast cancer mouse model confirmed specific targeting. In addition, therapeutic in vivo experiments using doxorubicin-conjugated bisphosphonate NPs demonstrated a 40% greater inhibition of tumor growth in Saos-2 human osteosarcoma xenograft mouse model when compared to free doxorubicin. In this research we have shown the potential use of doxorubicin-conjugated BP NPs for the targeting and treatment of primary and metastatic bone tumors. The targeted delivery of doxorubicin to the tumor significantly increased the efficacy of the anti-cancer drug, thus enabling the effective use of a lower concentration of doxorubicin. Furthermore, the targeting ability of the BP NPs in an orthotopic xenograft mouse model reinforced our findings that these BP NPs have the potential to be

  3. Conditions Simulating Primary Bone Neoplasms.

    PubMed

    Carter, Jodi M; Howe, Benjamin Matthew; Inwards, Carrie Y

    2017-09-01

    A number of nonneoplastic conditions can mimic tumors of bone. Some of the more common mimics of primary bone tumors include infectious, inflammatory, periosteal, and degenerative joint disease-associated lesions that produce tumorlike bone surface-based or intraosseous lesions. This article considers a spectrum of reactive and nonreactive processes including stress fracture, subchondral cysts, osteonecrosis, heterotopic ossification, osteomyelitis, sarcoidosis, and amyloidoma that can present in such a way that they are mistaken for a tumor arising primary in bone. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Identifying Differentiation Stage of Individual Primary Hematopoietic Cells from Mouse Bone Marrow by Multivariate Analysis of TOF-Secondary Ion Mass Spectrometry Data

    PubMed Central

    Frisz, Jessica F.; Choi, Ji Sun; Wilson, Robert L.; Harley, Brendan A. C.; Kraft, Mary L.

    2014-01-01

    The ability to self-renew and differentiate into multiple types of blood and immune cells renders hematopoietic stem and progenitor cells (HSPCs) valuable for clinical treatment of hematopoietic pathologies and as models of stem cell differentiation for tissue engineering applications. To study directed HSC differentiation and identify the conditions that recreate the native bone marrow environment, combinatorial biomaterials that exhibit lateral variations in chemical and mechanical properties are employed. New experimental approaches are needed to facilitate correlating cell differentiation stage with location in the culture system. We demonstrate that multivariate analysis of time-of-flight secondary ion mass spectrometry (TOF-SIMS) data can be used to identify the differentiation state of individual hematopoietic cells (HCs) isolated from mouse bone marrow. Here, we identify primary HCs from three distinct stages of B cell lymphopoiesis at the single cell level: HSPCs, common lymphoid progenitors, and mature B cells. The differentiation state of individual HCs in a test set could be identified with a partial least squares discriminant analysis (PLS-DA) model that was constructed with calibration spectra from HCs of known differentiation status. The lowest error of identification was obtained when the intra-population spectral variation between the cells in the calibration and test sets was minimized. This approach complements the traditional methods that are used to identify HC differentiation stage. Further, the ability to gather mass spectrometry data from single HSCs cultured on graded biomaterial substrates may provide significant new insight into how HSPCs respond to extrinsic cues as well as the molecular changes that occur during cell differentiation. PMID:22507202

  5. Effects of Cerium Oxide Nanoparticles on the Proliferation, Osteogenic Differentiation and Adipogenic Differentiation of Primary Mouse Bone Marrow Stromal Cells In Vitro.

    PubMed

    Zhang, Qun; Ge, Kun; Ren, Huihui; Zhang, Cuimiao; Zhang, Jinchao

    2015-09-01

    The effects of cerium oxide nanoparticles (nanoceria) on the proliferation, osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells (BMSCs) were studied by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny tetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, collagen production, alizarin red-S (ARS) and oil red o stain assays. The results indicated that nanoceria increased the viability of BMSCs at all tested concentrations with evident dose dependence for 24 and 72 h. On day 14, nanoceria inhibited the osteogenic differentiation of BMSCs at all tested concentrations. On day 19 and 24, nanoceria inhibited the formation of mineralized matrix nodules of BMSCs at all tested concentrations. On day 17, nanoceria inhibited the adipogenic differentiation of BMSCs at all tested concentrations. This suggests that the effects of nanoceria on the proliferation, osteogenic differentiation and adipogenic differentiation of BMSCs are very complicated. Both the concentration and culture time have significant influence on the proliferation, osteogenic differentiation and adipogenic differentiation of BMSCs. These results will be helpful for rational applications of nanoceria in the future.

  6. Bone disease in primary hyperparathyroidism

    PubMed Central

    Bandeira, Francisco; Cusano, Natalie E.; Silva, Barbara C.; Cassibba, Sara; Almeida, Clarissa Beatriz; Machado, Vanessa Caroline Costa; Bilezikian, John P.

    2015-01-01

    Bone disease in severe primary hyperparathyroidism (PHPT) is described classically as osteitis fibrosa cystica (OFC). Bone pain, skeletal deformities and pathological fractures are features of OFC. Bone mineral density is usually extremely low in OFC, but it is reversible after surgical cure. The signs and symptoms of severe bone disease include bone pain, pathologic fractures, proximal muscle weakness with hyperreflexia. Bone involvement is typically characterized as salt-and-pepper appearance in the skull, bone erosions and bone resorption of the phalanges, brown tumors and cysts. In the radiography, diffuse demineralization is observed, along with pathological fractures, particularly in the long bones of the extremities. In severe, symptomatic PHPT, marked elevation of the serum calcium and PTH concentrations are seen and renal involvement is manifested by nephrolithiasis and nephrocalcinosis. A new technology, recently approved for clinical use in the United States and Europe, is likely to become more widely available because it is an adaptation of the lumbar spine DXA image. Trabecular bone score (TBS) is a gray-level textural analysis that provides an indirect index of trabecular microarchitecture. Newer technologies, such as high-resolution peripheral quantitative computed tomography (HR-pQCT), have provided further understanding of the microstructural skeletal features in PHPT. PMID:25166047

  7. Primary bone marrow oedema syndromes.

    PubMed

    Patel, Sanjeev

    2014-05-01

    MRI scanning in patients with rheumatological conditions often shows bone marrow oedema, which can be secondary to inflammatory, degenerative, infective or malignant conditions but can also be primary. The latter condition is of uncertain aetiology and it is also uncertain whether it represents a stage in the progression to osteonecrosis in some patients. Patients with primary bone marrow oedema usually have lower limb pain, commonly the hip, knee, ankle or feet. The diagnosis is one of exclusion with the presence of typical MRI findings. Treatment is usually conservative and includes analgesics and staying off the affected limb. The natural history is that of gradual resolution of symptoms over a number of months. Evidence for medical treatment is limited, but open-label studies suggest bisphosphonates may help in the resolution of pain and improve radiological findings. Surgical decompression is usually used as a last resort.

  8. Mouse Models of Bone Marrow Transplantation

    PubMed Central

    Reddy, Pavan; Negrin, Robert; Hill, Geoffrey R.

    2010-01-01

    Over the last 50 years, mouse models of bone marrow transplantation have provided the critical links between graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) pathophysiology and clinical practice. The initial insight from mouse models that GVHD and GVL were T cell dependent has long been confirmed clinically. More recent translations from mouse models have included the important role of inflammatory cytokines in GVHD. Newly developed concepts relating to the ability of antigen presenting cell (APC) and T cell subsets to mediate GVHD now promise significant clinical advances. The ability to use knockout and transgenic approaches to dissect mechanisms of GVHD and GVL mean that mouse systems will continue as the predominant preclinical platform. The basic transplant approach in these models, coupled with modern “real-time” immunologic imaging of GVHD and GVL is discussed. PMID:18162233

  9. Tracking Mouse Bone Marrow Monocytes In Vivo

    PubMed Central

    Hamon, Pauline; Rodero, Mathieu Paul; Combadière, Christophe; Boissonnas, Alexandre

    2015-01-01

    Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions. PMID:25867540

  10. [Classification of primary bone tumors].

    PubMed

    Dominok, G W; Frege, J

    1986-01-01

    An expanded classification for bone tumors is presented based on the well known international classification as well as earlier systems. The current status and future trends in this area are discussed.

  11. Mouse models for studying prostate cancer bone metastasis

    PubMed Central

    Dai, Jinlu; Hensel, Janine; Wang, Ning; Kruithof-de Julio, Marianna; Shiozawa, Yusuke

    2016-01-01

    Once tumor cells metastasize to the bone, the prognosis for prostate cancer patients is generally very poor. The mechanisms involved in bone metastasis, however, remain elusive, because of lack of relevant animal models. In this manuscript, we describe step-by-step protocols for the xenograft mouse models that are currently used for studying prostate cancer bone metastasis. The different routes of tumor inoculation (intraosseous, intracardiac, intravenous and orthotopic) presented are useful for exploring the biology of bone metastasis. PMID:26916039

  12. Regional bone change in intramuscular haemangioma mimicking primary bone tumour.

    PubMed

    Shikhare, Sumer; Chacko, Julio K; Chuah, Khoon L

    2015-04-01

    Intramuscular haemangiomas are benign soft-tissue tumours, commonly located in the extremities. We present a right-leg intramuscular haemangioma with florid periosteal reaction in adjacent tibia, mimicking a primary bone tumour. Plain radiograph and magnetic resonance imaging features are illustrated with the surgical and histopathological findings. Radiologists need to be familiar with reactive bone changes secondary to deep-seated intramuscular haemangiomas to avoid potential misdiagnosis.

  13. Primary bone tumors of the spine.

    PubMed

    Cañete, A Navas; Bloem, H L; Kroon, H M

    2016-04-01

    Primary bone tumors of the spine are less common than metastases or multiple myeloma. Based on the patient's age and the radiologic pattern and topography of the tumor, a very approximate differential diagnosis can be established for an osseous vertebral lesion. This article shows the radiologic manifestations of the principal primary bone tumors of the spine from a practical point of view, based on our personal experience and a review of the literature. If bone metastases, multiple myeloma, lymphomas, hemangiomas, and enostoses are excluded, only eight types of tumors account for 80% of all vertebral tumors. These are chordomas, osteoblastomas, chondrosarcomas, giant-cell tumors, osteoid osteomas, Ewing's sarcomas, osteosarcomas, and aneurysmal bone cysts.

  14. Europium-doped Gd2O3 nanotubes cause the necrosis of primary mouse bone marrow stromal cells through lysosome and mitochondrion damage.

    PubMed

    Jin, Yi; Chen, Shizhu; Duan, Jianlei; Jia, Guang; Zhang, Jinchao

    2015-05-01

    With the wide applications of europium-doped Gd2O3 nanoparticles (Gd2O3:Eu(3+) NPs) in biomedical fields, it will inevitably increase the chance of human exposure. It was reported that Gd2O3:Eu(3+) NPs could accumulate in bone. However, there have been few reports about the potential effect of Gd2O3:Eu(3+) NPs on bone marrow stromal cells (BMSCs). In this study, the Gd2O3:Eu(3+) nanotubes were prepared and characterized by powder X-ray diffraction (XRD), photoluminescence (PL) excitation and emission spectra, scanning electron microscope (SEM), and transmission electron microscopy (TEM). The cytotoxicity of Gd2O3:Eu(3+) nanotubes on BMSCs and the associated mechanisms were further studied. The results indicated that they could be uptaken into BMSCs by an energy-dependent and macropinocytosis-mediated endocytosis process, and primarily localized in lysosome. Gd2O3:Eu(3+) nanotubes effectively inhibited the viability of BMSCs in concentration and time-dependent manners. A significant increase in the percentage of late apoptotic/necrotic cells, lactate dehydrogenase (LDH) leakage and the number of PI-stained cells was found after BMSCs were treated by 10, 20, and 40μg/mL of Gd2O3:Eu(3+) nanotubes for 12h. No obvious DNA ladders were detected, but a dispersed band was observed. The above results revealed that Gd2O3:Eu(3+) nanotubes could trigger cell death by necrosis instead of apoptosis. Two mechanisms were involved in Gd2O3:Eu(3+) nanotube-induced BMSCs necrosis: lysosomal rupture and release of cathepsins B; and the overproduction of reactive oxygen species (ROS) injury to the mitochondria and DNA. The study provides novel evidence to elucidate the toxicity mechanisms and may be beneficial to more rational applications of these nanomaterials in the future.

  15. Primary extra nodal Hodgkin disease: Bone presentation.

    PubMed

    Nikolica, Goran; Badnjar, Zorka; Cadjenovic, Tanja; Raceta-Masic, Dijana

    2014-01-01

    Extra nodal and extra lymphatic propagation of Hodgkin's disease is a characteristic of the fourth stage of disease when the organs are affected. Primary appearances of the disease outside the lymph node is a rare event. Therefore, it makes diagnostic problem. Skeletal system is possible localization of primary extra nodal Hodgkin's disease. Women, 42-years-old, was admitted to hospital because of swelling and pain in the right shoulder. After imaging and histological examination diagnosed Hodgkin's nodular sclerosing histological subtype disease has been established. The patient starts to receive chemotherapy. Primary extra nodal Hodgkin's disease of bone is manifested with painful swelling in geared area. Imaging method shows destruction of the affected bone, with swelling of the soft tissues. Propagation in soft tissue is not accompanied by their destruction, but rather manifested swelling of the surrounding soft tissue.

  16. [Surgical management of primary bone cancer].

    PubMed

    Anract, Philippe

    2009-01-01

    Patients with primary bone malignancies must be treated by specialized multidisciplinary teams composed of pathologists, surgeons, orthopedists, oncologists, radiologists and radiotherapists, all with experience in the diagnosis and treatment of these tumors. If a malignancy is suspected, the biopsy must also be performed in such a center. Biopsy is part of the treatment and must be done by a senior surgeon, before starting specific treatment. Indeed, inappropriate biopsy can compromise the patient's functional prognosis and sometimes the vital outcome. The biopsy can be done percutaneously under radiological control with a True-cut needle or a trocart to obtain cores of pathological tissue. The pathologist must be well-versed in bone disorders. Open surgical biopsy is preferable for primary bone tumors, especially when a cartilaginous tumor is suspected. A short incision is used, situated on the same approach as that which will be used for surgical resection of the tumor, so that the biopsy scar is excised along with the tumor, in a single block. Surgical treatment of primary bone malignancies requires extensive resection, i.e. excision of the affected bone segment and any invaded soft tissues, as a single block, without breaching the tumor, and preserving a peripheral margin of healthy tissue. In most cases, reconstruction is necessary to preserve the function of the resected region. It is based on standard orthopedic techniques, namely osteosynthesis, bone grafts (autografts and allografts), prostheses of variable size, or a combination of prostheses and allografts (composite reconstruction). Amputation is only indicated if conservative resection is impossible. It has been shown that conservative resection, now possible in about 80% of cases, does not reduce the survival chances of patients with osteosarcoma. The indications for amputation include massive tumors invading vessels and nerves, resection of which would leave the limb non functional, as sell as tumor

  17. Primary Ameloblastoma of the Temporal Bone.

    PubMed

    Košec, Andro; Ajduk, Jakov; Ries, Mihael; Trotić, Robert

    2017-06-01

    Ameloblastoma is a locally aggressive tumor derived from odontogenic epithelium. Although benign, its clinical behavior can often exhibit malignant characteristics. It is marked by slow and persistent growth with infiltration of adjacent tissues. Almost 70% occur in the mandible in patients older than 30 years. Recurrence of ameloblastoma from inadequate treatment is frequent. Because of its slow growth, recurrences can present decades after primary surgery. A primary ameloblastoma in an area outside the mandibular, maxillary, and infratemporal fossa regions has not been described in detail to date, with only 1 possible case mentioned in the literature. The authors present a case of primary temporal bone ameloblastoma in a 17-year-old boy. The tumor originated in the left mastoid, infiltrated the lateral semicircular canal, facial nerve, and cochlea, and adhered to the sigmoid sinus and posterior cranial fossa dura. Although invasion of multiple structures in the infratemporal fossa and temporal bone leads to variable disease presentation, this case is unique because the first symptom of disease was sudden and recurring unilateral sensorineural hearing loss. Surgery required transection of the facial nerve. Histopathology confirmed primary temporal bone ameloblastoma. The difficulties in achieving wide surgical margins, diagnostics, and further management are addressed. Copyright © 2017 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

  18. URB expression in human bone marrow stromal cells and during mouse development.

    PubMed

    Liu, Yi; Monticone, Massimiliano; Tonachini, Laura; Mastrogiacomo, Maddalena; Marigo, Valeria; Cancedda, Ranieri; Castagnola, Patrizio

    2004-09-17

    Seven genes preferentially expressed in undifferentiated human bone marrow stromal cells (BMSC) with respect to BMSC-derived osteoblasts were previously identified by differential display. Here we characterize the expression of one of these genes, URB, belonging to the sushi-repeat-containing protein superfamily. In culture, URB is expressed in both human primary and cloned BMSC, and is drastically downregulated during osteoblastic differentiation of these cells. Here we show that in mouse tissues a single 3.8kb Urb transcript is detected and that the mouse Urb protein is secreted as a 150kDa glycoprotein. During mouse development Urb RNA is barely detectable in 9dpc embryos and increases at later stages. Both in situ hybridization and immunohistochemistry analysis show Urb expression in mouse embryos starting from 14dpc mostly in cartilage. The temporal and spatial expression pattern of Urb suggests its role in mouse skeletogenesis.

  19. Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model.

    PubMed

    Alexander, Kylie A; Chang, Ming K; Maylin, Erin R; Kohler, Thomas; Müller, Ralph; Wu, Andy C; Van Rooijen, Nico; Sweet, Matthew J; Hume, David A; Raggatt, Liza J; Pettit, Allison R

    2011-07-01

    Bone-lining tissues contain a population of resident macrophages termed osteomacs that interact with osteoblasts in vivo and control mineralization in vitro. The role of osteomacs in bone repair was investigated using a mouse tibial bone injury model that heals primarily through intramembranous ossification and progresses through all major phases of stabilized fracture repair. Immunohistochemical studies revealed that at least two macrophage populations, F4/80(+) Mac-2(-/low) TRACP(-) osteomacs and F4/80(+) Mac-2(hi) TRACP(-) inflammatory macrophages, were present within the bone injury site and persisted throughout the healing time course. In vivo depletion of osteomacs/macrophages (either using the Mafia transgenic mouse model or clodronate liposome delivery) or osteoclasts (recombinant osteoprotegerin treatment) established that osteomacs were required for deposition of collagen type 1(+) (CT1(+)) matrix and bone mineralization in the tibial injury model, as assessed by quantitative immunohistology and micro-computed tomography. Conversely, administration of the macrophage growth factor colony-stimulating factor 1 (CSF-1) increased the number of osteomacs/macrophages at the injury site significantly with a concurrent increase in new CT1(+) matrix deposition and enhanced mineralization. This study establishes osteomacs as participants in intramembranous bone healing and as targets for primary anabolic bone therapies. Copyright © 2011 American Society for Bone and Mineral Research.

  20. Mouse Models in Bone Marrow Transplantation and Adoptive Cellular Therapy

    PubMed Central

    Arber, Caroline; Brenner, Malcolm K.; Reddy, Pavan

    2014-01-01

    Mouse models of transplantation have been indispensable to the development of bone marrow transplantation (BMT). Their role in the generation of basic science knowledge is invaluable and is subject to discussion below. However, this article focuses on the direct role and relevance of mouse models towards the clinical development and advances in BMT and adoptive T-cell therapy for human diseases. The authors aim to present a thoughtful perspective on the pros and cons of mouse models while noting that despite imperfections these models are obligatory for the development of science-based medicine. PMID:24216170

  1. Cilia/Ift protein and motor -related bone diseases and mouse models.

    PubMed

    Yuan, Xue; Yang, Shuying

    2015-01-01

    Primary cilia are essential cellular organelles projecting from the cell surface to sense and transduce developmental signaling. They are tiny but have complicated structures containing microtubule (MT)-based internal structures (the axoneme) and mother centriole formed basal body. Intraflagellar transport (Ift) operated by Ift proteins and motors are indispensable for cilia formation and function. Mutations in Ift proteins or Ift motors cause various human diseases, some of which have severe bone defects. Over the last few decades, major advances have occurred in understanding the roles of these proteins and cilia in bone development and remodeling by examining cilia/Ift protein-related human diseases and establishing mouse transgenic models. In this review, we describe current advances in the understanding of the cilia/Ift structure and function. We further summarize cilia/Ift-related human diseases and current mouse models with an emphasis on bone-related phenotypes, cilia morphology, and signaling pathways.

  2. Cilia/Ift protein and motor-related bone diseases and mouse models

    PubMed Central

    Yuan, Xue; Yang, Shuying

    2015-01-01

    Primary cilia are essential cellular organelles projecting from the cell surface to sense and transduce developmental signaling. They are tiny but have complicated structures containing microtubule (MT)-based internal structures (the axoneme) and mother centriole formed basal body. Intraflagellar transport (Ift) operated by Ift proteins and motors are indispensable for cilia formation and function. Mutations in Ift proteins or Ift motors cause various human diseases, some of which have severe bone defects. Over the last few decades, major advances have occurred in understanding the roles of these proteins and cilia in bone development and remodeling by examining cilia/Ift protein-related human diseases and establishing mouse transgenic models. In this review, we describe current advances in the understanding of the cilia/Ift structure and function. We further summarize cilia/Ift-related human diseases and current mouse models with an emphasis on bone-related phenotypes, cilia morphology, and signaling pathways. PMID:25553465

  3. The bone diagnostic instrument III: Testing mouse femora

    NASA Astrophysics Data System (ADS)

    Randall, Connor; Mathews, Phillip; Yurtsev, Eugene; Sahar, Nadder; Kohn, David; Hansma, Paul

    2009-06-01

    Here we describe modifications that allow the bone diagnostic instrument (BDI) [P. Hansma et al., Rev. Sci. Instrum. 79, 064303 (2008); Rev. Sci. Instrum. 77, 075105 (2006)], developed to test human bone, to test the femora of mice. These modifications include reducing the effective weight of the instrument on the bone, designing and fabricating new probe assemblies to minimize damage to the small bone, developing new testing protocols that involve smaller testing forces, and fabricating a jig for securing the smaller bones for testing. With these modifications, the BDI was used to test the hypothesis that short-term running has greater benefit on the mechanical properties of the femur for young growing mice compared to older, skeletally mature mice. We measured elastic modulus, hardness, and indentation distance increase (IDI), which had previously been shown to be the best discriminators in model systems known to exhibit differences in mechanical properties at the whole bone level. In the young exercised murine femora, the IDI was significantly lower than in young control femora. Since IDI has a relation to postyield properties, these results suggest that exercise during bone development increases post yield mechanical competence. We were also able to measure effects of aging on bone properties with the BDI. There was a significant increase in the IDI, and a significant decrease in the elastic modulus and hardness between the young and old groups. Thus, with the modifications described here, the BDI can take measurements on mouse bones and obtain statistically significant results.

  4. Primary Hyperparathyroidism: Effects on Bone Health.

    PubMed

    Zanocco, Kyle A; Yeh, Michael W

    2017-03-01

    Primary hyperparathyroidism (PHPT) is the most common cause of chronic hypercalcemia. With the advent of routine calcium screening, the classic presentation of renal and osseous symptoms has been largely replaced with mild, asymptomatic disease. In hypercalcemia caused by PHPT, serum parathyroid hormone levels are either high, or inappropriately normal. A single-gland adenoma is responsible for 80% of PHPT cases. Less frequent causes include 4-gland hyperplasia and parathyroid carcinoma. Diminished bone mineral density and nephrolithiasis are the major current clinical sequelae. Parathyroidectomy is the only definitive treatment for PHPT, and in experienced hands, cure rates approach 98%.

  5. A severe combined immunodeficient-hu in vivo mouse model of human primary mantle cell lymphoma.

    PubMed

    Wang, Michael; Zhang, Liang; Han, Xiaohong; Yang, Jing; Qian, Jianfei; Hong, Sungyoul; Lin, Pei; Shi, Yuankai; Romaguera, Jorge; Kwak, Larry W; Yi, Qing

    2008-04-01

    To establish a severe combined immunodeficient (SCID)-hu in vivo mouse model of human primary mantle cell lymphoma (MCL) for the study of the biology and novel therapy of human MCL. Primary MCL cells were isolated from spleen, lymph node, bone marrow aspirates, or peripheral blood of six different patients and injected respectively into human bone chips, which had been s.c. implanted in SCID-hu. Circulating human beta(2)-microglobulin in mouse serum was used to monitor the engraftment and growth of patient's MCL cells. H&E staining and immunohistochemical staining with anti-human CD20 and cyclin D1 antibodies were used to confirm the tumor growth and migration. Increasing levels of circulating human beta(2)-microglobulin in mouse serum indicated that the patient's MCL cells were engrafted successfully into human bone chip of SCID-hu mice. The engraftment and growth of patient's MCL cells were dependent on human bone marrow microenvironment. Immunohistochemical staining with anti-human CD20 and cyclin D1 antibodies confirmed that patient's MCL cells were able to not only survive and propagate in the bone marrow microenvironment of the human fetal bone chips, but also similar to the human disease, migrate to lymph nodes, spleen, bone marrow, and gastrointestinal tract of host mice. Treatment of MCL-bearing SCID-hu mice with atiprimod, a novel antitumor compound against the protection of bone marrow stromal cells, induced tumor regression. This is the first human primary MCL animal model that should be useful for the biological and therapeutic research on MCL.

  6. Genetic mouse models for bone studies—Strengths and limitations

    PubMed Central

    Elefteriou, Florent; Yang, Xiangli

    2012-01-01

    Mice have become a preferred model system for bone research because of their genetic and pathophysiological similarities to humans: a relatively short reproductive period, leading to relatively low cost of maintenance and the availability of the entire mouse genome sequence information. The success in producing the first transgenic mouse line that expressed rabbit β-globin protein in mouse erythrocytes three decades ago marked the beginning of the use of genetically engineered mice as model system to study human diseases. Soon afterward the development of cultured pluripotent embryonic stem cells provided the possibility of gene replacement or gene deletion in mice. These technologies have been critical to identify new genes involved in bone development, growth, remodeling, repair, and diseases, but like many other approaches, they have limitations. This review will introduce the approaches that allow the generation of transgenic mice and global or conditional (tissue-specific and inducible) mutant mice. A list of the various promoters used to achieve bone-specific gene deletion or overexpression is included. The limitations of these approaches are discussed, and general guidelines related to the analysis of genetic mouse models are provided. PMID:21907838

  7. Regulation of vitamin C transporter in the type 1 diabetic mouse bone and bone marrow.

    PubMed

    Sangani, Rajnikumar; Naime, Mohammad; Zakhary, Ibrahim; Ahmad, Saif; Chutkan, Norman; Zhu, Andy; Ha, Yonju; Hamrick, Mark; Isales, Carlos; Elsalanty, Mohammed; Smith, Sylvia; Liou, Gregory I; Fulzele, Sadanand

    2013-12-01

    A number of studies have revealed that Type I diabetes (T1D) is associated with bone loss and an increased risk of fractures. T1D induces oxidative stress in various tissues and organs. Vitamin C plays an important role in the attenuation of oxidative stress; however, little is known about the effect of T1D induced oxidative stress on the regulation of vitamin C transporter in bone and bone marrow cells. To investigate this, T1D was induced in mice by multiple low dose injections of streptozotocin. We have demonstrated that endogenous antioxidants, glutathione peroxidase (GPx) and superoxide dismutase (SOD) are down-regulated in the bone and bone marrow of T1D. The vitamin C transporter isoform SVCT2, the only known transporter expressed in bone and bone marrow stromal cells (BMSCs), is negatively regulated in the bone and bone marrow of T1D. The μCT imaging of the bone showed significantly lower bone quality in the 8 week T1D mouse. The in-vitro study in BMSCS showed that the knockdown of SVCT2 transporter decreases ascorbic acid (AA) uptake, and increases oxidative stress. The significant reversing effect of antioxidant vitamin C is only possible in control cells, not in knockdown cells. This study suggested that T1D induces oxidative stress and decreases SVCT2 expression in the bone and bone marrow environment. Furthermore, this study confirms that T1D increases bone resorption, decreases bone formation and changes the microstructure of bones. This study has provided evidence that the regulation of the SVCT2 transporter plays an important role not only in T1D osteoporosis but also in other oxidative stress-related musculoskeletal complications.

  8. New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis.

    PubMed

    Sabrautzki, Sibylle; Rubio-Aliaga, Isabel; Hans, Wolfgang; Fuchs, Helmut; Rathkolb, Birgit; Calzada-Wack, Julia; Cohrs, Christian M; Klaften, Matthias; Seedorf, Hartwig; Eck, Sebastian; Benet-Pagès, Ana; Favor, Jack; Esposito, Irene; Strom, Tim M; Wolf, Eckhard; Lorenz-Depiereux, Bettina; Hrabě de Angelis, Martin

    2012-08-01

    Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.

  9. Genotoxicity of ibuprofen in mouse bone marrow cells in vivo.

    PubMed

    Tripathi, Rina; Pancholi, Shyam S; Tripathi, Pankaj

    2012-10-01

    Genotoxicity of ibuprofen was evaluated by employing the mouse in vivo chromosomal aberration (CA) test. Ibuprofen administered orally at doses of 10, 20, 40, and 60 mg/kg body weight to mice resulted in mitotic depression and induction of CAs. A dose-related decrease in mitotic index (MI) and an increase in the frequencies of chromosomal aberrations per cell (CAs/cell) were recorded in bone marrow cells. However, a statistically significant reduction in MI and an increase in CAs/cell were found for both the higher doses. The results obtained indicate that ibuprofen is capable of inducing dose-dependent genotoxicity in bone marrow cells of mice.

  10. New mouse primary retinal degeneration (rd-3)

    SciTech Connect

    Chang, B.; Hawes, N.L.; Roderick, T.H. ); Heckenlively, J.R. )

    1993-04-01

    A new mouse retinal degeneration that appears to be an excellent candidate for modeling human retinitis pigmentosa is reported. In this degeneration, called rd-3, differentiation proceeds postnatally through 2 weeks, and photoreceptor degeneration starts by 3 weeks. The rod photoreceptor loss is essentially complete by 5 weeks, whereas remnant cone cells are seen through 7 weeks. This is the only mouse homozygous retinal degeneration reported to date in which photoreceptors are initially normal. Crosses with known mouse retinal degenerations rd, Rds, nr, and pcd are negative for retinal degeneration in offspring, and linkage analysis places rd-3 on mouse chromosome 1 at 10 [+-]2.5 cM distal to Akp-1. Homology mapping suggests that the homologous human locus should be on chromosome 1q. 32 refs., 3 figs., 3 tabs.

  11. Effects of suspension-induced osteopenia on the mechanical behaviour of mouse long bones

    NASA Technical Reports Server (NTRS)

    Simske, S. J.; Greenberg, A. R.; Luttges, M. W.; Spooner, B. S. (Principal Investigator)

    1991-01-01

    Whereas most studies of tail-suspension induced osteopenia have utilized rat femora, the present study investigated the effects of a 14 day tail-suspension on the mechanical behaviour of mice femora, tibiae and humeri. Force-deflection properties were obtained via three-point bending for long bones from suspended and control mice. Whole bone behaviour was characterized by converting the force-deflection values to stiffness, strength, ductility and energy parameters which were not normalized for specimen geometry. The effects of a systematic variation in the deflection rate over the range 0.1-10 mm min-1 were also evaluated. Statistical analysis indicated that the primary effect of the tail-suspension period was lowered bone mass which was manifested mechanically through lower values of the bone strength parameters. These effects were similar in the bones of both the fore and hind limbs. The results also demonstrated that the stiffness, ductility and energy characteristics were much less influenced by the tail-suspension. Whereas a significant dependence of the bone strength values upon deflection rate was observed for the femora and humeri, the other mechanical parameters were less sensitive. Based upon the nature of the physical and mechanical changes observed in the long bones following tail-suspension, the mouse appears to be a suitable animal model for the study of osteopenia.

  12. A Computational Analysis of Bone Formation in the Cranial Vault in the Mouse

    PubMed Central

    Lee, Chanyoung; Richtsmeier, Joan T.; Kraft, Reuben H.

    2015-01-01

    Bones of the cranial vault are formed by the differentiation of mesenchymal cells into osteoblasts on a surface that surrounds the brain, eventually forming mineralized bone. Signaling pathways causative for cell differentiation include the actions of extracellular proteins driven by information from genes. We assume that the interaction of cells and extracellular molecules, which are associated with cell differentiation, can be modeled using Turing’s reaction–diffusion model, a mathematical model for pattern formation controlled by two interacting molecules (activator and inhibitor). In this study, we hypothesize that regions of high concentration of an activator develop into primary centers of ossification, the earliest sites of cranial vault bone. In addition to the Turing model, we use another diffusion equation to model a morphogen (potentially the same as the morphogen associated with formation of ossification centers) associated with bone growth. These mathematical models were solved using the finite volume method. The computational domain and model parameters are determined using a large collection of experimental data showing skull bone formation in mouse at different embryonic days in mice carrying disease causing mutations and their unaffected littermates. The results show that the relative locations of the five ossification centers that form in our model occur at the same position as those identified in experimental data. As bone grows from these ossification centers, sutures form between the bones. PMID:25853124

  13. Effects of suspension-induced osteopenia on the mechanical behaviour of mouse long bones

    NASA Technical Reports Server (NTRS)

    Simske, S. J.; Greenberg, A. R.; Luttges, M. W.; Spooner, B. S. (Principal Investigator)

    1991-01-01

    Whereas most studies of tail-suspension induced osteopenia have utilized rat femora, the present study investigated the effects of a 14 day tail-suspension on the mechanical behaviour of mice femora, tibiae and humeri. Force-deflection properties were obtained via three-point bending for long bones from suspended and control mice. Whole bone behaviour was characterized by converting the force-deflection values to stiffness, strength, ductility and energy parameters which were not normalized for specimen geometry. The effects of a systematic variation in the deflection rate over the range 0.1-10 mm min-1 were also evaluated. Statistical analysis indicated that the primary effect of the tail-suspension period was lowered bone mass which was manifested mechanically through lower values of the bone strength parameters. These effects were similar in the bones of both the fore and hind limbs. The results also demonstrated that the stiffness, ductility and energy characteristics were much less influenced by the tail-suspension. Whereas a significant dependence of the bone strength values upon deflection rate was observed for the femora and humeri, the other mechanical parameters were less sensitive. Based upon the nature of the physical and mechanical changes observed in the long bones following tail-suspension, the mouse appears to be a suitable animal model for the study of osteopenia.

  14. Nanoindentation analysis of the micromechanical anisotropy in mouse cortical bone.

    PubMed

    Casanova, Michele; Balmelli, Anna; Carnelli, Davide; Courty, Diana; Schneider, Philipp; Müller, Ralph

    2017-02-01

    Studies investigating micromechanical properties in mouse cortical bone often solely focus on the mechanical behaviour along the long axis of the bone. Therefore, data on the anisotropy of mouse cortical bone is scarce. The aim of this study is the first-time evaluation of the anisotropy ratio between the longitudinal and transverse directions of reduced modulus and hardness in mouse femurs by using the nanoindentation technique. For this purpose, nine 22-week-old mice (C57BL/6) were sacrificed and all femurs extracted. A total of 648 indentations were performed with a Berkovich tip in the proximal (P), central (C) and distal (D) regions of the femoral shaft in the longitudinal and transverse directions. Higher values for reduced modulus are obtained for indentations in the longitudinal direction, with anisotropy ratios of 1.72 ± 0.40 (P), 1.75 ± 0.69 (C) and 1.34 ± 0.30 (D). Hardness is also higher in the longitudinal direction, with anisotropic ratios of 1.35 ± 0.27 (P), 1.35 ± 0.47 (C) and 1.17 ± 0.19 (D). We observed a significant anisotropy in the micromechanical properties of the mouse femur, but the correlation for reduced modulus and hardness between the two directions is low (r(2) < 0.3) and not significant. Therefore, we highly recommend performing independent indentation testing in both the longitudinal and transverse directions when knowledge of the tissue mechanical behaviour along multiple directions is required.

  15. Nanoindentation analysis of the micromechanical anisotropy in mouse cortical bone

    PubMed Central

    Balmelli, Anna; Carnelli, Davide; Courty, Diana; Müller, Ralph

    2017-01-01

    Studies investigating micromechanical properties in mouse cortical bone often solely focus on the mechanical behaviour along the long axis of the bone. Therefore, data on the anisotropy of mouse cortical bone is scarce. The aim of this study is the first-time evaluation of the anisotropy ratio between the longitudinal and transverse directions of reduced modulus and hardness in mouse femurs by using the nanoindentation technique. For this purpose, nine 22-week-old mice (C57BL/6) were sacrificed and all femurs extracted. A total of 648 indentations were performed with a Berkovich tip in the proximal (P), central (C) and distal (D) regions of the femoral shaft in the longitudinal and transverse directions. Higher values for reduced modulus are obtained for indentations in the longitudinal direction, with anisotropy ratios of 1.72 ± 0.40 (P), 1.75 ± 0.69 (C) and 1.34 ± 0.30 (D). Hardness is also higher in the longitudinal direction, with anisotropic ratios of 1.35 ± 0.27 (P), 1.35 ± 0.47 (C) and 1.17 ± 0.19 (D). We observed a significant anisotropy in the micromechanical properties of the mouse femur, but the correlation for reduced modulus and hardness between the two directions is low (r2 < 0.3) and not significant. Therefore, we highly recommend performing independent indentation testing in both the longitudinal and transverse directions when knowledge of the tissue mechanical behaviour along multiple directions is required. PMID:28386450

  16. Remnant Woven Bone and Calcified Cartilage in Mouse Bone: Differences between Ages/Sex and Effects on Bone Strength

    PubMed Central

    Ip, Victoria; Toth, Zacharie; Chibnall, John; McBride-Gagyi, Sarah

    2016-01-01

    Introduction Mouse models are used frequently to study effects of bone diseases and genetic determinates of bone strength. Murine bones have an intracortical band of woven bone that is not present in human bones. This band is not obvious under brightfield imaging and not typically analyzed. Due to the band’s morphology and location it has been theorized to be remnant bone from early in life. Furthermore, lamellar and woven bone are well known to have differing mechanical strengths. The purpose of this study was to determine (i) if the band is from early life and (ii) if the woven bone or calcified cartilage contained within the band affect whole bone strength. Woven Bone Origin Studies In twelve to fourteen week old mice, doxycycline was used to label bone formed prior to 3 weeks old. Doxycycline labeling and woven bone patterns on contralateral femora matched well and encompassed an almost identical cross-sectional area. Also, we highlight for the first time in mice the presence of calcified cartilage exclusively within the band. However, calcified cartilage could not be identified on high resolution cone-beam microCT scans when examined visually or by thresholding methods. Mechanical Strength Studies Subsequently, three-point bending was used to analyze the effects of woven bone and calcified cartilage on whole bone mechanics in a cohort of male and female six and 13 week old Balb/C mice. Three-point bending outcomes were correlated with structural and compositional measures using multivariate linear regression. Woven bone composed a higher percent of young bones than older bones. However, calcified cartilage in older bones was twice that of younger bones, which was similar when normalized by area. Area and/or tissue mineral density accounted for >75% of variation for most strength outcomes. Percent calcified cartilage added significant predictive power to maximal force and bending stress. Calcified cartilage and woven bone could have more influence in genetic

  17. [Distribution of compact bone mesenchymal stem cells in lung tissue and bone marrow of mouse].

    PubMed

    Wang, Rui-Ping; Wu, Ren-Na; Guo, Yu-Qing; Zhang, Bin; Chen, Hu

    2014-02-01

    This study was aimed to investigate the distribution of compact bone mesenchymal stem cells(MSC) marked with lentiviral plasmid pGC FU-RFP-LV in lung tissue and bone marrow of mouse. The MSC were infected by lentivirus with infection efficiency 78%, the infected MSC were injected into BALB/c mice via tail veins in concentration of 1×10(6) /mouse. The mice were randomly divided into 4 group according to 4 time points as 1, 2, 5 and 7 days. The lung tissue and bone marrow were taken and made of frozen sections and smears respectively in order to observed the distributions of MSC. The results indicated that the lentiviral infected MSC displayed phenotypes and biological characteristics which conformed to MSC by immunophenotyping analysis and induction differentiation detection. After the MSC were infected with optimal viral titer MOI = 50, the cell growth no significantly changed; the fluorescent microscopy revealed that the distributions of MSC in bone marrow on day 1, 2, 5 and 7 were 0.50 ± 0.20, 0.67 ± 0.23, 0.53 ± 0.14, 0.33 ± 0.16; those in lung tissue were 0.55 ± 0.15, 0.47 ± 0.13, 0.29 ± 0.13, 0.26 ± 0.08. It is concluded that the distribution of MSC in lung tissue reaches a peak on day 1, while distribution of MSC in bone marrow reaches a peak on day 2. The distribution of mouse MSC relates with RFP gene expression and implantation of MSC in lung tissue and bone marrow.

  18. The effect of diet on bone shape in the mouse.

    PubMed Central

    Johnson, D R; O'Higgins, P; McAndrew, T J

    1990-01-01

    The effects of four deficient diets (oats, barley, wheat, buckwheat) on the shape of first and second cervical vertebrae and scapulae in C57BL mice have been measured using Fourier analysis. Bone shape was found to be robust, and only minimally affected by dietary change. The significance of this lack of change is discussed in the light of changes induced by diet in non-metrical variants in the skeleton. The study further emphasises the dangers of using certain non-metrical characters in taxonomic studies and indicates that the shapes of mouse bones are affected to a lesser degree by dietary influences than are the incidences of certain non-metrical character states. PMID:2272903

  19. Mouse bone marrow cytogenetic damage produced by residues of tequila.

    PubMed

    Madrigal-Bujaidar, E; Rojas, A; Ramos, A; Rosas, E; Díaz Barriga-Arceo, S

    1990-06-01

    Five concentrations (50-860 mg/kg) of residues obtained after distillation and lyophilization of commercial tequila were injected into mice for evaluation of chromosome aberrations, sister-chromatid exchanges, and proliferation kinetics in mouse bone marrow cells. Appropriate positive and negative controls were included. Our results showed significant dose-related increases of chromosomal aberrations starting at 50 mg/kg and for sister-chromatid exchanges at 430 mg/kg. Cellular proliferation kinetics showed no alterations. With these data we demonstrated that the residues of tequila are genotoxic in vivo.

  20. BONE FORMATION INDUCED IN MOUSE THIGH BY CULTURED HUMAN CELLS

    PubMed Central

    Anderson, H. Clarke; Coulter, P. R.

    1967-01-01

    Cultured FL human amnion cells injected intramuscularly into cortisone-conditioned mice proliferate to form discrete nodules which become surrounded by fibroblasts. Within 12 days, fibroblastic zones differentiate into cartilage which calcifies to form bone. Experiments were conducted to test the hypothesis that FL cells behave as an inductor of bone formation. In the electron microscope, FL cells were readily distinguished from surrounding fibroblasts. Transitional forms between the two cell types were not recognized. Stains for acid mucopolysaccharides emphasized the sharp boundary between metachromatic fibroblastic and cartilaginous zones and nonmetachromatic FL cells. 35S was taken up preferentially by fibroblasts and chondrocytes and then deposited extracellularly in a manner suggesting active secretion of sulfated mucopolysaccharides. FL cells showed negligible 35S utilization and secretion. FL cells, labeled in vitro with thymidine-3H, were injected and followed radioautographically, during bone formation. Nuclear label of injected FL cells did not appear in adjacent fibroblasts in quantities sufficient to indicate origin of the latter from FL cells. The minimal fibroblast nuclear labeling seen may represent reutilization of label from necrotic FL cells. It is suggested that FL cells injected into the mouse thigh induced cartilage and bone formation by host fibroblasts. PMID:4226746

  1. Bone formation induced in mouse thigh by cultured human cells.

    PubMed

    Anderson, H C; Coulter, P R

    1967-04-01

    Cultured FL human amnion cells injected intramuscularly into cortisone-conditioned mice proliferate to form discrete nodules which become surrounded by fibroblasts. Within 12 days, fibroblastic zones differentiate into cartilage which calcifies to form bone. Experiments were conducted to test the hypothesis that FL cells behave as an inductor of bone formation. In the electron microscope, FL cells were readily distinguished from surrounding fibroblasts. Transitional forms between the two cell types were not recognized. Stains for acid mucopolysaccharides emphasized the sharp boundary between metachromatic fibroblastic and cartilaginous zones and nonmetachromatic FL cells. (35)S was taken up preferentially by fibroblasts and chondrocytes and then deposited extracellularly in a manner suggesting active secretion of sulfated mucopolysaccharides. FL cells showed negligible (35)S utilization and secretion. FL cells, labeled in vitro with thymidine-(3)H, were injected and followed radioautographically, during bone formation. Nuclear label of injected FL cells did not appear in adjacent fibroblasts in quantities sufficient to indicate origin of the latter from FL cells. The minimal fibroblast nuclear labeling seen may represent reutilization of label from necrotic FL cells. It is suggested that FL cells injected into the mouse thigh induced cartilage and bone formation by host fibroblasts.

  2. Bone metabolism: a note on the significance of mouse models.

    PubMed

    Raska, O; Bernásková, K; Raska, I

    2009-01-01

    This minireview briefly surveys the complexity of regulations governing the bone metabolism. The impact of clinical studies devoted to osteoporosis is briefly summarized and the emphasis is put on the significance of experimental mouse models based on an extensive use of genetically modified animals. Despite possible arising drawbacks, the studies in mice are of prime importance for expanding our knowledge on bone metabolism. With respect to human physiology and medicine, one should be always aware of possible limitations as the experimental results may not be, or may be only to some extent, transposed to humans. If applicable to humans, results obtained in mice provide new clues for assessing unforeseen treatment strategies for patients. A recent publication representing in our opinion the important breakthrough in the field of bone metabolism in mice is commented in detail. It provides an evidence that skeleton is endocrine organ that affects energy metabolism and osteocalcin, a protein specifically synthesized and secreted by osteoblasts, is a hormone involved. If confirmed by other groups and applicable to humans, this study provides the awaited connection of long duration between bone disorders on one hand and obesity and diabetes on the other.

  3. Radioimmune imaging of bone marrow in patients with suspected bone metastases from primary breast cancer

    SciTech Connect

    Duncker, C.M.; Carrio, I.; Berna, L.; Estorch, M.; Alonso, C.; Ojeda, B.; Blanco, R.; Germa, J.R.; Ortega, V. )

    1990-09-01

    Radioimmune imaging of bone marrow was performed by technetium-99m- (99mTc) labeled antigranulocyte monoclonal antibody BW 250/183 (AGMoAb) scans in 32 patients with suspected bone metastases from primary breast cancer. AGMoAb scans showed bone marrow defects in 25/32 (78%) patients; bone invasion was subsequently confirmed in 23 (72%) patients. Conventional bone scans performed within the same week detected bone metastases in 17/32 (53%) patients (p less than 0.001). AGMoAb scans detected more sites indicating metastatic disease than bone scans in 12 of these 17 patients (71%). All patients with bone metastases in the axial skeleton had bone marrow defects at least at the sites of bone metastases. Of 15 patients with normal, or indicative of, benign disease bone scans, 8 patients (53%) presented with bone marrow defects in the AGMoAb scans. Bone invasion was confirmed in six of them. AGMoAb bone marrow scans provide a method for the early detection of bone metastatic invasion in patients with breast cancer and suspected bone metastases.

  4. [Pathological proximal femur fracture: consider also primary bone tumour].

    PubMed

    van de Sande, Michiel A J; van Rijswijk, Carla S P; Dijkstra, P D Sander; Taminiau, Antonie M H

    2010-01-01

    Two male and one female patient, aged 64, 70 and 51 respectively, were surgically treated for pathological fracture of the proximal femur without preoperative biopsy. In contrast to their benign radiological diagnosis, all three patients were finally diagnosed as having a malignant primary bone tumour. The proximal femur is the primary location of pathological fractures in the appendicular skeleton. Metastases to bone are the most common cause of a destructive lesion of the skeleton in an adult. Although rare, a primary bone tumour must be included in differential diagnosis of a pathological fracture. A systematic diagnostic strategy is critical to avoid complications that make curative treatment impossible. A solitary bone lesion seen on radiography should never be assumed to be a bone metastasis. Without further diagnostic research, surgical treatment for a pathological fracture should never be commenced before a definitive diagnosis is made.

  5. Thyroid hormone acting via TRβ induces expression of browning genes in mouse bone marrow adipose tissue.

    PubMed

    Lindsey, Richard C; Mohan, Subburaman

    2017-04-01

    Mutant hypothyroid mouse models have recently shown that thyroid hormone is critical for skeletal development during an important prepubertal growth period. Additionally, thyroid hormone negatively regulates total body fat, consistent with the well-established effects of thyroid hormone on energy and fat metabolism. Since bone marrow mesenchymal stromal cells differentiate into both adipocytes and osteoblasts and a relationship between bone marrow adipogenesis and osteogenesis has been predicted, we hypothesized thyroid hormone deficiency during the postnatal growth period increases marrow adiposity in mice. Marrow adiposity in TH-deficient (Tshr (-/-)) mice treated with T3/T4, TH receptor β-specific agonist GC-1, or vehicle control was evaluated via dual-energy X-ray absorptiometry and osmium micro-computed tomography. To further examine the mechanism for thyroid hormone regulation of marrow adiposity, we used real-time RT-PCR to measure the effects of thyroid hormone on adipocyte differentiation markers in primary mouse bone marrow mesenchymal stromal cells and two mouse cell lines in vitro and in Tshr (-/-) mice in vivo. Marrow adiposity increased >20% (P < 0.01) in Tshr (-/-) mice at 3 weeks of age, and treatment with T3/T4 when serum thyroid hormone normally increases (day 5-14) rescued this phenotype. Furthermore, GC-1 rescued this phenotype equally well, suggesting this thyroid hormone effect is in part mediated via TRβ signaling. Treatment of bone marrow mesenchymal stromal or ST2 cells with T3 or GC-1 significantly increased expression of several brown/beige fat markers. Moreover, injection of T3/T4 increased browning-specific markers in white fat of Tshr (-/-) mice. These data suggest that thyroid hormone regulation of marrow adiposity is mediated at least in part via activation of TRβ signaling.

  6. Staging of primary malignancies of bone.

    PubMed

    Heck, Robert K; Peabody, Terrance D; Simon, Michael A

    2006-01-01

    Staging of bone sarcomas is the process whereby patients are evaluated with regard to histology, as well as the local and distant extent, of disease. Bone sarcomas are staged based on grade, size, and the presence and location of metastases. The system is designed to help stratify patients according to known risk factors. Proper staging helps define the prognosis for patients and helps guide their treatment. Furthermore, staging allows meaningful comparisons to be done among groups of patients.

  7. Differentiation of lymphocytes in the mouse bone marrow

    PubMed Central

    Stocker, J. W.; Osmond, D. G.; Nossal, G. J. V.

    1974-01-01

    The response of mouse spleen cells to the T cell-independent antigen dinitrophenylated polymer of flagellin (DNP—POL), has been studied using an adoptive transfer system, and compared with the response of bone marrow cells. Spleen cells showed a complex cell dose—response relationship, with a markedly discontinuous curve, for assays performed before day 9 after transfer and antigen challenge. This discontinuity could be explained by a delay in attainment of the peak response for lower cell inocula. The curve became linear on a log—log scale when spleens were harvested on days 9 and 10 post-transfer. Bone marrow cells gave a lower response than would be expected from their lymphocyte content. This response increased progressively with a delay before antigen challenge in the irradiated recipient or in tissue culture prior to cell transfer, suggesting a functional maturation in this cell population, whereas the performance of spleen cells fell off under similar circumstances. The findings were consistent with, but could not prove, the hypothesis that the immediate precursors of anti-DNP antibody-forming cells in bone marrow were high surface immunoglobulin density small lymphocytes that had arisen locally from precursors lacking detectable surface immunoglobulin, by a non-mitotic maturation. PMID:4279889

  8. Surgically-induced mouse models in the study of bone regeneration: Current models and future directions

    PubMed Central

    Ning, Bin; Zhao, Yunpeng; Buza, John A.; Li, Wei; Wang, Wenzhao; Jia, Tanghong

    2017-01-01

    Bone regeneration has been extensively studied over the past several decades. The surgically-induced mouse model is the key animal model for studying bone regeneration, of the various research strategies used. These mouse models mimic the trauma and recovery processes in vivo and serve as carriers for tissue engineering and gene modification to test various therapies or associated genes in bone regeneration. The present review introduces a classification of surgically induced mouse models in bone regeneration, evaluates the application and value of these models and discusses the potential development of further innovations in this field in the future. PMID:28138711

  9. Bone and bone-marrow blood flow in chronic granulocytic leukemia and primary myelofibrosis

    SciTech Connect

    Lahtinen, R.; Lahtinen, T.; Romppanen, T.

    1982-03-01

    Blood flow in hematopoietic bone marrow and in nonhematopoietic bone has been measured with a Xe-133 washout method in 20 patients with chronic granulocytic leukemia (CGL) and in seven with primary myelofibrosis. Age-matched healthy persons served as controls. Bone-marrow blood flow in CGL was dependent upon the phase of the disease. In the metamorphosis phase, bone-marrow blood flow was high compared with that in the well-controlled phase. Apart from the initial phase, the mean values for bone blood flow in CGL were increased compared with the values of the healthy controls. In myelofibrosis the bone blood flow was also increased. Bone-marrow blood flow in these diseases was dependent upon the cellularity of bone marrow as measured morphometrically.

  10. Primary Splenic Angiosarcoma Revealed by Bone Marrow Metastasis

    PubMed Central

    Anoun, Soumaya; Marouane, Sofia; Quessar, Asmae; Benchekroun, Said

    2014-01-01

    Primary splenic angiosarcomas are the most common malignant non-hematopoietic tumors of the spleen. Metastatic diseases were found in 69% of patients in a reported series but the incidence of bone marrow involvement is unclear. We report a rare case of a 25-years-old Moroccan woman with unsuspected primary splenic angiosarcoma revealed by bone marrow metastasis. She presented with serious anemia and splenomegaly. Bone marrow biopsy revealed proliferating spindle cells. Computed tomography scanning showed an enlarged spleen with heterogeneous lesions. Splenectomy was performed and retrospective histological study of the spleen confirmed the diagnosis. She died 1 year after splenectomy. PMID:25541659

  11. Primary splenic angiosarcoma revealed by bone marrow metastasis.

    PubMed

    Anoun, Soumaya; Marouane, Sofia; Quessar, Asmae; Benchekroun, Said

    2014-12-05

    Primary splenic angiosarcomas are the most common malignant non-hematopoietic tumors of the spleen. Metastatic diseases were found in 69% of patients in a reported series but the incidence of bone marrow involvement is unclear. We report a rare case of a 25-years-old Moroccan woman with unsuspected primary splenic angiosarcoma revealed by bone marrow metastasis. She presented with serious anemia and splenomegaly. Bone marrow biopsy revealed proliferating spindle cells. Computed tomography scanning showed an enlarged spleen with heterogeneous lesions. Splenectomy was performed and retrospective histological study of the spleen confirmed the diagnosis. She died 1 year after splenectomy.

  12. IGF-1 and Bone: New Discoveries From Mouse Models

    PubMed Central

    Yakar, Shoshana; Courtland, Hayden-William; Clemmons, David

    2010-01-01

    Insulin-like growth factor-1 (IGF-1) plays a central role in cellular growth, differentiation, survival, and cell cycle progression. It is expressed early during development and its effects are mediated through binding to a tyrosine kinase receptor, the insulin-like growth factor-1 receptor (IGF-1R). In the circulation, the IGFs bind to IGF-binding proteins (IGFBPs), which determine their bioavailability and regulate the interaction between the IGFs and IGF-1R. Studies in animal models and in humans have established critical roles for IGFs in skeletal growth and development. In this review we present new and old findings from mouse models of the IGF system and discuss their clinical relevance to normal and pathological skeletal physiology. © 2010 American Society for Bone and Mineral Research. PMID:20836088

  13. Primary Cilia and Intraflagellar Transport Proteins in Bone and Cartilage.

    PubMed

    Yuan, X; Yang, S

    2016-11-01

    Primary cilia, present on most mammalian cells, function as a sensor to sense the environment change and transduce signaling. Loss of primary cilia causes a group of human pleiotropic syndromes called Ciliopathies. Some of the ciliopathies display skeletal dysplasias, implying the important role of primary cilia in skeletal development and homeostasis. Emerging evidence has shown that loss or malfunction of primary cilia or ciliary proteins in bone and cartilage is associated with developmental and function defects. Intraflagellar transport (IFT) proteins are essential for cilia formation and/or function. In this review, we discuss the role of primary cilia and IFT proteins in the development of bone and cartilage, as well as the differentiation and mechanotransduction of mesenchymal stem cells, osteoblasts, osteocytes, and chondrocytes. We also include the role of primary cilia in tooth development and highlight the current advance of primary cilia and IFT proteins in the pathogenesis of cartilage diseases, including osteoarthritis, osteosarcoma, and chondrosarcoma.

  14. Mobilization of bone marrow mesenchymal stem cells in vivo augments bone healing in a mouse model of segmental bone defect.

    PubMed

    Kumar, Sanjay; Ponnazhagan, Selvarangan

    2012-04-01

    Although the number of mesenchymal stem cells (MSC) in the bone marrow is sufficient to maintain skeletal homeostasis, in osteopenic pathology, aggravated osteoclast activity or insufficient osteoblast numbers ensue, affecting normal bone remodeling. Most of the currently available therapies are anti-resorptive with limited osteogenic potential. Since mobilization of stem/progenitors from the BM is a prerequisite for their participation in tissue repair, amplification of endogenous stem cells may provide an alternative approach in these conditions. The present study determined the potential of MSC mobilization in vivo, using combinations of different growth factors with the CXCR4 antagonist, AMD3100, in a mouse model of segmental bone defect. Results indicated that among several factors tested IGF1 had maximum proliferative ability of MSC in vitro. Results of the in vivo studies indicated that the combination of IGF1 and AMD3100 provided significant augmentation of bone growth as determined by DXA, micro-CT and histomorphometry in mice bearing segmental fractures. Further, characterization of MSC isolated from mice treated with IGF1 and AMD3100 indicated Akt/PI3K, MEK1/2-Erk1/2 and smad2/3 as key signaling pathways mediating this effect. These data indicate the potential of in vivo stem cell mobilization as a novel alternative for bone healing.

  15. Biomechanical properties of bone in a mouse model of Rett syndrome

    PubMed Central

    Kamal, Bushra; Russell, David; Payne, Anthony; Constante, Diogo; Tanner, K. Elizabeth; Isaksson, Hanna; Mathavan, Neashan; Cobb, Stuart R.

    2015-01-01

    Rett syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene are the primary cause of the disorder. Despite the dominant neurological phenotypes, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as scoliosis, reduced bone mineral density and skeletal fractures are also common and important clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any defects, we have conducted a series of histological, imaging and biomechanical tests of bone in a functional knockout mouse model of RTT. Both hemizygous Mecp2stop/y male mice in which Mecp2 is silenced in all cells and female Mecp2stop/+ mice in which Mecp2 is silenced in ~ 50% of cells as a consequence of random X-chromosome inactivation, revealed significant reductions in cortical bone stiffness, microhardness and tensile modulus. Microstructural analysis also revealed alterations in both cortical and cancellous femoral bone between wild-type and MeCP2-deficient mice. Furthermore, unsilencing of Mecp2 in adult mice cre-mediated stop cassette deletion resulted in a restoration of biomechanical properties (stiffness, microhardness) towards wild-type levels. These results show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies. PMID:25445449

  16. Biomechanical properties of bone in a mouse model of Rett syndrome.

    PubMed

    Kamal, Bushra; Russell, David; Payne, Anthony; Constante, Diogo; Tanner, K Elizabeth; Isaksson, Hanna; Mathavan, Neashan; Cobb, Stuart R

    2015-02-01

    Rett syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene are the primary cause of the disorder. Despite the dominant neurological phenotypes, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as scoliosis, reduced bone mineral density and skeletal fractures are also common and important clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any defects, we have conducted a series of histological, imaging and biomechanical tests of bone in a functional knockout mouse model of RTT. Both hemizygous Mecp2(stop/y) male mice in which Mecp2 is silenced in all cells and female Mecp2(stop/+) mice in which Mecp2 is silenced in ~50% of cells as a consequence of random X-chromosome inactivation, revealed significant reductions in cortical bone stiffness, microhardness and tensile modulus. Microstructural analysis also revealed alterations in both cortical and cancellous femoral bone between wild-type and MeCP2-deficient mice. Furthermore, unsilencing of Mecp2 in adult mice cre-mediated stop cassette deletion resulted in a restoration of biomechanical properties (stiffness, microhardness) towards wild-type levels. These results show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies.

  17. Mouse models of primary Sjögren’s syndrome

    PubMed Central

    Park, Young-Seok; Gauna, Adrienne E.; Cha, Seunghee

    2015-01-01

    Sjögren’s syndrome (SjS) is a chronic autoimmune disorder characterized by immune cell infiltration and progressive injury to the salivary and lacrimal glands. As a consequence, patients with SjS develop xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). SjS is the third most common rheumatic autoimmune disorder, affecting 4 million Americans with over 90% of patients being female. Current diagnostic criteria for SjS frequently utilize histological examinations of minor salivary glands for immune cell foci, serology for autoantibodies, and dry eye evaluation by corneal or conjunctival staining. SjS can be classified as primary or secondary SjS, depending on whether it occurs alone or in association with other systemic rheumatic conditions, respectively. Clinical manifestations typically become apparent when the disease is relatively advanced in SjS patients, which poses a challenge for early diagnosis and treatment of SjS. Therefore, SjS mouse models, because of their close resemblance to the human SjS, have been extremely valuable to identify early disease markers and to investigate underlying biological and immunological dysregulations. However, it is important to bear in mind that no single mouse model has duplicated all aspects of SjS pathogenesis and clinical features, mainly due to the multifactorial etiology of SjS that includes numerous susceptibility genes and environmental factors. As such, various mouse models have been developed in the field to try to recapitulate SjS. In this review, we focus on recent mouse models of primary SjS and describe them under three categories of spontaneous, genetically engineered, and experimentally induced development of SjS-like disease. In addition, we discuss future perspectives of SjS mouse models highlighting pros and cons of utilizing mouse models and demands for improved models. PMID:25777752

  18. Spatial clustering of tuning in mouse primary visual cortex

    PubMed Central

    Ringach, Dario L.; Mineault, Patrick J.; Tring, Elaine; Olivas, Nicholas D.; Garcia-Junco-Clemente, Pablo; Trachtenberg, Joshua T.

    2016-01-01

    The primary visual cortex of higher mammals is organized into two-dimensional maps, where the preference of cells for stimulus parameters is arranged regularly on the cortical surface. In contrast, the preference of neurons in the rodent appears to be arranged randomly, in what is termed a salt-and-pepper map. Here we revisited the spatial organization of receptive fields in mouse primary visual cortex by measuring the tuning of pyramidal neurons in the joint orientation and spatial frequency domain. We found that the similarity of tuning decreases as a function of cortical distance, revealing a weak but statistically significant spatial clustering. Clustering was also observed across different cortical depths, consistent with a columnar organization. Thus, the mouse visual cortex is not strictly a salt-and-pepper map. At least on a local scale, it resembles a degraded version of the organization seen in higher mammals, hinting at a possible common origin. PMID:27481398

  19. [Primary cancellous bone formation around micro-chambered beads].

    PubMed

    Draenert, M E; Draenert, Y; Draenert, K; Pohlemann, T; Erler, M

    2014-01-01

    The question has been raised whether benign bone defects in patients can be treated with bone forming osteoconductive ceramics achieving primarily a cancellous bone scaffold, which is under load from the beginning. Ten reconstructions were performed in 9patients (6women and 3male), with a mean age of 49 (25-65)years, suffering a high variety of epi- and metaphyseal defects, four tibial fractures, two calcaneal fractures, one pathological phalangeal fracture, one chondroma of the distal femur and two open-wedge osteotomies were filled with micro-chambered ceramic beads of 4 and 6mm in diameter. The mean follow up was 22 (7- 8)months. X-rays and CT-scans formed the basis for the evaluation of the reconstruction of the cancellous bone scaffolds. All cancellous structures were rebuilt, if completely filled with bone-forming elements. If the filling was incomplete, no physiological cancellous bone scaffold resulted. The β-TCP micro-chambered beads were completely reabsorbed or sandwich-like incorporated at the time of evaluation. The HA micro-chambered beads revealed a contrast enhancement and were integrated in the osseous construction of the bone scaffold. Primary cancellous bone formation can be achieved with osteoconductive ceramic micro-chambered beads and can be combined with any osteosynthesis for stable fixation. Copyright © 2013 SECOT. Published by Elsevier Espana. All rights reserved.

  20. [Primary osteolytic intraosseous meningioma of the occipital bone].

    PubMed

    Bernal-García, Luis Miguel; Cabezudo-Artero, José Manuel; Marcelo-Zamorano, María Bella; Fernández-Alarcón, Luis; Gilete-Tejero, Ignacio

    2015-01-01

    Primary intraosseous meningiomas are considered extradural meningiomas when no dural attachment is present. Most of them arise from the cranial bones and can present either as an osteoblastic or an osteolytic lesion. Osteolytic intraosseous meningiomas are the rarest and very few cases have been reported. Given that many of these may develop signs of malignancy, early histological confirmation is important in order to ensure appropriate treatment. The recommended therapy is surgery, with complete resection whenever possible. We present the case of a large primary intraosseous osteolytic meningioma within the occipital bone, which was completely excised five years ago, currently presenting no signs of recurrence.

  1. Development, validation and characterization of a novel mouse model of Adynamic Bone Disease (ABD).

    PubMed

    Ng, Adeline H; Willett, Thomas L; Alman, Benjamin A; Grynpas, Marc D

    2014-11-01

    The etiology of Adynamic Bone Disease (ABD) is poorly understood but the hallmark of ABD is a lack of bone turnover. ABD occurs in renal osteodystrophy (ROD) and is suspected to occur in elderly patients on long-term anti-resorptive therapy. A major clinical concern of ABD is diminished bone quality and an increased fracture risk. To our knowledge, experimental animal models for ABD other than ROD-ABD have not been developed or studied. The objectives of this study were to develop a mouse model of ABD without the complications of renal ablation, and to characterize changes in bone quality in ABD relative to controls. To re-create the adynamic bone condition, 4-month old female Col2.3Δtk mice were treated with ganciclovir to specifically ablate osteoblasts, and pamidronate was used to inhibit osteoclastic resorption. Four groups of animals were used to characterize bone quality in ABD: Normal bone controls, No Formation controls, No Resorption controls, and an Adynamic group. After a 6-week treatment period, the animals were sacrificed and the bones were harvested for analyses. Bone quality assessments were conducted using established techniques including bone histology, quantitative backscattered electron imaging (qBEI), dual energy X-ray absorptiometry (DXA), microcomputed tomography (microCT), and biomechanical testing. Histomorphometry confirmed osteoblast-related hallmarks of ABD in our mouse model. Bone formation was near complete suppression in the No Formation and Adynamic specimens. Inhibition of bone resorption in the Adynamic group was confirmed by tartrate-resistant acid phosphatase (TRAP) stain. Normal bone mineral density and architecture were maintained in the Adynamic group, whereas the No Formation group showed a reduction in bone mineral content and trabecular thickness relative to the Adynamic group. As expected, the No Formation group had a more hypomineralized profile and the Adynamic group had a higher mean mineralization profile that is

  2. Reference point indentation is not indicative of whole mouse bone measures of stress intensity fracture toughness

    PubMed Central

    Carriero, Alessandra; Bruse, Jan L.; Oldknow, Karla J.; Millán, José Luis; Farquharson, Colin; Shefelbine, Sandra J.

    2014-01-01

    Bone fragility is a concern for aged and diseased bone. Measuring bone toughness and understanding fracture properties of the bone are critical for predicting fracture risk associated with age and disease and for preclinical testing of therapies. A reference point indentation technique (BioDent) has recently been developed to determine bone's resistance to fracture in a minimally invasive way by measuring the indentation distance increase (IDI) between the first and last indentations over cyclic indentations in the same position. In this study, we investigate the relationship between fracture toughness KC and reference point indentation parameters (i.e. IDI, total indentation distance (TID) and creep indentation distance (CID)) in bones from 38 mice from six types (C57Bl/6, Balb, oim/oim, oim/+, Phospho1−/− and Phospho1 wild type counterpart). These mice bone are models of healthy and diseased bone spanning a range of fracture toughness from very brittle (oim/oim) to ductile (Phospho1−/−). Left femora were dissected, notched and tested in 3-point bending until complete failure. Contralateral femora were dissected and indented in 10 sites of their anterior and posterior shaft surface over 10 indentation cycles. IDI, TID and CID were measured. Results from this study suggest that reference point indentation parameters are not indicative of stress intensity fracture toughness in mouse bone. In particular, the IDI values at the anterior mid-diaphysis across mouse types overlapped, making it difficult to discern differences between mouse types, despite having extreme differences in stress intensity based toughness measures. When more locations of indentation were considered, the normalised IDIs could distinguish between mouse types. Future studies should investigate the relationship of the reference point indentation parameters for mouse bone in other material properties of the bone tissue in order to determine their use for measuring bone quality. PMID:25280470

  3. Reference point indentation is not indicative of whole mouse bone measures of stress intensity fracture toughness.

    PubMed

    Carriero, Alessandra; Bruse, Jan L; Oldknow, Karla J; Millán, José Luis; Farquharson, Colin; Shefelbine, Sandra J

    2014-12-01

    Bone fragility is a concern for aged and diseased bone. Measuring bone toughness and understanding fracture properties of the bone are critical for predicting fracture risk associated with age and disease and for preclinical testing of therapies. A reference point indentation technique (BioDent) has recently been developed to determine bone's resistance to fracture in a minimally invasive way by measuring the indentation distance increase (IDI) between the first and last indentations over cyclic indentations in the same position. In this study, we investigate the relationship between fracture toughness KC and reference point indentation parameters (i.e. IDI, total indentation distance (TID) and creep indentation distance (CID)) in bones from 38 mice from six types (C57Bl/6, Balb, oim/oim, oim/+, Phospho1(-/-) and Phospho1 wild type counterpart). These mice bone are models of healthy and diseased bone spanning a range of fracture toughness from very brittle (oim/oim) to ductile (Phospho1(-/-)). Left femora were dissected, notched and tested in 3-point bending until complete failure. Contralateral femora were dissected and indented in 10 sites of their anterior and posterior shaft surface over 10 indentation cycles. IDI, TID and CID were measured. Results from this study suggest that reference point indentation parameters are not indicative of stress intensity fracture toughness in mouse bone. In particular, the IDI values at the anterior mid-diaphysis across mouse types overlapped, making it difficult to discern differences between mouse types, despite having extreme differences in stress intensity based toughness measures. When more locations of indentation were considered, the normalised IDIs could distinguish between mouse types. Future studies should investigate the relationship of the reference point indentation parameters for mouse bone in other material properties of the bone tissue in order to determine their use for measuring bone quality.

  4. Primary Ewing's Sarcoma of the temporal bone in an infant.

    PubMed

    Goudarzipour, Kourosh; Shamsian, Shahin; Alavi, Samin; Nourbakhsh, Kazem; Aghakhani, Roxana; Eydian, Zahra; Arzanian, Mohammad Taghi

    2015-04-01

    Introduction : Ewing's sarcoma is the second most common primary malignant tumor of bone found in children after Osteosarcoma. It accounts for 4-9% of primary malignant bone tumors and it affects bones of the skull or face in only 1-4% of cases. Hence it rarely affects the head and neck. Subject and Method : In this case report, we describe a case of primary Ewing's sarcoma occurring in the temporal bone. The tumor was surgically excised, and the patient underwent chemotherapy for ten months. Results : Neither recurrence nor distant metastasis was noted in these 10 months after surgery but about 18 months after surgery our patient was expired. Conclusion : Although the prognosis of Ewing's sarcoma is generally poor because of early metastasis to the lungs and to other bones, a review of the article suggested that Ewing's sarcoma occurring in the skull can often be successfully managed by intensive therapy with radical excision and chemotherapy. This result was supported by the case reported here.

  5. Imaging of primary bone tumors in veterinary medicine: which differences?

    PubMed

    Vanel, Maïa; Blond, Laurent; Vanel, Daniel

    2013-12-01

    Veterinary medicine is most often a mysterious world for the human doctors. However, animals are important for human medicine thanks to the numerous biological similarities. Primary bone tumors are not uncommon in veterinary medicine and especially in small domestic animals as dogs and cats. As in human medicine, osteosarcoma is the most common one and especially in the long bones extremities. In the malignant bone tumor family, chondrosarcoma, fibrosarcoma and hemangiosarcoma are following. Benign bone tumors as osteoma, osteochondroma and bone cysts do exist but are rare and of little clinical significance. Diagnostic modalities used depend widely on the owner willing to treat his animal. Radiographs and bone biopsy are the standard to make a diagnosis but CT, nuclear medicine and MRI are more an more used. As amputation is treatment number one in appendicular bone tumor in veterinary medicine, this explains on the one hand why more recent imaging modalities are not always necessary and on the other hand, that prognostic on large animals is so poor that it is not much studied. Chemotherapy is sometimes associated with the surgery procedure, depending on the aggressivity of the tumor. Although, the strakes differs a lot between veterinary and human medicine, biological behavior are almost the same and should led to a beneficial team work between all.

  6. Binocular integration and disparity selectivity in mouse primary visual cortex

    PubMed Central

    Scholl, Benjamin; Burge, Johannes

    2013-01-01

    Signals from the two eyes are first integrated in primary visual cortex (V1). In many mammals, this binocular integration is an important first step in the development of stereopsis, the perception of depth from disparity. Neurons in the binocular zone of mouse V1 receive inputs from both eyes, but it is unclear how that binocular information is integrated and whether this integration has a function similar to that found in other mammals. Using extracellular recordings, we demonstrate that mouse V1 neurons are tuned for binocular disparities, or spatial differences, between the inputs from each eye, thus extracting signals potentially useful for estimating depth. The disparities encoded by mouse V1 are significantly larger than those encoded by cat and primate. Interestingly, these larger disparities correspond to distances that are likely to be ecologically relevant in natural viewing, given the stereo-geometry of the mouse visual system. Across mammalian species, it appears that binocular integration is a common cortical computation used to extract information relevant for estimating depth. As such, it is a prime example of how the integration of multiple sensory signals is used to generate accurate estimates of properties in our environment. PMID:23515794

  7. CURRENT PROTOCOLS IN TOXICOLOGY: Evaluation of toxicity in mouse bone marrow progenitor cells

    PubMed Central

    Ezeh, Peace C.; Xu, Huan; Wang, Shu Chun; Medina, Sebastian; Burchiel, Scott W.

    2016-01-01

    Development of blood cells through hematopoiesis occurs in the bone marrow (BM), and can be adversely impacted by various substances and/or conditions ranging from known therapeutic, intentionally administered xenobiotics to unintentional food additives and exposure to environmental chemicals. The principles underlying the techniques for evaluating toxicity to BM progenitors (erythroid, myeloid, and lymphoid) exploit changes in the normal hematopoietic process, biochemical cell surface and intracellular markers, as well as components of the BM microenvironment. Toxicological investigations following in vivo exposures of mice or in vitro exposures of mouse primary BM cell cultures allow the assessment of the developmental and functional integrity of BM cells, cell population shifts, and adverse biochemical effects due to toxicity. Colony forming unit (CFU) assays and flow cytometry are indispensable techniques in these toxicity studies. PMID:26828331

  8. Longitudinal assessment of in vivo bone dynamics in a mouse tail model of postmenopausal osteoporosis.

    PubMed

    Lambers, Floor M; Kuhn, Gisela; Schulte, Friederike A; Koch, Kathleen; Müller, Ralph

    2012-02-01

    Recently, it has been shown that transient bone biology can be observed in vivo using time-lapse micro-computed tomography (μCT) in the mouse tail bone. Nevertheless, in order for the mouse tail bone to be a model for human disease, the hallmarks of any disease must be mimicked. The aim of this study was to investigate whether postmenopausal osteoporosis could be modeled in caudal vertebrae of C57Bl/6 mice, considering static and dynamic bone morphometry as well as mechanical properties, and to describe temporal changes in bone remodeling rates. Twenty C57Bl/6 mice were ovariectomized (OVX, n = 11) or sham-operated (SHM, n = 9) and monitored with in vivo μCT on the day of surgery and every 2 weeks after, up to 12 weeks. There was a significant decrease in bone volume fraction for OVX (-35%) compared to SHM (+16%) in trabecular bone (P < 0.001). For OVX, high-turnover bone loss was observed, with the bone resorption rate exceeding the bone formation rate (P < 0.001). Furthermore there was a significant decrease in whole-bone stiffness for OVX (-16%) compared to SHM (+11%, P < 0.001). From these results we conclude that the mouse tail vertebra mimics postmenopausal bone loss with respect to these parameters and therefore might be a suitable model for postmenopausal osteoporosis. When evaluating temporal changes in remodeling rates, we found that OVX caused an immediate increase in bone resorption rate (P < 0.001) and a delayed increase in bone formation rate (P < 0.001). Monitoring transient bone biology is a promising method for future research.

  9. The Relevance of Mouse Models for Investigating Age-Related Bone Loss in Humans

    PubMed Central

    2013-01-01

    Mice are increasingly used for investigation of the pathophysiology of osteoporosis because their genome is easily manipulated, and their skeleton is similar to that of humans. Unlike the human skeleton, however, the murine skeleton continues to grow slowly after puberty and lacks osteonal remodeling of cortical bone. Yet, like humans, mice exhibit loss of cancellous bone, thinning of cortical bone, and increased cortical porosity with advancing age. Histologic evidence in mice and humans alike indicates that inadequate osteoblast-mediated refilling of resorption cavities created during bone remodeling is responsible. Mouse models of progeria also show bone loss and skeletal defects associated with senescence of early osteoblast progenitors. Additionally, mouse models of atherosclerosis, which often occurs in osteoporotic participants, also suffer bone loss, suggesting that common diseases of aging share pathophysiological pathways. Knowledge of the causes of skeletal fragility in mice should therefore be applicable to humans if inherent limitations are recognized. PMID:23689830

  10. The relevance of mouse models for investigating age-related bone loss in humans.

    PubMed

    Jilka, Robert L

    2013-10-01

    Mice are increasingly used for investigation of the pathophysiology of osteoporosis because their genome is easily manipulated, and their skeleton is similar to that of humans. Unlike the human skeleton, however, the murine skeleton continues to grow slowly after puberty and lacks osteonal remodeling of cortical bone. Yet, like humans, mice exhibit loss of cancellous bone, thinning of cortical bone, and increased cortical porosity with advancing age. Histologic evidence in mice and humans alike indicates that inadequate osteoblast-mediated refilling of resorption cavities created during bone remodeling is responsible. Mouse models of progeria also show bone loss and skeletal defects associated with senescence of early osteoblast progenitors. Additionally, mouse models of atherosclerosis, which often occurs in osteoporotic participants, also suffer bone loss, suggesting that common diseases of aging share pathophysiological pathways. Knowledge of the causes of skeletal fragility in mice should therefore be applicable to humans if inherent limitations are recognized.

  11. [Unknown primary bone metastasis: therapeutic and diagnostic strategies].

    PubMed

    Bortolus, R; Roncadin, M; Arcicasa, M; Boz, G; Franchin, G; De Paoli, A; Falchi, A M; Trovó, M G

    1993-04-01

    The primary site remains unknown in 0.5-15% of patients with multiple metastases. In 5-20% of these patients bone metastases are the first sign of disease. Survival after diagnosis ranges 2 to 6 months and 1-year survival is less than 25%. First of all, this paper reviews the current diagnostic and therapeutic approaches to patients with bone metastases from an unknown primary site and then reports the 10-year experience with radiotherapy at the Radiotherapy Division of the Centro di Riferimento Oncologico (Aviano) and of the Pordenone General Hospital in this patient population. Seventy-nine patients with bone metastases were evaluated. In 33 patients histology confirmed the original diagnosis and in 46 patients diagnosis was based on radiological and clinical findings. With respect to pain relief, differences in irradiated areas and in the ages of the patients were not significant. The patient's sex, however, did make a difference both to the degree of pain relief achieved and to survival (more favorable in women). To conclude, this paper suggests the diagnostic procedures necessary for the correct follow-up of patients with bone metastases from an unknown primary site.

  12. Resection arthrodesis for primary bone tumour about the knee.

    PubMed

    Kwan, M K; Ng, E S; Penafort, R; Saw, A; Sengupta, S

    2005-07-01

    Bone defect following en bloc resection of primary bone tumor around the knee can be reconstructed by allograft or prothesis or combination of both. Resection-arthrodesis is an alternative option for young vigorous patients facing circumstances of financial constrain or limited supply of allograft. This study was undertaken to determine the outcome and complications associated with resection-arthrodesis of 22 primary bone tumors (13 giant cell tumors and 9 osteosarcomas) around the knee treated between 1990 and 2003 at the University Malaya Medical Center. The mean follow-up was 6 years (range 1-13 years). hree patients with osteosarcoma died of lung metastasis, 3 required above knee amputation and 2 defaulted follow-up. Local complications of the procedure include infection in 8 cases (36.4%), non-union 7 (31.8%) and mal-union. Of 14 patients who returned for final evaluation, 79.8% had satisfactory outcomes according to the Musculoskeletal Tumor Society grading system. In conclusion, resection-arthrodesis of the knee is a viable treatment option for selected patients with primary bone tumor around the knee, and good functional outcome can be expected in the presence of short-term local complications.

  13. Curcumin analogue UBS109 prevents bone loss in breast cancer bone metastasis mouse model: involvement in osteoblastogenesis and osteoclastogenesis.

    PubMed

    Yamaguchi, Masayoshi; Zhu, Shijun; Zhang, Shumin; Wu, Daqing; Moore, Terry M; Snyder, James P; Shoji, Mamoru

    2014-07-01

    Bone metastasis of breast cancer typically leads to osteolysis, which causes severe pathological bone fractures and hypercalcemia. Bone homeostasis is skillfully regulated through osteoblasts and osteoclasts. Bone loss with bone metastasis of breast cancer may be due to both activation of osteoclastic bone resorption and suppression of osteoblastic bone formation. This study was undertaken to determine whether the novel curcumin analogue UBS109 has preventive effects on bone loss induced by breast cancer cell bone metastasis. Nude mice were inoculated with breast cancer MDA-MB-231 bone metastatic cells (10(6) cells/mouse) into the head of the right and left tibia. One week after inoculation, the mice were treated with control (vehicle), oral administration (p.o.) of UBS109 (50 or 150 mg/kg body weight), or intraperitoneal administration (i.p.) of UBS109 (10 or 20 mg/kg body weight) once daily for 5 days per week for 7 weeks. After UBS109 administration for 7 weeks, hind limbs were assessed using an X-ray diagnosis system and hematoxylin and eosion staining to determine osteolytic destruction. Bone marrow cells obtained from the femurs and tibias were cultured to estimate osteoblastic mineralization and osteoclastogenesis ex vivo and in vitro. Remarkable bone loss was demonstrated in the tibias of mice inoculated with breast cancer MDA-MB-231 bone metastatic cells. This bone loss was prevented by p.o. administration of UBS109 (50 and 150 mg/kg body weight) and i.p. treatment of UBS109 (10 and 20 mg/kg) in vivo. Culture of bone marrow cells obtained from the bone tissues of mice with breast cancer cell bone metastasis showed suppressed osteoblastic mineralization and stimulated osteoclastogenesis ex vivo. These changes were not seen after culture of the bone marrow cells obtained from mice treated with UBS109. Moreover, UBS109 was found to stimulate osteoblastic mineralization and suppress lipopolysaccharide (LPS)-induced osteoclastogenesis in bone marrow

  14. Bone Windows for Distinguishing Malignant from Benign Primary Bone Tumors on FDG PET/CT

    PubMed Central

    Costelloe, Colleen M.; Chuang, Hubert H.; Chasen, Beth A.; Pan, Tinsu; Fox, Patricia S.; Bassett, Roland L.; Madewell, John E.

    2013-01-01

    Objective. The default window setting on PET/CT workstations is soft tissue. This study investigates whether bone windowing and hybrid FDG PET/CT can help differentiate between malignant and benign primary bone tumors. Materials and methods. A database review included 98 patients with malignant (n=64) or benign primary bone (n=34) tumors. The reference standard was biopsy for malignancies and biopsy or >1 year imaging follow-up of benign tumors. Three radiologists and/or nuclear medicine physicians blinded to diagnosis and other imaging viewed the lesions on CT with bone windows (CT-BW) without and then with PET (PET/CT-BW), and separate PET-only images for malignancy or benignity. Three weeks later the tumors were viewed on CT with soft tissue windows (CT-STW) without and then with PET (PET/CT-STW). Results. Mean sensitivity and specificity for identifying malignancies included: CT-BW: 96%, 90%; CT-STW: 90%, 90%; PET/CT-BW: 95%, 85%, PET/CT-STW: 95%, 86% and PET-only: 96%, 75%, respectively. CT-BW demonstrated higher specificity than PET-only and PET/CT-BW (p=0.0005 and p=0.0103, respectively) and trended toward higher sensitivity than CT-STW (p=0.0759). Malignant primary bone tumors were more avid than benign lesions overall (p<0.0001) but the avidity of benign aggressive lesions (giant cell tumors and Langerhans Cell Histiocytosis) trended higher than the malignancies (p=0.08). Conclusion. Bone windows provided high specificity for distinguishing between malignant and benign primary bone tumors and are recommended when viewing FDG PET/CT. PMID:23983816

  15. Bone Windows for Distinguishing Malignant from Benign Primary Bone Tumors on FDG PET/CT.

    PubMed

    Costelloe, Colleen M; Chuang, Hubert H; Chasen, Beth A; Pan, Tinsu; Fox, Patricia S; Bassett, Roland L; Madewell, John E

    2013-01-01

    Objective. The default window setting on PET/CT workstations is soft tissue. This study investigates whether bone windowing and hybrid FDG PET/CT can help differentiate between malignant and benign primary bone tumors. Materials and methods. A database review included 98 patients with malignant (n=64) or benign primary bone (n=34) tumors. The reference standard was biopsy for malignancies and biopsy or >1 year imaging follow-up of benign tumors. Three radiologists and/or nuclear medicine physicians blinded to diagnosis and other imaging viewed the lesions on CT with bone windows (CT-BW) without and then with PET (PET/CT-BW), and separate PET-only images for malignancy or benignity. Three weeks later the tumors were viewed on CT with soft tissue windows (CT-STW) without and then with PET (PET/CT-STW). Results. Mean sensitivity and specificity for identifying malignancies included: CT-BW: 96%, 90%; CT-STW: 90%, 90%; PET/CT-BW: 95%, 85%, PET/CT-STW: 95%, 86% and PET-only: 96%, 75%, respectively. CT-BW demonstrated higher specificity than PET-only and PET/CT-BW (p=0.0005 and p=0.0103, respectively) and trended toward higher sensitivity than CT-STW (p=0.0759). Malignant primary bone tumors were more avid than benign lesions overall (p<0.0001) but the avidity of benign aggressive lesions (giant cell tumors and Langerhans Cell Histiocytosis) trended higher than the malignancies (p=0.08). Conclusion. Bone windows provided high specificity for distinguishing between malignant and benign primary bone tumors and are recommended when viewing FDG PET/CT.

  16. Targeting a novel bone degradation pathway in primary bone cancer by inactivation of the collagen receptor uPARAP/Endo180.

    PubMed

    Engelholm, Lars H; Melander, Maria C; Hald, Andreas; Persson, Morten; Madsen, Daniel H; Jürgensen, Henrik J; Johansson, Kristina; Nielsen, Christoffer; Nørregaard, Kirstine S; Ingvarsen, Signe Z; Kjaer, Andreas; Trovik, Clement S; Laerum, Ole D; Bugge, Thomas H; Eide, Johan; Behrendt, Niels

    2016-01-01

    In osteosarcoma, a primary mesenchymal bone cancer occurring predominantly in younger patients, invasive tumour growth leads to extensive bone destruction. This process is insufficiently understood, cannot be efficiently counteracted and calls for novel means of treatment. The endocytic collagen receptor, uPARAP/Endo180, is expressed on various mesenchymal cell types and is involved in bone matrix turnover during normal bone growth. Human osteosarcoma specimens showed strong expression of this receptor on tumour cells, along with the collagenolytic metalloprotease, MT1-MMP. In advanced tumours with ongoing bone degeneration, sarcoma cells positive for these proteins formed a contiguous layer aligned with the degradation zones. Remarkably, osteoclasts were scarce or absent from these regions and quantitative analysis revealed that this scarcity marked a strong contrast between osteosarcoma and bone metastases of carcinoma origin. This opened the possibility that sarcoma cells might directly mediate bone degeneration. To examine this question, we utilized a syngeneic, osteolytic bone tumour model with transplanted NCTC-2472 sarcoma cells in mice. When analysed in vitro, these cells were capable of degrading the protein component of surface-labelled bone slices in a process dependent on MMP activity and uPARAP/Endo180. Systemic treatment of the sarcoma-inoculated mice with a mouse monoclonal antibody that blocks murine uPARAP/Endo180 led to a strong reduction of bone destruction. Our findings identify sarcoma cell-resident uPARAP/Endo180 as a central player in the bone degeneration of advanced tumours, possibly following an osteoclast-mediated attack on bone in the early tumour stage. This points to uPARAP/Endo180 as a promising therapeutic target in osteosarcoma, with particular prospects for improved neoadjuvant therapy.

  17. Even Your Bones Can Get Fat, Mouse Study Suggests

    MedlinePlus

    ... More Health News on Bone Density Exercise and Physical Fitness Obesity Recent Health News Related MedlinePlus Health Topics Bone Density Exercise and Physical Fitness Obesity About MedlinePlus Site Map FAQs Customer Support ...

  18. Modularity in the Organization of Mouse Primary Visual Cortex

    PubMed Central

    Ji, Weiqing; Gămănuţ, Răzvan; Bista, Pawan; D’Souza, Rinaldo D.; Wang, Quanxin; Burkhalter, Andreas

    2015-01-01

    SUMMARY Layer 1 (L1) of primary visual cortex (V1) is the target of projections from many brain regions outside of V1. We found that inputs to the non-columnar mouse V1 from the dorsal lateral geniculate nucleus and feedback projections from multiple higher cortical areas to L1 are patchy. The patches are matched to a pattern of M2 muscarinic acetylcholine receptor expression at fixed locations of mouse, rat and monkey V1. Neurons in L2/3 aligned with M2-rich patches have high spatial acuity whereas cells in M2-poor zones exhibited high temporal acuity. Together M2+ and M2− zones form constant-size domains that are repeated across V1. Domains map subregions of the receptive field, such that multiple copies are contained within the point image. The results suggest that the modular network in mouse V1 selects spatiotemporally distinct clusters of neurons within the point image for top-down control and differential routing of inputs to cortical streams. PMID:26247867

  19. Mechanical loading, damping, and load-driven bone formation in mouse tibiae.

    PubMed

    Dodge, Todd; Wanis, Mina; Ayoub, Ramez; Zhao, Liming; Watts, Nelson B; Bhattacharya, Amit; Akkus, Ozan; Robling, Alexander; Yokota, Hiroki

    2012-10-01

    Mechanical loads play a pivotal role in the growth and maintenance of bone and joints. Although loading can activate anabolic genes and induce bone remodeling, damping is essential for preventing traumatic bone injury and fracture. In this study we investigated the damping capacity of bone, joint tissue, muscle, and skin using a mouse hindlimb model of enhanced loading in conjunction with finite element modeling to model bone curvature. Our hypothesis was that loads were primarily absorbed by the joints and muscle tissue, but that bone also contributed to damping through its compression and natural bending. To test this hypothesis, fresh mouse distal lower limb segments were cyclically loaded in axial compression in sequential bouts, with each subsequent bout having less surrounding tissue. A finite element model was generated to model effects of bone curvature in silico. Two damping-related parameters (phase shift angle and energy loss) were determined from the output of the loading experiments. Interestingly, the experimental results revealed that the knee joint contributed to the largest portion of the damping capacity of the limb, and bone itself accounted for approximately 38% of the total phase shift angle. Computational results showed that normal bone curvature enhanced the damping capacity of the bone by approximately 40%, and the damping effect grew at an accelerated pace as curvature was increased. Although structural curvature reduces critical loads for buckling in beam theory, evolution apparently favors maintaining curvature in the tibia. Histomorphometric analysis of the tibia revealed that in response to axial loading, bone formation was significantly enhanced in the regions that were predicted to receive a curvature-induced bending moment. These results suggest that in addition to bone's compressive damping capacity, surrounding tissues, as well as naturally-occurring bone curvature, also contribute to mechanical damping, which may ultimately affect

  20. Bone Marrow Stromal Cells Contribute to Bone Formation Following Infusion into Femoral Cavities of a Mouse Model of Osteogenesis Imperfecta

    PubMed Central

    Li, Feng; Wang, Xujun; Niyibizi, Christopher

    2010-01-01

    Currently, there are conflicting data in literature regarding contribution of bone marrow stromal cells (BMSCs) to bone formation when the cells are systemically delivered in recipient animals. To understand if BMSCs contribute to bone cell phenotype and bone formation in osteogenesis imperfecta bones (OI), MSCs marked with GFP were directly infused into the femurs of a mouse model of OI (oim). The contribution of the cells to the cell phenotype and bone formation was assessed by histology, immunohistochemistry and biomechanical loading of recipient bones. Two weeks following infusion of BMSCs, histological examination of the recipient femurs demonstrated presence of new bone when compared to femurs injected with saline which showed little or no bone formation. The new bone contained few donor cells as demonstrated by GFP fluorescence. At six weeks following cell injection, new bone was still detectable in the recipient femurs but was enhanced by injection of the cells suspended in pepsin solublized type I collagen. Immunofluorescence and immunohistochemical staining showed that donor GFP positive cells in the new bone were localized with osteocalcin expressing cells suggesting that the cells differentiated into osteoblasts in vivo. Biomechanical loading to failure in thee point bending, revealed that, femurs infused with BMSCs in PBS or in soluble type I collagen were biomechanically stronger than those injected with PBS or type I collagen alone. Taken together, the results indicate that transplanted cells differentiated into osteoblasts in vivo and contributed to bone formation in vivo; we also speculate that donor cells induced differentiation or recruitment of endogenous cells to initiate reparative process at early stages following transplantation. PMID:20570757

  1. Probiotic L. reuteri treatment prevents bone loss in a menopausal ovariectomized mouse model

    PubMed Central

    Britton, Robert A.; Irwin, Regina; Quach, Darin; Schaefer, Laura; Zhang, Jing; Lee, Taehyung; Parameswaran, Narayanan; McCabe, Laura R.

    2014-01-01

    Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post-menopausal bone loss. Recent studies suggest an important role for gut-bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri treated mice. Consistent with this, L. reuteri suppressed Ovx-induced increases in bone marrow CD4+ T-lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identif ied that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost-effective approach to reduce post-menopausal bone loss. PMID:24677054

  2. Positive effects of bisphosphonates on bone and muscle in a mouse model of Duchenne muscular dystrophy.

    PubMed

    Yoon, Sung-Hee; Sugamori, Kim S; Grynpas, Marc D; Mitchell, Jane

    2016-01-01

    Patients with Duchenne muscular dystrophy are at increased risk of decreased bone mineral density and bone fracture as a result of inactivity. To determine if antiresorptive bisphosphonates could improve bone quality and their effects on muscle we studied the Mdx mouse, treated with pamidronate during peak bone growth at 5 and 6 weeks of age, and examined the outcome at 13 weeks of age. Pamidronate increased cortical bone architecture and strength in femurs with increased resistance to fracture. While overall long bone growth was not affected by pamidronate, there was significant inhibition of remodeling in metaphyseal trabecular bone with evidence of residual calcified cartilage. Pamidronate treatment had positive effects on skeletal muscle in the Mdx mice with decreased serum and muscle creatine kinase and evidence of improved muscle histology and grip strength.

  3. Primary cutaneous aspergillosis and idiopathic bone marrow aplasia*

    PubMed Central

    Furlan, Karina Colossi; Pires, Mario Cezar; Kakizaki, Priscila; Chartuni, Juliana Cabral Nunes; Valente, Neusa Yuriko Sakai

    2016-01-01

    We describe the case of a 9-year-old boy with idiopathic bone marrow aplasia and severe neutropenia, who developed skin ulcers under cardiac monitoring electrodes. The diagnosis of primary cutaneous aspergillosis was made after the second biopsy and culture. Imaging investigation did not reveal internal fungal infection. The child was treated, but did not improve and died 3 months after admission. The report highlights and discusses the preventable risk of aspergillus skin infection in immunocompromised patients. PMID:27438213

  4. Bone

    NASA Astrophysics Data System (ADS)

    Helmberger, Thomas K.; Hoffmann, Ralf-Thorsten

    The typical clinical signs in bone tumours are pain, destruction and destabilization, immobilization, neurologic deficits, and finally functional impairment. Primary malignant bone tumours are a rare entity, accounting for about 0.2% of all malignancies. Also benign primary bone tumours are in total rare and mostly asymptomatic. The most common symptomatic benign bone tumour is osteoid osteoma with an incidence of 1:2000.

  5. Primary Osteosarcoma of the Bone with Rhabdoid Features: A Rare, Previously Undescribed Primary Malignant Tumor of Bone

    PubMed Central

    Al Maaieh, Motasem; Rosenberg, Andrew; Conway, Sheila

    2016-01-01

    Primary osteosarcoma of the bone with rhabdoid features is a rare malignant tumor of bone, not previously described in the literature. Here we report a 69-year-old female who originally presented with a right femur pathologic fracture. Radiographs of the injury showed an aggressive-appearing lesion of the distal femur. Initial biopsy was done, which was not diagnostic; additional advanced imaging studies were performed, which failed to show any other site within the body with detectable disease process. Accordingly, the patient underwent radical resection of the distal femur and reconstruction with endoprosthesis. Histopathology obtained from the operative specimen showed osteosarcoma with rhabdoid features. Two months after surgery, the patient is symptom-free and doing well; she is currently pending adjuvant chemotherapy. Although rhabdoid features have been described in extraskeletal osteosarcoma, this appears to be the first mention of osteosarcoma of bone with rhabdoid features in the literature. PMID:28058126

  6. Endochondral bone formation in embryonic mouse pre-metatarsals

    NASA Technical Reports Server (NTRS)

    Klement, B. J.; Spooner, B. S.

    1992-01-01

    Long term exposure to a reduced gravitational environment has a deleterious effect on bone. The developmental events which occur prior to initial bone deposition will provide insight into the regulation of mature bone physiology. We have characterized a system in which the events preceding bone formation take place in an isolated in vitro organ culture environment. We show that cultured pre-metatarsal tissue parallels development of pre-metatarsal tissue in the embryo. Both undergo mesenchyme differentiation and morphogenesis to form a cartilage rod, which resembles the future bone, followed by terminal chondrocyte differentiation in a definite morphogenetic pattern. These sequential steps occur prior to osteoblast maturation and bone matrix deposition in the developing organism. Alkaline phosphatase (ALP) activity is a distinctive enzymatic marker for mineralizing tissues. We have measured this activity throughout pre-metatarsal development and show (a) where in the tissue it is predominantly found, and (b) that this is indeed the mineralizing isoform of the enzyme.

  7. Differential Bone Loss in Mouse Models of Colon Cancer Cachexia

    PubMed Central

    Bonetto, Andrea; Kays, Joshua K.; Parker, Valorie A.; Matthews, Ryan R.; Barreto, Rafael; Puppa, Melissa J.; Kang, Kyung S.; Carson, James A.; Guise, Theresa A.; Mohammad, Khalid S.; Robling, Alexander G.; Couch, Marion E.; Koniaris, Leonidas G.; Zimmers, Teresa A.

    2017-01-01

    Cachexia is a distinctive feature of colorectal cancer associated with body weight loss and progressive muscle wasting. Several mechanisms responsible for muscle and fat wasting have been identified, however it is not known whether the physiologic and molecular crosstalk between muscle and bone tissue may also contribute to the cachectic phenotype in cancer patients. The purpose of this study was to clarify whether tumor growth associates with bone loss using several experimental models of colorectal cancer cachexia, namely C26, HT-29, and ApcMin/+. The effects of cachexia on bone structure and strength were evaluated with dual energy X-ray absorptiometry (DXA), micro computed tomography (μCT), and three-point bending test. We found that all models showed tumor growth consistent with severe cachexia. While muscle wasting in C26 hosts was accompanied by moderate bone depletion, no loss of bone strength was observed. However, HT-29 tumor bearing mice showed bone abnormalities including significant reductions in whole-body bone mineral density (BMD), bone mineral content (BMC), femoral trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), but no declines in strength. Similarly, cachexia in the ApcMin/+ mice was associated with significant decreases in BMD, BMC, BV/TV, Tb.N, and Tb.Th as well as decreased strength. Our data suggest that colorectal cancer is associated with muscle wasting and may be accompanied by bone loss dependent upon tumor type, burden, stage and duration of the disease. It is clear that preserving muscle mass promotes survival in cancer cachexia. Future studies will determine whether strategies aimed at preventing bone loss can also improve outcomes and survival in colorectal cancer cachexia. PMID:28123369

  8. Differential Bone Loss in Mouse Models of Colon Cancer Cachexia.

    PubMed

    Bonetto, Andrea; Kays, Joshua K; Parker, Valorie A; Matthews, Ryan R; Barreto, Rafael; Puppa, Melissa J; Kang, Kyung S; Carson, James A; Guise, Theresa A; Mohammad, Khalid S; Robling, Alexander G; Couch, Marion E; Koniaris, Leonidas G; Zimmers, Teresa A

    2016-01-01

    Cachexia is a distinctive feature of colorectal cancer associated with body weight loss and progressive muscle wasting. Several mechanisms responsible for muscle and fat wasting have been identified, however it is not known whether the physiologic and molecular crosstalk between muscle and bone tissue may also contribute to the cachectic phenotype in cancer patients. The purpose of this study was to clarify whether tumor growth associates with bone loss using several experimental models of colorectal cancer cachexia, namely C26, HT-29, and Apc(Min/+). The effects of cachexia on bone structure and strength were evaluated with dual energy X-ray absorptiometry (DXA), micro computed tomography (μCT), and three-point bending test. We found that all models showed tumor growth consistent with severe cachexia. While muscle wasting in C26 hosts was accompanied by moderate bone depletion, no loss of bone strength was observed. However, HT-29 tumor bearing mice showed bone abnormalities including significant reductions in whole-body bone mineral density (BMD), bone mineral content (BMC), femoral trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), but no declines in strength. Similarly, cachexia in the Apc(Min/+) mice was associated with significant decreases in BMD, BMC, BV/TV, Tb.N, and Tb.Th as well as decreased strength. Our data suggest that colorectal cancer is associated with muscle wasting and may be accompanied by bone loss dependent upon tumor type, burden, stage and duration of the disease. It is clear that preserving muscle mass promotes survival in cancer cachexia. Future studies will determine whether strategies aimed at preventing bone loss can also improve outcomes and survival in colorectal cancer cachexia.

  9. Genetics of primary and timing effects in the mnd mouse

    SciTech Connect

    Messer, A.; Plummer, J.; MacMillen, M.C.

    1995-06-05

    The mnd mouse shows a spontaneous adult-onset hereditary neurological disease, with motor abnormality by 6 months of age, progressing to severe spastic paralysis and premature death. The disease is autosomal recessive, with heterozygote effects seen under stress. It maps to mouse chromosome (chr) 8. Histopathology with Nissl stains documents substantial abnormalities of upper and lower motor neurons, and there is retinal degeneration beginning in the first month, even without light exposure. Increasing levels of autofluorescent lipopigment are found in both neuronal and non-neuronal tissues as the mnd mice age. Recently, NCL-like inclusions and accumulating subunit c have also been described. When mnd is outcrossed to the AKR/J genetic background, ca. 40% of the mnd/mnd F2 progeny show early onset (onset by 4.5-5 months and death by 7 months). This accelerated timing effect seems to be strain-specific, and unlinked to the mnd gene itself. Our current working hypothesis is that the timing effect is due to 2 or 3 unlinked dominant genes with incomplete penetrance at any single locus. In a combined RFLP/PCR fragment genetic analysis, the strongest deviation from the expected ratio of AKR vs B6 alleles occurs with markers on proximal half of chr 1. Additional loci on chrs 5 and 10 may also be involved. The mechanism of interaction of these modifying genes with the primary mnd gene may offer new therapeutic avenues. 22 refs., 2 tabs.

  10. Cadmium stimulates osteoclast-like multinucleated cell formation in mouse bone marrow cell cultures

    SciTech Connect

    Miyahara, Tatsuro; Takata, Masakazu; Miyata, Masaki; Nagai, Miyuki; Sugure, Akemi; Kozuka, Hiroshi; Kuze, Shougo )

    1991-08-01

    Most of cadmium (Cd)-treated animals have been reported to show osteoporosis-like changes in bones. This suggests that Cd may promote bone loss by a direct action on bone. It was found that Cd stimulated prostaglandin E{sub 2}(PGE{sub 2}) production in the osteoblast-like cell, MC3T3-E1. Therefore, Cd stimulates bone resorption by increasing PGE{sub 2} production. Recently, several bone marrow cell culture systems have been developed for examining the formation of osteoclast-like multinucleated cells in vitro. As osteoblasts produce PGE{sub 2} by Cd-induced cyclooxygenase and may play an important role in osteoclast formation, the present study was undertaken to clarify the possibility that Cd might stimulate osteoclast formation in a mouse bone marrow culture system.

  11. A method for isolating high quality RNA from mouse cortical and cancellous bone.

    PubMed

    Kelly, Natalie H; Schimenti, John C; Patrick Ross, F; van der Meulen, Marjolein C H

    2014-11-01

    The high incidence of fragility fractures in cortico-cancellous bone locations, plus the fact that individual skeletal sites exhibit different responsiveness to load and disease, emphasizes the need to document separately gene expression in cortical and cancellous bone. A further confounding factor is marrow contamination since its high cellularity may effect gene expression measurements. We isolated RNA from cortical and cancellous bone of intact mouse tibiae, and also after marrow removal by flushing or centrifugation. RNA isolated from cancellous bone by each method was sufficient for gene expression analysis. Centrifugation removed contaminating cells more efficiently than flushing, as indexed by histology and decreased expression of Icam4, a highly expressed erythroid gene. In contrast, centrifuged cortical bone had 12- and 13- fold higher expression of the bone-related genes Col1a1 and Bglap, while levels in marrow-free cancellous bone were 30- and 31-fold higher when compared to bone where marrow was left intact. Furthermore, cortical bone had higher expression of Col1a1 and Bglap than cancellous bone. Thus, RNA isolated by this novel approach can reveal site-specific changes in gene expression in cortical and cancellous bone sites.

  12. Mineral metabolism in isolated mouse long bones: Opposite effects of microgravity on mineralization and resorption

    NASA Technical Reports Server (NTRS)

    Veldhuijzen, Jean Paul; Vanloon, Jack J. W. A.

    1994-01-01

    An experiment using isolated skeletal tissues under microgravity, is reported. Fetal mouse long bones (metatarsals) were cultured for 4 days in the Biorack facility of Spacelab during the IML-1 (International Microgravity Laboratory) mission of the Space Shuttle. Overall growth was not affected, however glucose consumption was significantly reduced under microgravity. Mineralization of the diaphysis was also strongly reduced under microgravity as compared to the on-board 1 g group. In contrast, mineral resorption by osteoclasts was signficantly increased. These results indicate that these fetal mouse long bones are a sensitive and useful model to further study the cellular mechanisms involved in the changed mineral metabolism of skeletal tissues under microgravity.

  13. Primary bone tumors and pseudotumors of the lumbosacral spine.

    PubMed

    Leone, A; Costantini, A; Guglielmi, G; Settecasi, C; Priolo, F

    2000-01-01

    Primary tumors of the spine are relatively infrequent lesions compared with metastatic disease, multiple myeloma, and lymphoma which are the more frequent neoplasms of the spine and usually manifest with multifocal lesions and thus pose little diagnostic dilemma. However, in the presence of a solitary spinal lesion, the more uncommon primary tumors of the spine represent an important group of entities for diagnostic consideration. The most common benign and malignant primary tumors of the spine are enostosis, osteoid osteoma, osteoblastoma, giant cell tumor, aneurysmal bone cyst, osteochondroma, chordoma, chondrosarcoma, Ewing sarcoma, primitive neuroectodermal tumor, and osteosarcoma. The imaging features of these lesions are often characteristic. Radiologists should be aware of the appearance of these unusual tumors in order to provide a complete differential diagnosis.

  14. Primary homologies of the circumorbital bones of snakes.

    PubMed

    Palci, Alessandro; Caldwell, Michael W

    2013-09-01

    Some snakes have two circumorbital ossifications that in the current literature are usually referred to as the postorbital and supraorbital. We review the arguments that have been proposed to justify this interpretation and provide counter-arguments that reject those conjectures of primary homology based on the observation of 32 species of lizards and 81 species of snakes (both extant and fossil). We present similarity arguments, both topological and structural, for reinterpretation of the primary homologies of the dorsal and posterior orbital ossifications of snakes. Applying the test of similarity, we conclude that the posterior orbital ossification of snakes is topologically consistent as the homolog of the lacertilian jugal, and that the dorsal orbital ossification present in some snakes (e.g., pythons, Loxocemus, and Calabaria) is the homolog of the lacertilian postfrontal. We therefore propose that the terms postorbital and supraorbital should be abandoned as reference language for the circumorbital bones of snakes, and be replaced with the terms jugal and postfrontal, respectively. The primary homology claim for the snake "postorbital" fails the test of similarity, while the term "supraorbital" is an unnecessary and inaccurate application of the concept of a neomorphic ossification, for an element that passes the test of similarity as a postfrontal. This reinterpretation of the circumorbital bones of snakes is bound to have important repercussions for future phylogenetic analyses and consequently for our understanding of the origin and evolution of snakes. Copyright © 2013 Wiley Periodicals, Inc.

  15. Polarization in Raman spectroscopy helps explain bone brittleness in genetic mouse models

    PubMed Central

    Makowski, Alexander J.; Pence, Isaac J.; Uppuganti, Sasidhar; Zein-Sabatto, Ahbid; Huszagh, Meredith C.; Mahadevan-Jansen, Anita; Nyman, Jeffry S.

    2014-01-01

    Abstract. Raman spectroscopy (RS) has been extensively used to characterize bone composition. However, the link between bone biomechanics and RS measures is not well established. Here, we leveraged the sensitivity of RS polarization to organization, thereby assessing whether RS can explain differences in bone toughness in genetic mouse models for which traditional RS peak ratios are not informative. In the selected mutant mice—activating transcription factor 4 (ATF4) or matrix metalloproteinase 9 (MMP9) knock-outs—toughness is reduced but differences in bone strength do not exist between knock-out and corresponding wild-type controls. To incorporate differences in the RS of bone occurring at peak shoulders, a multivariate approach was used. Full spectrum principal components analysis of two paired, orthogonal bone orientations (relative to laser polarization) improved genotype classification and correlation to bone toughness when compared to traditional peak ratios. When applied to femurs from wild-type mice at 8 and 20 weeks of age, the principal components of orthogonal bone orientations improved age classification but not the explanation of the maturation-related increase in strength. Overall, increasing polarization information by collecting spectra from two bone orientations improves the ability of multivariate RS to explain variance in bone toughness, likely due to polarization sensitivity to organizational changes in both mineral and collagen. PMID:25402627

  16. Polarization in Raman spectroscopy helps explain bone brittleness in genetic mouse models

    NASA Astrophysics Data System (ADS)

    Makowski, Alexander J.; Pence, Isaac J.; Uppuganti, Sasidhar; Zein-Sabatto, Ahbid; Huszagh, Meredith C.; Mahadevan-Jansen, Anita; Nyman, Jeffry S.

    2014-11-01

    Raman spectroscopy (RS) has been extensively used to characterize bone composition. However, the link between bone biomechanics and RS measures is not well established. Here, we leveraged the sensitivity of RS polarization to organization, thereby assessing whether RS can explain differences in bone toughness in genetic mouse models for which traditional RS peak ratios are not informative. In the selected mutant mice-activating transcription factor 4 (ATF4) or matrix metalloproteinase 9 (MMP9) knock-outs-toughness is reduced but differences in bone strength do not exist between knock-out and corresponding wild-type controls. To incorporate differences in the RS of bone occurring at peak shoulders, a multivariate approach was used. Full spectrum principal components analysis of two paired, orthogonal bone orientations (relative to laser polarization) improved genotype classification and correlation to bone toughness when compared to traditional peak ratios. When applied to femurs from wild-type mice at 8 and 20 weeks of age, the principal components of orthogonal bone orientations improved age classification but not the explanation of the maturation-related increase in strength. Overall, increasing polarization information by collecting spectra from two bone orientations improves the ability of multivariate RS to explain variance in bone toughness, likely due to polarization sensitivity to organizational changes in both mineral and collagen.

  17. How tough is Brittle Bone? Investigating Osteogenesis Imperfecta in Mouse Bone††

    PubMed Central

    Carriero, A.; Zimmermann, E. A.; Paluszny, A.; Tang, S. Y.; Bale, H.; Busse, B.; Alliston, T.; Kazakia, G.

    2015-01-01

    The multiscale hierarchical structure of bone is naturally optimized to resist fractures. In osteogenesis imperfecta, or brittle bone disease, genetic mutations affect the quality and/or quantity of collagen, dramatically increasing bone fracture risk. Here we reveal how the collagen defect results in bone fragility in a mouse model of osteogenesis imperfecta (oim), which has homotrimeric α1(I) collagen. At the molecular level we attribute the loss in toughness to a decrease in the stabilizing enzymatic crosslinks and an increase in non-enzymatic crosslinks, which may break prematurely inhibiting plasticity. At the tissue level, high vascular canal density reduces the stable crack growth, and extensive woven bone limits the crack-deflection toughening during crack growth. This demonstrates how modifications at the bone molecular level have ramifications at larger length scales affecting the overall mechanical integrity of the bone; thus, treatment strategies have to address multiscale properties in order to regain bone toughness. In this regard, findings from the heterozygous oim bone, where defective as well as normal collagen are present, suggest that increasing the quantity of healthy collagen in these bones helps to recover toughness at the multiple length scales. PMID:24420672

  18. The primary structure of genetic variants of mouse hemoglobin

    SciTech Connect

    Popp, R.A.; Bailiff, E.G.; Skow, L.C.; Whitney, J.B. III

    1982-01-01

    The primary structures of the ..cap alpha.. globins from CE/J, DBA/2J, and a stock of Potter's mice were determined to identify the amino acid substitutions associated with the unique isoelectric focusing patterns of these hemoglobins. In addition, the primary structures of the ..cap alpha.. globins from MOL III and PERU mice were studied in search of amino acid substitutions that may not be detected by isoelectric focusing. CE/J hemoglobin contains a unique kind of ..cap alpha.. globin called chain 5. It differs from the single kind of ..cap alpha.. globin (chain 1) in C57BL/6 by having alanine rather than glycine at position 78. DBA/2J hemoglobin has two kinds of ..cap alpha.. globins: one half is like chain 5 and the other half is like chain 1. The hemoglobin from Potter's stock of Mus musculus molossinus also contains chains 1 and 5, but they are expressed at different levels (i.e., 80% chain 1 and 20% chain 5). MOL III hemoglobin has a single kind of ..cap alpha.. globin identical to that in C57BL/6, and PERU hemoglobin contains approximately 40% chain 1 and 60% chain 4. Chains 1 and 4 have different amino acids at positions 25, 62, and 68. These studies confirm that mouse hemoglobins separable by isoelectric focusing, but not by other means of electrophoresis, have substitutions of neutrally charged amino acids in their ..cap alpha.. chains.

  19. Transgenic Mouse Model for Reducing Oxidative Damage in Bone

    NASA Technical Reports Server (NTRS)

    Schreurs, A.-S.; Torres, S.; Truong, T.; Kumar, A.; Alwood, J. S.; Limoli, C. L.; Globus, R. K.

    2014-01-01

    Exposure to musculoskeletal disuse and radiation result in bone loss; we hypothesized that these catabolic treatments cause excess reactive oxygen species (ROS), and thereby alter the tight balance between bone resorption by osteoclasts and bone formation by osteoblasts, culminating in bone loss. To test this, we used transgenic mice which over-express the human gene for catalase, targeted to mitochondria (MCAT). Catalase is an anti-oxidant that converts the ROS hydrogen peroxide into water and oxygen. MCAT mice were shown previously to display reduced mitochondrial oxidative stress and radiosensitivity of the CNS compared to wild type controls (WT). As expected, MCAT mice expressed the transgene in skeletal tissue, and in marrow-derived osteoblasts and osteoclast precursors cultured ex vivo, and also showed greater catalase activity compared to wildtype (WT) mice (3-6 fold). Colony expansion in marrow cells cultured under osteoblastogenic conditions was 2-fold greater in the MCAT mice compared to WT mice, while the extent of mineralization was unaffected. MCAT mice had slightly longer tibiae than WT mice (2%, P less than 0.01), although cortical bone area was slightly lower in MCAT mice than WT mice (10%, p=0.09). To challenge the skeletal system, mice were treated by exposure to combined disuse (2 wk Hindlimb Unloading) and total body irradiation Cs(137) (2 Gy, 0.8 Gy/min), then bone parameters were analyzed by 2-factor ANOVA to detect possible interaction effects. Treatment caused a 2-fold increase (p=0.015) in malondialdehyde levels of bone tissue (ELISA) in WT mice, but had no effect in MCAT mice. These findings indicate that the transgene conferred protection from oxidative damage caused by treatment. Unexpected differences between WT and MCAT mice emerged in skeletal responses to treatment.. In WT mice, treatment did not alter osteoblastogenesis, cortical bone area, moment of inertia, or bone perimeter, whereas in MCAT mice, treatment increased these

  20. Changes in Mouse Bone Turnover in Response to Microgravity

    NASA Technical Reports Server (NTRS)

    Cheng-Campbell, M.; Blaber, E.; Almeida, E.

    2016-01-01

    Mechanical unloading during spaceflight is known to adversely affect mammalian physiology. Our previous studies using the Animal Enclosure Module on short duration Shuttle missions enabled us to identify a deficit in stem cell based-tissue regeneration as being a significant concern for long-duration spaceflight. Specifically, we found that mechanical unloading in microgravity resulted in inhibition of differentiation of mesenchymal and hematopoietic stem cells in the bone marrow compartment. Also, we observed overexpression of a cell cycle arrest molecule, CDKN1a/p21, in osteoprecursor cells on the bone surface, chondroprogenitors in the articular cartilage, and in myofibers attached to bone tissue. Specifically in bone tissue during both short (15-day) and long (30-day) microgravity experiments, we observed significant loss of bone tissue and structure in both the pelvis and the femur. After 15-days of microgravity on STS-131, pelvic ischium displayed a 6.23% decrease in bone fraction (p=0.005) and 11.91% decrease in bone thickness (p=0.002). Furthermore, during long-duration spaceflight we observed onset of an accelerated aging-like phenotype and osteoarthritic disease state indicating that stem cells within the bone tissue fail to repair and regenerate tissues in a normal manner, leading to drastic tissue alterations in response to microgravity. The Rodent Research Hardware System provides the capability to investigate these effects during long-duration experiments on the International Space Station. During the Rodent Research-1 mission 10 16-week-old female C57Bl/6J mice were exposed to 37-days of microgravity. All flight animals were euthanized and frozen on orbit for future dissection. Ground (n=10) and vivarium controls (n=10) were housed and processed to match the flight animal timeline. During this study we collected pelvis, femur, and tibia from all animal groups to test the hypothesis that stem cell-based tissue regeneration is significantly altered

  1. Preclinical mouse models for assessing axial compression of long bones during exercise

    PubMed Central

    Stadelmann, Vincent A; Brun, Julia; Bonnet, Nicolas

    2015-01-01

    The aim of this laboratory method is to describe two approaches for the investigation of bone responses to mechanical loading in mice in vivo. The first is running exercise, because it is easily translatable clinically, and the second is axial compression of the tibia, because it is precisely controllable. The effects of running exercise, and in general physical activity, on bone tissue have been shown to be both direct through mechanical loading (ground impact and muscle tension) and indirect through metabolic changes. Therefore, running exercise has been considered the most convenient preclinical model for demonstrating the general idea that exercise is good for bone health, either early in age for increasing peak bone mass or later in age by slowing down bone loss. However, numerous combinations of protocols have been reported, which makes it difficult to formulate a simple take-home message. This laboratory method also provides a detailed description of in vivo direct mechanical axial compression of the mouse tibia. The effects of mechanical loading depend on the force (strain), frequency, waveform and duration of application, and they range from bone anabolism with low bone remodeling, inducing lamellar bone accumulation, to bone catabolism with high bone remodeling, leading to microdamage, woven bone formation and bone loss. Direct in vivo loading models are extensively used to study mechanotransduction pathways, and contribute by this way to the development of new bone anabolism treatments. Although it is particularly difficult to assemble an internationally adopted protocol description, which would give reproducible bone responses, here we have attempted to provide a comprehensive guide for best practice in performing running exercise and direct in vivo mechanical loading in the laboratory. PMID:26788286

  2. Preclinical mouse models for assessing axial compression of long bones during exercise.

    PubMed

    Stadelmann, Vincent A; Brun, Julia; Bonnet, Nicolas

    2015-01-01

    The aim of this laboratory method is to describe two approaches for the investigation of bone responses to mechanical loading in mice in vivo. The first is running exercise, because it is easily translatable clinically, and the second is axial compression of the tibia, because it is precisely controllable. The effects of running exercise, and in general physical activity, on bone tissue have been shown to be both direct through mechanical loading (ground impact and muscle tension) and indirect through metabolic changes. Therefore, running exercise has been considered the most convenient preclinical model for demonstrating the general idea that exercise is good for bone health, either early in age for increasing peak bone mass or later in age by slowing down bone loss. However, numerous combinations of protocols have been reported, which makes it difficult to formulate a simple take-home message. This laboratory method also provides a detailed description of in vivo direct mechanical axial compression of the mouse tibia. The effects of mechanical loading depend on the force (strain), frequency, waveform and duration of application, and they range from bone anabolism with low bone remodeling, inducing lamellar bone accumulation, to bone catabolism with high bone remodeling, leading to microdamage, woven bone formation and bone loss. Direct in vivo loading models are extensively used to study mechanotransduction pathways, and contribute by this way to the development of new bone anabolism treatments. Although it is particularly difficult to assemble an internationally adopted protocol description, which would give reproducible bone responses, here we have attempted to provide a comprehensive guide for best practice in performing running exercise and direct in vivo mechanical loading in the laboratory.

  3. Activity of the human carcinogen MeCCNU in the mouse bone marrow micronucleus assay

    SciTech Connect

    Tinwell, H.; Ashby, J. )

    1991-01-01

    The nitrosourea mustard MeCCNU is the most recent organic chemical to be classified as a human carcinogen by IARC. MeCCNU gave a strong positive response when tested in the mouse bone marrow micronucleus assay. Activity was evident using either ip injection or oral gavage of the test chemical. These results further support the correlation between human carcinogens and their genotoxicity.

  4. Decreased Bone Formation Explains Osteoporosis in a Genetic Mouse Model of Hemochromatosiss

    PubMed Central

    Doyard, Mathilde; Chappard, Daniel; Leroyer, Patricia; Roth, Marie-Paule; Loréal, Olivier; Guggenbuhl, Pascal

    2016-01-01

    Osteoporosis may complicate iron overload diseases such as genetic hemochromatosis. However, molecular mechanisms involved in the iron-related osteoporosis remains poorly understood. Recent in vitro studies support a role of osteoblast impairment in iron-related osteoporosis. Our aim was to analyse the impact of excess iron in Hfe-/- mice on osteoblast activity and on bone microarchitecture. We studied the bone formation rate, a dynamic parameter reflecting osteoblast activity, and the bone phenotype of Hfe−/− male mice, a mouse model of human hemochromatosis, by using histomorphometry. Hfe−/− animals were sacrificed at 6 months and compared to controls. We found that bone contains excess iron associated with increased hepatic iron concentration in Hfe−/− mice. We have shown that animals with iron overload have decreased bone formation rate, suggesting a direct impact of iron excess on active osteoblasts number. For bone mass parameters, we showed that iron deposition was associated with bone loss by producing microarchitectural impairment with a decreased tendency in bone trabecular volume and trabecular number. A disorganization of trabecular network was found with marrow spaces increased, which was confirmed by enhanced trabecular separation and star volume of marrow spaces. These microarchitectural changes led to a loss of connectivity and complexity in the trabecular network, which was confirmed by decreased interconnectivity index and increased Minkowski’s fractal dimension. Our results suggest for the first time in a genetic hemochromatosis mouse model, that iron overload decreases bone formation and leads to alterations in bone mass and microarchitecture. These observations support a negative effect of iron on osteoblast recruitment and/or function, which may contribute to iron-related osteoporosis. PMID:26829642

  5. Transgenic Mouse Model for Reducing Oxidative Damage in Bone

    NASA Technical Reports Server (NTRS)

    Schreurs, Ann-Sofie; Torres, S.; Truong, T.; Moyer, E. L.; Kumar, A.; Tahimic, Candice C. G.; Alwood, J. S.; Limoli, C. L.; Globus, R. K.

    2016-01-01

    Bone loss can occur due to many challenges such age, radiation, microgravity, and Reactive Oxygen Species (ROS) play a critical role in bone resorption by osteoclasts (Bartell et al. 2014). We hypothesize that suppression of excess ROS in skeletal cells, both osteoblasts and osteoclasts, regulates skeletal growth and remodeling. To test our hypothesis, we used transgenic mCAT mice which overexpress the human anti-oxidant catalase gene targeted to the mitochondria, the main site for endogenous ROS production. mCAT mice have a longer life-span than wildtype controls and have been used to study various age-related disorders. To stimulate remodeling, 16 week old mCAT mice or wildtype mice were exposed to treatment (hindlimb-unloading and total body-irradiation) or sham treatment conditions (control). Tissues were harvested 2 weeks later for skeletal analysis (microcomputed tomography), biochemical analysis (gene expression and oxidative damage measurements), and ex vivo bone marrow derived cell culture (osteoblastogenesis and osteoclastogenesis). mCAT mice expressed the transgene and displayed elevated catalase activity in skeletal tissue and marrow-derived osteoblasts and osteoclasts grown ex vivo. In addition, when challenged with treatment, bone tissues from wildtype mice showed elevated levels of malondialdehyde (MDA), indicating oxidative damage) whereas mCAT mice did not. Correlation analysis revealed that increased catalase activity significantly correlated with decreased MDA levels and that increased oxidative damage correlated with decreased percent bone volume (BVTV). In addition, ex-vivo cultured osteoblast colony growth correlated with catalase activity in the osteoblasts. Thus, we showed that these transgenic mice can be used as a model to study the relationship between markers of oxidative damage and skeletal properties. mCAT mice displayed reduced BVTV and trabecular number relative to wildtype mice, as well as increased structural model index in the

  6. Primary lymphoma of bone originating in a rib.

    PubMed

    Nishiyama, N; Nakatani, S; Inoue, K; Katoh, T; Kinoshita, H

    2000-03-01

    Malignant lymphoma originating in the bone is rare and is now recognized as being an independent clinicopathologic entity known as primary lymphoma of bone. A 60-year-old man complaining of right chest and back pain consulted our hospital for further examination. Chest X-ray and computed tomogram revealed osteolysis and a surrounding soft tissue mass in the sixth right rib. An ultrasonically-guided needle biopsy of the tumor was performed, and histologic examination indicated the dense proliferation of similar-sized atypical cells with nucleoli and an irregular nuclear border. A diagnosis of diffuse, medium-sized non-Hodgkin's lymphoma, B-cell type was made. En block resection of the tumor and chest wall was performed. Macroscopically, the tumor measured 7.5 x 4.8 x 3.0 cm in diameter, and the histologic findings were similar to those of the preoperative needle biopsy. Unfortunately, postoperative treatment with radiation therapy and chemotherapy was ultimately unsuccessful, and a local recurrence and metastatic lesions appeared in the stomach and para-aortic abdominal lymph nodes 7 months after the first symptom appeared. The patient died 3 months later. Surgery was chosen as the initial therapy as it was considered that a rib resection would not result in serious respiratory compromise and the complete resection of the tumor would be superior to radiation therapy for local control. Some authors have reported that the surgical resection of a primary lymphoma of the bone originating in a rib can yield a good prognosis. However, it is a systemic disease and a more effective therapeutic strategy should be developed.

  7. Effects of 810 nm laser on mouse primary cortical neurons

    NASA Astrophysics Data System (ADS)

    Kharkwal, Gitika B.; Sharma, Sulbha K.; Huang, Ying-Ying; De Taboada, Luis; McCarthy, Thomas; Hamblin, Michael R.

    2011-03-01

    In the past four decades numerous studies have reported the efficacy of low level light (laser) therapy (LLLT) as a treatment for diverse diseases and injuries. Recent studies have shown that LLLT can biomodulate processes in the central nervous system and has been extensively studied as a stroke treatment. However there is still a lack of knowledge on the effects of LLLT at the cellular level in neurons. The present study aimed to study the effect of 810 nm laser on several cellular processes in primary cortical neurons cultured from mouse embryonic brains. Neurons were irradiated with light dose of 0.03, 0.3, 3, 10 and 30 J/cm2 and intracellular levels of reactive oxygen species, nitric oxide and calcium were measured. The changes in mitochondrial function in response to light were studied in terms of adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP). Light induced a significant increase in calcium, ATP and MMP at lower fluences and a decrease at higher fluence. ROS was induced significantly by light at all light doses. Nitric oxide levels also showed an increase on treatment with light. The results of the present study suggest that LLLT at lower fluences is capable of inducing mediators of cell signaling process which in turn may be responsible for the biomodulatory effects of the low level laser. At higher fluences beneficial mediators are reduced but potentially harmful mediators are increased thus offering an explanation for the biphasic dose response.

  8. Effects of mouse genotype on bone wound healing and irradiation-induced delay of healing.

    PubMed

    Glowacki, Julie; Mizuno, Shuichi; Kung, Jason; Goff, Julie; Epperly, Michael; Dixon, Tracy; Wang, Hong; Greenberger, Joel S

    2014-01-01

    We tested the effects of mouse genotype (C57BL/6NHsd, NOD/SCID, SAMR1, and SAMP6) and ionizing irradiation on bone wound healing. Unicortical wounds were made in the proximal tibiae, and the time course of spontaneous healing and effects of irradiation were monitored radiographically and histologically. There was reproducible healing beginning with intramedullary osteogenesis, subsequent bone resorption by osteoclasts, gradual bridging of the cortical wound, and re-population of medullary hematopoietic cells. The most rapid wound closure was noted in SAMR1 mice, followed by SAMP6, C57BL/6NHsd, and NOD/SCID. Ionizing irradiation (20 Gy) to the leg significantly delayed bone wound healing in mice of all four genotypes. Mice with genetically-determined predisposition to early osteopenia (SAMP6) or with immune deficiency (NOD/SCID) had impairments in bone wound healing. These mouse models should be valuable for determining the effects of irradiation on bone healing and also for the design and testing of novel bone growth-enhancing drugs and mitigators of ionizing irradiation.

  9. In vivo longitudinal visualization of bone marrow engraftment process in mouse calvarium bone marrow with two-photon microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Le, Viet Hoan; Lee, Seunghun; Kim, Ki Hean; Lee, Seungwon; Lee, Seung-Woo

    2017-02-01

    Bone marrow transplantation became the standard choice for treatment of many leukemias, tumors and metabolic diseases. Understanding the dynamic behavior of bone marrow niches, especially in case of bone marrow transplantation is critical to improve the efficiency of the treatment. Intravital microscopy was demonstrated to be a powerful tool to study physiological structure of bone marrow niche. However, current method of intravital microscopy has difficulty in longitudinal monitoring the same bone marrow niche site due to the invasion of the prior-imaging surgery. In this study, we introduce a method to improve the bone marrow niche imaging process and enable the longitudinal imaging of murine calvarium bone marrow. Mouse model for calvarium bone marrow imaging was made by attaching cover glass window to the calvarium bone. Longitudinal imaging of whole bone marrow engraftment process was carried out to demonstrate the advantage of our mouse model. Qualitative and quantitative analysis were also executed on the image data. The result provided a dynamic and full visualization of the bone marrow engraftment process. The study was expected to provide helpful tool for bone marrow studies and useful information for bone marrow transplantation in future.

  10. Comparative analyses of B cell populations in trout kidney and mouse bone marrow; establishing “B cell signatures”

    PubMed Central

    Zwollo, Patty; Mott, Katrina; Barr, Maggie

    2010-01-01

    This study aimed to identify the frequency and distribution of developing B cell populations in the kidney of the rainbow trout, using four molecular B cell markers that are highly conserved between species, including two transcription factors, Pax5 and EBF1, recombination activating gene RAG1, and the immunoglobulin heavy chain mu. Three distinct B cell stages were defined: early developing B cells (CLP, pro-B, and early pre-B cells), late developing B cell (late pre-B, immature B, and mature B cells), and IgM-secreting cells. Developmental stage-specific, combinatorial expression of Pax5, EBF1, RAG1 and immunoglobulin mu was determined in trout anterior kidney cells by flow cytometry. Trout staining patterns were compared to a well-defined primary immune tissue, mouse bone marrow, and using mouse surface markers B220 and CD43. A remarkable level of similarity was uncovered between the primary immune tissues of both species. Subsequent analysis of the entire trout kidney, divided into five contiguous segments K1-K5, revealed a complex pattern of early developing, late developing, and IgM-secreting B cells. Patterns in anterior kidney segment K1 were most similar to those of mouse bone marrow, while the most posterior part of the kidney, K5, had many IgM-secreting cells, but lacked early developing B cells. A potential second B lymphopoiesis site was uncovered in segment K4 of the kidney. The B cell patterns, or “B cell signatures” described here provide information on the relative abundance of distinct developing B cell populations in the trout kidney, and can be used in future studies on B cell development in other vertebrate species. PMID:20705088

  11. Effect of cyclophosphamide and electromagnetic fields on mouse bone marrow

    SciTech Connect

    Cadossi, R.; Zucchini, P.; Emilia, G.; Torelli, G. )

    1990-02-26

    The authors have previously shown that the exposure to low frequency pulsing electromagnetic fields (PEMF) of mice X-ray irradiated resulted in an increased damage to the bone marrow. The series of experiments here reported were designed to investigate the effect of PEMF exposure after intraperitoneum injection of 200mg/kg of cyclophosphamide (CY). Control mice were CY injected only; experimental mice were CY injected and then exposed to PEMF. Exposure to PEMF (24 hours/day) increased the rate of decline of white blood cells in peripheral blood. Spleen weight was statistically higher among control mice than among mice exposed to PEMF at day 6, 8 and 10 after CY injection. Spleen autoradiography proved to be higher among PEMF exposed mice than among controls at day 8 and 9 after CY injection. The grafting efficiency of the bone marrow obtained from control mice was higher than the grafting efficiency of the bone marrow recovered from mice exposed to PEMF. All these data indicate that the exposure to PEMF increases the cytotoxic effect of CY.

  12. Periodontal Disease: Linking the Primary Inflammation to Bone Loss

    PubMed Central

    Di Benedetto, Adriana; Gigante, Isabella; Colucci, Silvia; Grano, Maria

    2013-01-01

    Periodontal disease (PD), or periodontitis, is defined as a bacterially induced disease of the tooth-supporting (periodontal) tissues. It is characterized by inflammation and bone loss; therefore understanding how they are linked would help to address the most efficacious therapeutic approach. Bacterial infection is the primary etiology but is not sufficient to induce the disease initiation or progression. Indeed, bacteria-derived factors stimulate a local inflammatory reaction and activation of the innate immune system. The innate response involves the recognition of microbial components by host cells, and this event is mediated by toll-like receptors (TLRs) expressed by resident cells and leukocytes. Activation of these cells leads to the release of proinflammatory cytokines and recruitment of phagocytes and lymphocytes. Activation of T and B cells initiates the adaptive immunity with Th1 Th2 Th17 Treg response and antibodies production respectively. In this inflammatory scenario, cytokines involved in bone regulation and maintenance have considerable relevance because tissue destruction is believed to be the consequence of host inflammatory response to the bacterial challenge. In the present review, we summarize host factors including cell populations, cytokines, and mechanisms involved in the destruction of the supporting tissues of the tooth and discuss treatment perspectives based on this knowledge. PMID:23762091

  13. Ex vivo determination of bone tissue strains for an in vivo mouse tibial loading model.

    PubMed

    Carriero, Alessandra; Abela, Lisa; Pitsillides, Andrew A; Shefelbine, Sandra J

    2014-07-18

    Previous studies introduced the digital image correlation (DIC) as a viable technique for measuring bone strain during loading. In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations on the strain contour maps measured with a DIC system. We loaded tibiae of C57BL/6 mice (12 and 18 weeks old male) up to 12 N at 8 N/min. Images of speckles on the bone surface were recorded at 1N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps. This work provides a better understanding of parameters affecting full field strain measurements from DIC in ex vivo murine tibial loading tests.

  14. Predicting cortical bone adaptation to axial loading in the mouse tibia

    PubMed Central

    Pereira, A. F.; Javaheri, B.; Pitsillides, A. A.; Shefelbine, S. J.

    2015-01-01

    The development of predictive mathematical models can contribute to a deeper understanding of the specific stages of bone mechanobiology and the process by which bone adapts to mechanical forces. The objective of this work was to predict, with spatial accuracy, cortical bone adaptation to mechanical load, in order to better understand the mechanical cues that might be driving adaptation. The axial tibial loading model was used to trigger cortical bone adaptation in C57BL/6 mice and provide relevant biological and biomechanical information. A method for mapping cortical thickness in the mouse tibia diaphysis was developed, allowing for a thorough spatial description of where bone adaptation occurs. Poroelastic finite-element (FE) models were used to determine the structural response of the tibia upon axial loading and interstitial fluid velocity as the mechanical stimulus. FE models were coupled with mechanobiological governing equations, which accounted for non-static loads and assumed that bone responds instantly to local mechanical cues in an on–off manner. The presented formulation was able to simulate the areas of adaptation and accurately reproduce the distributions of cortical thickening observed in the experimental data with a statistically significant positive correlation (Kendall's τ rank coefficient τ = 0.51, p < 0.001). This work demonstrates that computational models can spatially predict cortical bone mechanoadaptation to a time variant stimulus. Such models could be used in the design of more efficient loading protocols and drug therapies that target the relevant physiological mechanisms. PMID:26311315

  15. Measuring the dynamic mechanical response of hydrated mouse bone by nanoindentation

    PubMed Central

    Pathak, Siddhartha; Swadener, J. Gregory; Kalidindi, Surya R.; Courtland, Hayden-William; Jepsen, Karl J.; Goldman, Haviva M.

    2011-01-01

    This study demonstrates a novel approach to characterizing hydrated bone’s viscoelastic behavior at the lamellar length scales using dynamic indentation techniques. We studied the submicron-level viscoelastic response of bone tissue from two different inbred mouse strains, A/J and B6, with known differences in whole bone and tissue-level mechanical properties. Our results show that bone having a higher collagen content or a lower mineral-to-matrix ratio demonstrates a trend towards a larger viscoelastic response. When normalized for anatomical location relative to biological growth patterns in the antero-medial (AM) cortex, bone tissue from B6 femora, known to have a lower mineral-to-matrix ratio, is shown to exhibit a significantly higher viscoelastic response compared to A/J tissue. Newer bone regions with a higher collagen content (closer to the endosteal edge of the AM cortex) showed a trend towards a larger viscoelastic response. Our study demonstrates the feasibility of this technique to be used to study local composition-property relationships in bone. Further, this technique of viscoelastic nanoindentation mapping of the bone surface at these submicron length scales is shown to be highly advantageous in studying sub-surface features, such as porosity, of wet hydrated biological specimens, which are difficult to identify using other methods. PMID:21094478

  16. Primary cilia act as mechanosensors during bone healing around an implant

    PubMed Central

    Leucht, P.; Monica, S.D.; Temiyasathit, S.; Lenton, K.; Manu, A.; Longaker, M.T.; Jacobs, C.R.; Spilker, R.L.; Guo, H.; Brunski, J.B.; Helms, J.A.

    2012-01-01

    The primary cilium is an organelle that senses cues in a cell’s local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3afl/fl mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective. PMID:22784673

  17. Neuronal Representation of Ultraviolet Visual Stimuli in Mouse Primary Visual Cortex

    PubMed Central

    Tan, Zhongchao; Sun, Wenzhi; Chen, Tsai-Wen; Kim, Douglas; Ji, Na

    2015-01-01

    The mouse has become an important model for understanding the neural basis of visual perception. Although it has long been known that mouse lens transmits ultraviolet (UV) light and mouse opsins have absorption in the UV band, little is known about how UV visual information is processed in the mouse brain. Using a custom UV stimulation system and in vivo calcium imaging, we characterized the feature selectivity of layer 2/3 neurons in mouse primary visual cortex (V1). In adult mice, a comparable percentage of the neuronal population responds to UV and visible stimuli, with similar pattern selectivity and receptive field properties. In young mice, the orientation selectivity for UV stimuli increased steadily during development, but not direction selectivity. Our results suggest that, by expanding the spectral window through which the mouse can acquire visual information, UV sensitivity provides an important component for mouse vision. PMID:26219604

  18. Neuronal Representation of Ultraviolet Visual Stimuli in Mouse Primary Visual Cortex.

    PubMed

    Tan, Zhongchao; Sun, Wenzhi; Chen, Tsai-Wen; Kim, Douglas; Ji, Na

    2015-07-29

    The mouse has become an important model for understanding the neural basis of visual perception. Although it has long been known that mouse lens transmits ultraviolet (UV) light and mouse opsins have absorption in the UV band, little is known about how UV visual information is processed in the mouse brain. Using a custom UV stimulation system and in vivo calcium imaging, we characterized the feature selectivity of layer 2/3 neurons in mouse primary visual cortex (V1). In adult mice, a comparable percentage of the neuronal population responds to UV and visible stimuli, with similar pattern selectivity and receptive field properties. In young mice, the orientation selectivity for UV stimuli increased steadily during development, but not direction selectivity. Our results suggest that, by expanding the spectral window through which the mouse can acquire visual information, UV sensitivity provides an important component for mouse vision.

  19. An investigation of the mineral in ductile and brittle cortical mouse bone.

    PubMed

    Rodriguez-Florez, Naiara; Garcia-Tunon, Esther; Mukadam, Quresh; Saiz, Eduardo; Oldknow, Karla J; Farquharson, Colin; Millán, José Luis; Boyde, Alan; Shefelbine, Sandra J

    2015-05-01

    Bone is a strong and tough material composed of apatite mineral, organic matter, and water. Changes in composition and organization of these building blocks affect bone's mechanical integrity. Skeletal disorders often affect bone's mineral phase, either by variations in the collagen or directly altering mineralization. The aim of the current study was to explore the differences in the mineral of brittle and ductile cortical bone at the mineral (nm) and tissue (µm) levels using two mouse phenotypes. Osteogenesis imperfecta model, oim(-/-) , mice have a defect in the collagen, which leads to brittle bone; PHOSPHO1 mutants, Phospho1(-/-) , have ductile bone resulting from altered mineralization. Oim(-/-) and Phospho1(-/-) were compared with their respective wild-type controls. Femora were defatted and ground to powder to measure average mineral crystal size using X-ray diffraction (XRD) and to monitor the bulk mineral to matrix ratio via thermogravimetric analysis (TGA). XRD scans were run after TGA for phase identification to assess the fractions of hydroxyapatite and β-tricalcium phosphate. Tibiae were embedded to measure elastic properties with nanoindentation and the extent of mineralization with backscattered electron microscopy (BSE SEM). Results revealed that although both pathology models had extremely different whole-bone mechanics, they both had smaller apatite crystals, lower bulk mineral to matrix ratio, and showed more thermal conversion to β-tricalcium phosphate than their wild types, indicating deviations from stoichiometric hydroxyapatite in the original mineral. In contrast, the degree of mineralization of bone matrix was different for each strain: brittle oim(-/-) were hypermineralized, whereas ductile Phospho1(-/-) were hypomineralized. Despite differences in the mineralization, nanoscale alterations in the mineral were associated with reduced tissue elastic moduli in both pathologies. Results indicated that alterations from normal crystal size

  20. Primary malignant mixed tumor of bone: a case report

    PubMed Central

    Su, Zhansan; Li, Zhi; Liu, Baoan

    2015-01-01

    Background: An extremely rare primary mixed tumor occurring in left proximal femurs of a 47-year old female is reported. Case report: She had left hip pain for three months in April 2004. Radiological examinations revealed that a translucent expansive lesion in the left greater trochanter. She received the curettage of lesion and bone graft surgery. Curettage specimens were diagnosed as malignant mixed tumor, considered to be metastatic. Five months late the lesion recurred. She underwent obturator neurotomy plus total hip replacement of left hip. A long-term of more than ten years follow-up showed there were no evidence of disease recurrence or metastasis and no any signs of other tumor in her body. Discussion: The tumor contained myoepithelial component with positive immunostain of S-100 protein, p63, CK-pan, and vimentin, epithelial component confirmed by CK-pan, CK-LMW and cartilage, which indicated the tumor was a mixed tumor. Cellular atypia, relative high mitosis index, cartilage consistent with grade I chordrosarcoma, focal coagulative necrosis, and infiltration between trabeculae found in the tumor indicated that the tumor had a low grade malignant nature. During long-time follow-up there were no signs of any tumor found in the patient, which strongly suggested that the tumor be a primary one. PMID:26339414

  1. Primary Ewing Sarcoma of Sphenoid Bone with Intracranial Extension: A Common Tumour at an Uncommon Location

    PubMed Central

    Singh, Guddi Rani; Choudhary, Vijayanand

    2017-01-01

    Primary Ewing Sarcoma of the cranial bone is rare, contributing to only 1% of all Ewing Sarcomas. Primary cranial Ewing Sarcoma occurs most commonly in temporal bone followed by parietal and occipital bones. Sphenoid bone is less commonly involved. We report a case of Ewing Sarcoma of the sphenoid bone with intra-cranial extension in a 20-month-old boy. On CT scan a provisional diagnosis of rhabdomyosarcoma was made. Fine Needle Aspiration Cytology (FNAC) and histopathological examination of core needle biopsy showed small round cell tumour. On Immunohistochemistry (IHC), CD99 (MIC2) and FLI 1 were strongly positive and final diagnosis of Ewing Sarcoma was made. Considering the rarity of this unusual site, we report a case of primary Ewing Sarcoma arising in the sphenoid bone with erosion of adjacent bones and intra-cranial extension. PMID:28384876

  2. Expression and methylation analysis of p15 and p16 in mouse bone marrow cells exposed to 1,4-benzoquinone.

    PubMed

    Tian, J-F; Peng, C-H; Yu, X-Y; Yang, X-J; Yan, Hong-Tao

    2012-07-01

    Benzene is an important industrial chemical. It is also an environmental pollutant recognized as a human carcinogen. Both prenatal and adult exposures to benzene are associated with the development of leukemia. To understand the mechanism of benzene-induced epigenetic variations, we investigated the expression and methylation patterns of CpG (phosphodiester bond between cytosine and guanine) islands in p15 and p16 promoter regions in 1,4-benzoquinone (1,4-BQ)-treated primary cultivated C57BL/6J mouse bone marrow cells in vitro. The cell toxicity of 1,4-BQ was evaluated by cell viability test, real-time PCR was used to measure the mRNA expression levels, and bisulfite sequencing PCR (BSP) was used to look into the methylation patterns. The cell viability test indicates that 1,4-BQ exhibited a dose-dependent toxicity to mouse bone marrow cells. After a 24-h exposure to 1,4-BQ at final concentrations of 0, 0.1, 1, and 10 μmol/L, the mRNA expression of p15 and p16 decreased with the increase in 1,4-BQ concentration. The BSP results gathered from the exposure and the control groups were the same. In summary, despite the observation that short-term exposure to 1,4-BQ primary cultivated mouse bone marrow cells decreased the p15 and p16 transcripts, with no influence by their gene promoter methylation.

  3. Abnormal bone formation induced by implantation of osteosarcoma-derived bone-inducing substance in the X-linked hypophosphatemic mouse

    SciTech Connect

    Yoshikawa, H.; Masuhara, K.; Takaoka, K.; Ono, K.; Tanaka, H.; Seino, Y.

    1985-01-01

    The X-linked hypophosphatemic mouse (Hyp) has been proposed as a model for the human familial hypophosphatemia (the most common form of vitamin D-resistant rickets). An osteosarcoma-derived bone-inducing substance was subcutaneously implanted into the Hyp mouse. The implant was consistently replaced by cartilage tissue at 2 weeks after implantation. The cartilage matrix seemed to be normal, according to the histological examination, and 35sulphur (TVS) uptake was also normal. Up to 4 weeks after implantation the cartilage matrix was completely replaced by unmineralized bone matrix and hematopoietic bone marrow. Osteoid tissue arising from the implantation of bone inducing substance in the Hyp mouse showed no radiologic or histologic sign of calcification. These findings suggest that the abnormalities of endochondral ossification in the Hyp mouse might be characterized by the failure of mineralization in cartilage and bone matrix. Analysis of the effects of bone-inducing substance on the Hyp mouse may help to give greater insight into the mechanism and treatment of human familial hypophosphatemia.

  4. Automated Cell Detection and Morphometry on Growth Plate Images of Mouse Bone

    PubMed Central

    Ascenzi, Maria-Grazia; Du, Xia; Harding, James I; Beylerian, Emily N; de Silva, Brian M; Gross, Ben J; Kastein, Hannah K; Wang, Weiguang; Lyons, Karen M; Schaeffer, Hayden

    2014-01-01

    Microscopy imaging of mouse growth plates is extensively used in biology to understand the effect of specific molecules on various stages of normal bone development and on bone disease. Until now, such image analysis has been conducted by manual detection. In fact, when existing automated detection techniques were applied, morphological variations across the growth plate and heterogeneity of image background color, including the faint presence of cells (chondrocytes) located deeper in tissue away from the image’s plane of focus, and lack of cell-specific features, interfered with identification of cell. We propose the first method of automated detection and morphometry applicable to images of cells in the growth plate of long bone. Through ad hoc sequential application of the Retinex method, anisotropic diffusion and thresholding, our new cell detection algorithm (CDA) addresses these challenges on bright-field microscopy images of mouse growth plates. Five parameters, chosen by the user in respect of image characteristics, regulate our CDA. Our results demonstrate effectiveness of the proposed numerical method relative to manual methods. Our CDA confirms previously established results regarding chondrocytes’ number, area, orientation, height and shape of normal growth plates. Our CDA also confirms differences previously found between the genetic mutated mouse Smad1/5CKO and its control mouse on fluorescence images. The CDA aims to aid biomedical research by increasing efficiency and consistency of data collection regarding arrangement and characteristics of chondrocytes. Our results suggest that automated extraction of data from microscopy imaging of growth plates can assist in unlocking information on normal and pathological development, key to the underlying biological mechanisms of bone growth. PMID:25525552

  5. Cytogenetic effects of pesticides. II. Induction of micronuclei in mouse bone marrow by the insecticide gardona.

    PubMed

    Amer, S M; Fahmy, M A

    1983-01-01

    The induction of micronuclei in mouse bone marrow by the organophosphorus insecticide gardona (also known as tetrachlorvinphos) was tested. 3 routes of administration were used for the pure insecticide: intraperitoneal, oral and dermal. The different routes of treatment with gardona caused toxicity of marrow indicated as significant increases in the percentage of polychromatic erythrocytes over that of the control. Intraperitoneal and oral treatments induced a statistically significant percentage of micronucleated PE.

  6. Primary Ewing's sarcoma of the greater wing of the sphenoid bone.

    PubMed

    Sharma, R R; Netalkar, A; Lad, S D

    2000-02-01

    Primary Ewing's sarcoma is an uncommon lethal tumour of the long bones and pelvic girdle mainly affecting children and young adults. An origin in the cranial bones is extremely rare. We report a unique case of primary involvement of the greater wing of sphenoid bone in a 16-year-old patient. Aggressive management using microsurgical resection, radiotherapy and chemotherapy was curative. Localized, primary Ewing's sarcoma of the cranial bones should be considered as a distinct clinicopathological entity with an extremely low rate of dural penetration and metastases, and with a relatively better prognosis as compared with those of long bones and pelvic girdle. In neurosurgical practice, primary Ewing's sarcoma of the cranial bones requires early aggressive management to achieve adequate long-term prognosis and cure.

  7. Genetic Toxicity Evaluation of Iodotrifluoromethane (CFsub3I). Volume 2. Results of In Vivo Mouse Bone Marrow Erythrocyte Micronucleus Testing

    DTIC Science & Technology

    1995-01-01

    the mouse micronucleus test which measures the clastogenic (chromosomes breaking) action of chemicals by the induction of micronuclei in bone marrow...preliminary toxicity information obtained by ManTech, a mouse bone marrow micronucleus test of CF 3I was conducted using 2.6, 5.0, and 7.5% CF 3I...the mice sacrificed 24 hours after the third exposure. Erythrocytes from mice exposed to the test material, and to the negative and positive controls

  8. Primary Hyperparathyroidism: The Influence of Bone Marrow Adipose Tissue on Bone Loss and of Osteocalcin on Insulin Resistance

    PubMed Central

    Mendonça, Maira L.; Batista, Sérgio L.; Nogueira-Barbosa, Marcello H.; Salmon, Carlos E.G.; de Paula, Francisco J.A.

    2016-01-01

    OBJECTIVES: Bone marrow adipose tissue has been associated with low bone mineral density. However, no data exist regarding marrow adipose tissue in primary hyperparathyroidism, a disorder associated with bone loss in conditions of high bone turnover. The objective of the present study was to investigate the relationship between marrow adipose tissue, bone mass and parathyroid hormone. The influence of osteocalcin on the homeostasis model assessment of insulin resistance was also evaluated. METHODS: This was a cross-sectional study conducted at a university hospital, involving 18 patients with primary hyperparathyroidism (PHPT) and 21 controls (CG). Bone mass was assessed by dual-energy x-ray absorptiometry and marrow adipose tissue was assessed by 1H magnetic resonance spectroscopy. The biochemical evaluation included the determination of parathyroid hormone, osteocalcin, glucose and insulin levels. RESULTS: A negative association was found between the bone mass at the 1/3 radius and parathyroid hormone levels (r = -0.69; p<0.01). Marrow adipose tissue was not significantly increased in patients (CG = 32.8±11.2% vs PHPT = 38.6±12%). The serum levels of osteocalcin were higher in patients (CG = 8.6±3.6 ng/mL vs PHPT = 36.5±38.4 ng/mL; p<0.005), but no associations were observed between osteocalcin and insulin or between insulin and both marrow adipose tissue and bone mass. CONCLUSION: These results suggest that the increment of adipogenesis in the bone marrow microenvironment under conditions of high bone turnover due to primary hyperparathyroidism is limited. Despite the increased serum levels of osteocalcin due to primary hyperparathyroidism, these patients tend to have impaired insulin sensitivity. PMID:27626477

  9. Identification of the vitamin D receptor in osteoblasts and chondrocytes but not osteoclasts in mouse bone.

    PubMed

    Wang, Yongji; Zhu, Jinge; DeLuca, Hector F

    2014-03-01

    Bone is clearly a target of vitamin D and as expected, the vitamin D receptor (VDR) is expressed in osteoblasts. However, the presence of VDR in other cells such as osteocytes, osteoclasts, chondroclasts, and chondrocytes is uncertain. Because of difficulties in obtaining sections of undecalcified adult bone, identification of the site of VDR expression in adult bone tissue has been problematic. In addition, the antibodies to VDR used in previous studies lacked specificity, a property crucial for unambiguous conclusions. In the present study, VDR in the various cells from neonatal and adult mouse bone tissues was identified by a highly specific and sensitive immunohistochemistry method following bone decalcification with EGTA. For accurate evaluation of weak immunosignals, samples from Demay VDR knockout mice were used as negative control. Molecular markers were used to identify cell types. Our results showed that EGTA-decalcification of bone tissue had no detectable effect on the immunoreactivity of VDR. VDR was found in osteoblasts and hypertrophic chondrocytes but not in the multinucleated osteoclasts, chondroclasts, and bone marrow stromal cells. Of interest is the finding that immature osteoblasts contain large amounts of VDR, whereas the levels are low or undetectable in mature osteoblasts including bone lining cells and osteocytes. Proliferating chondrocytes appear devoid of VDR, although low levels were found in the hypertrophic chondrocytes. These data demonstrate that osteoblasts and chondrocytes are major targets of 1α,25-dihydroxyvitamin D, but osteoclasts and chondroclasts are minor targets or not at all. A high level of VDR was found in the immature osteoblasts located in the cancellous bone, indicating that they are major targets of 1α,25-dihydroxyvitamin D. Thus, the immature osteoblasts are perhaps responsible for the vitamin D hormone signaling resulting in calcium mobilization and in osteogenesis.

  10. Impact of estrogens on atherosclerosis and bone in the apolipoprotein E-deficient mouse model.

    PubMed

    Fernández-Murga, María Leonor; Vinué, Ángela; Caeiro, José Ramón; Guede, David; Tarín, Juan J; Andrés, Vicente; Cano, Antonio

    2015-04-01

    The common inflammatory pathophysiology has nourished the hypothesis of a relationship between osteoporosis and cardiovascular disease. Estrogens are key agents in the modulation of both processes. We investigated whether induction of atherosclerosis affects bone and whether estrogens modulate both processes. Female apolipoprotein E-deficient mice (a well-established model of atherogenesis) were ovariectomized or falsely operated and fed either standard diet or high-fat diet (HFD). Six animals were included in each of the four groups. To clarify mechanisms, we treated preosteoblastic MC3T3-E1 cells with mouse serum. Physiological levels of estrogens in falsely operated mice limited atherosclerotic burden in the thoracic aorta, but not in the aortic arch. Bone resorption, as assessed by C-telopeptides, was increased by ovariectomy in animals fed standard diet, but not in animals fed HFD. Bone microstructural properties at the distal femur showed deteriorated trabecular architecture in bone volumetric fraction and trabecular number after ovariectomy, but trabecular pattern factor, trabecular thickness, trabecular spacing, or the structural model index remained unchanged. Changes in cortical parameters were not significant. Volumetric bone mineral density was reduced in trabecular bone, but not in cortical bone, in ovariectomized mice fed standard diet. Preosteoblastic MC3T3-E1 cells exhibited increased cellular proliferation and viability and alkaline phosphatase activity after treatment with sera from animals fed HFD. Endogenous estrogens partially reduce atherogenic burden in female apolipoprotein E-deficient mice. Ovariectomy increases bone resorption, but not under exacerbated proatherogenic conditions induced by HFD. The absence of apolipoprotein E might have an influence on the asymmetric responses of atherogenesis and bone resorption.

  11. Dendritic Cells Cause Bone Lesions in a New Mouse Model of Histiocytosis

    PubMed Central

    Grosjean, Frédéric; Nasi, Sonia; Schneider, Pascal; Chobaz, Véronique; Liu, Alexandra; Mordasini, Vanessa; Moullec, Kristell; Vezzoni, Paolo; Lavanchy, Christine; Busso, Nathalie; Acha-Orbea, Hans; Ehirchiou, Driss

    2015-01-01

    Langerhans cell histiocytosis (LCH) is a rare disease caused by the clonal accumulation of dendritic Langerhans cells, which is often accompanied by osteolytic lesions. It has been reported that osteoclast-like cells play a major role in the pathogenic bone destruction seen in patients with LCH and these cells are postulated to originate from the fusion of DCs. However, due to the lack of reliable animal models the pathogenesis of LCH is still poorly understood. In this study, we have established a mouse model of histiocytosis- recapitulating human disease for osteolytic lesions seen in LCH patients. At 12 weeks after birth, severe bone lesions were observed in our multisystem histiocytosis (Mushi) model, when CD8α conventional dendritic cells (DCs) are transformed (MuTuDC) and accumulate. Most importantly, our study demonstrates that bone loss in LCH can be accounted for the transdifferentiation of MuTuDCs into functional osteoclasts both in vivo and in vitro. Moreover, we have shown that injected MuTuDCs reverse the osteopetrotic phenotype of oc/oc mice in vivo. In conclusion, our results support a crucial role of DCs in bone lesions in histiocytosis patients. Furthermore, our new model of LCH based on adoptive transfer of MuTuDC lines, leading to bone lesions within 1–2 weeks, will be an important tool for investigating the pathophysiology of this disease and ultimately for evaluating the potential of anti-resorptive drugs for the treatment of bone lesions. PMID:26247358

  12. Accretion of Bone Quantity and Quality in the Developing Mouse Skeleton

    SciTech Connect

    Miller,L.; Little, W.; Schirmer, A.; Sheik, F.; Busa, B.; Judex, S.

    2007-01-01

    To meet the mechanical challenges during early development, the skeleton requires the rapid accretion of bone quality and bone quantity. Here, we describe early bone development in the mouse skeleton and test the hypothesis that specific compositional properties determine the stiffness of the tissue. Tibias of female BALB mice were harvested at eight time-points (n = 4 each) distributed between 1 and 40 days of age and subjected to morphometric ({mu}CT), chemical (Fourier transform infrared microscpectroscopy), and mechanical (nanoindentation) analyses. Tibias of 450-day-old mice served as fully mineralized control specimens. In this work, we found that bone mineral formation proceeded very rapidly in mice by 1 day of age, where the degree of mineralization, the tissue mineral density, and the mineral crystallinity reached 36%, 51%, and 87% of the adult values, respectively. However, even though significant mineralization had occurred, the elastic modulus of 1-day-old bone was only 14% of its adult value, indicating that the intrinsic stiffening of the bone lags considerably behind the initial mineral formation.

  13. Bone Marrow Transplantation Confers Modest Benefits in Mouse Models of Huntington’s Disease

    PubMed Central

    Kwan, Wanda; Magnusson, Anna; Chou, Austin; Adame, Anthony; Carson, Monica J.; Kohsaka, Shinichi; Masliah, Eliezer; Möller, Thomas; Ransohoff, Richard; Tabrizi, Sarah J.; Björkqvist, Maria; Muchowski, Paul J.

    2013-01-01

    Huntington’s disease (HD) is caused by an expanded polyglutamine tract in the protein huntingtin (htt). Although HD has historically been viewed as a brain-specific disease, htt is expressed ubiquitously, and recent studies indicate that mutant htt might cause changes to the immune system that could contribute to pathogenesis. Monocytes from HD patients and mouse models are hyperactive in response to stimulation, and increased levels of inflammatory cytokines and chemokines are found in pre-manifest patients that correlate with pathogenesis. In this study, wild-type (WT) bone marrow cells were transplanted into two lethally irradiated transgenic mouse models of HD that ubiquitously express full-length htt (YAC128 and BACHD mice). Bone marrow transplantation partially attenuated hypokinetic and motor deficits in HD mice. Increased levels of synapses in the cortex were found in HD mice that received bone marrow transplants. Importantly, serum levels of interleukin-6, interleukin-10, CXC chemokine ligand 1, and interferon-γ were significantly higher in HD than WT mice but were normalized in mice that received a bone marrow transplant. These results suggest that immune cell dysfunction might be an important modifier of pathogenesis in HD. PMID:22219276

  14. COLLAGEN MUTATION CAUSES CHANGES OF THE MICRODAMAGE MORPHOLOGY IN BONE OF AN OI MOUSE MODEL

    PubMed Central

    Dong, X. Neil; Zoghi, Mahyar; Ran, Qitao; Wang, Xiaodu

    2010-01-01

    Previous studies have postulated that ultrastructural changes may alter the pattern and capacity of microdamage accumulation in bone. Using an osteogenesis imperfecta (OI) mouse model, this study was performed to investigate the correlation of collagen mutation with the microdamage morphology and the associated brittleness of bone. In this study, femurs from mild OI and wild type mice were fatigued under four-point bending to create microdamage in the specimens. Then, the microdamage morphology of these specimens was examined using the bulk-staining technique with basic fuchsin. Similar with the results of previous studies, it was observed that linear microcracks were formed more easily in compression, whereas diffuse damage was induced more readily in tension for both wild-type and mild-type mice. However, less diffuse damage was found in the tensile side of mild OI mouse femurs (collagen mutation) compared with those of wild type mice, showing that the microdamage morphology is correlated to the brittleness of bone. The results of this study provide direct evidence that supports the prediction made by the previous numerical simulation studies, suggesting that microdamage morphology in bone is significantly correlated with the integrity of the collagen phase. PMID:20736092

  15. Magnetic assembly-mediated enhancement of differentiation of mouse bone marrow cells cultured on magnetic colloidal assemblies

    NASA Astrophysics Data System (ADS)

    Sun, Jianfei; Liu, Xuan; Huang, Jiqing; Song, Lina; Chen, Zihao; Liu, Haoyu; Li, Yan; Zhang, Yu; Gu, Ning

    2014-05-01

    Here we reported an interesting phenomenon that the field-induced assemblies of magnetic nanoparticles can promote the differentiation of primary mouse bone marrow cells into osteoblasts. The reason was thought to lie in the remnant magnetic interaction inside the assemblies which resulted from the magnetic field-directed assembly. Influence of the assemblies on the cells was realized by means of interface effect rather than the internalization effect. We fabricated a stripe-like assemblies array on the glass plate and cultured cells on this surface. We characterized the morphology of assemblies and measured the mechanic property as well as the magnetic property. The cellular differentiation was measured by staining and quantitative PCR. Finally, Fe uptake was excluded as the reason to cause the phenomenon.

  16. Magnetic assembly-mediated enhancement of differentiation of mouse bone marrow cells cultured on magnetic colloidal assemblies

    PubMed Central

    Sun, Jianfei; Liu, Xuan; Huang, Jiqing; Song, Lina; Chen, Zihao; Liu, Haoyu; Li, Yan; Zhang, Yu; Gu, Ning

    2014-01-01

    Here we reported an interesting phenomenon that the field-induced assemblies of magnetic nanoparticles can promote the differentiation of primary mouse bone marrow cells into osteoblasts. The reason was thought to lie in the remnant magnetic interaction inside the assemblies which resulted from the magnetic field-directed assembly. Influence of the assemblies on the cells was realized by means of interface effect rather than the internalization effect. We fabricated a stripe-like assemblies array on the glass plate and cultured cells on this surface. We characterized the morphology of assemblies and measured the mechanic property as well as the magnetic property. The cellular differentiation was measured by staining and quantitative PCR. Finally, Fe uptake was excluded as the reason to cause the phenomenon. PMID:24874764

  17. Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

    PubMed

    Chung, Hwa-Jin; Kyung Kim, Won; Joo Park, Hyen; Cho, Lan; Kim, Me-Riong; Kim, Min Jeong; Shin, Joon-Shik; Ho Lee, Jin; Ha, In-Hyuk; Kook Lee, Sang

    2016-02-17

    Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Use of a calcium sulfate-calcium phosphate synthetic bone graft composite in the surgical management of primary bone tumors.

    PubMed

    Evaniew, Nathan; Tan, Victoria; Parasu, Naveen; Jurriaans, Erik; Finlay, Karen; Deheshi, Benjamin; Ghert, Michelle

    2013-02-01

    Benign primary bone tumors are commonly treated with intralesional curettage with or without the use of surgical adjuvants. The reconstructive approach to the resulting contained bone defects is controversial, and clinical practice is varied. Synthetic bone substitutes may provide early mechanical support while minimizing the risks of disease transmission, nonunion, infection, and donor-site morbidity. Limited data exists regarding the use of calcium sulfate-calcium phosphate composite bone substitute for this purpose. The authors retrospectively reviewed the clinical outcomes of 24 patients with benign primary bone tumors who underwent intralesional curettage followed by reconstruction with a calcium sulfate-calcium phosphate composite bone substitute. Mean follow-up was 23 months. The most common diagnosis was giant cell tumor of bone. Six patients had upper-extremity tumors and 18 had lower-extremity tumors. Mean preoperative radiographic tumor volume was 41.0 cm(3). Mean volume of PRO-DENSE (Wright Medical Technology, Arlington, Tennessee) used in each patient was 15.6 cm(3). Mean time to full weight bearing for all patients was 7.3 weeks. Two patients sustained local tumor recurrences. No postoperative fractures occurred, and no complications occurred related to the use of the calcium sulfate-calcium phosphate composite. One case of deep infection occurred secondary to wound breakdown. The use of a calcium sulfate-calcium phosphate composite was associated with rapid biological integration and an early return to activities of daily living, with no composite-related complications. This technique is a viable option in the reconstruction of cavitary bone defects following intralesional curettage of primary benign bone tumors. Copyright 2013, SLACK Incorporated.

  19. Onlay bone augmentation on mouse calvarial bone using a hydroxyapatite/collagen composite material with total blood or platelet-rich plasma.

    PubMed

    Ohba, Seigo; Sumita, Yoshinori; Umebayashi, Mayumi; Yoshimura, Hitoshi; Yoshida, Hisato; Matsuda, Shinpei; Kimura, Hideki; Asahina, Izumi; Sano, Kazuo

    2016-01-01

    The aim of this study was to assess newly formed onlay bone on mouse calvarial bone using a new artificial bone material, a hydroxyapatite/collagen composite, with total blood or platelet-rich plasma. The hydroxyapatite/collagen composite material with normal saline, total blood or platelet-rich plasma was transplanted on mouse calvarial bone. The mice were sacrificed and the specimens were harvested four weeks after surgery. The newly formed bone area was measured on hematoxylin and eosin stained specimens using Image J software. The hydroxyapatite/collagen composite materials with total blood or platelet-rich plasma induced a significantly greater amount of newly formed bone than that with normal saline. Moreover, bone marrow was observed four weeks after surgery in the transplanted materials with total blood or platelet-rich plasma but not with normal saline. However, there were no significant differences in the amount of newly formed bone between materials used with total blood versus platelet-rich plasma. The hydroxyapatite/collagen composite material was valid for onlay bone augmentation and this material should be soaked in total blood or platelet-rich plasma prior to transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant.

    PubMed

    Song, Ningxia; Gao, Lei; Qiu, Huiying; Huang, Chongmei; Cheng, Hui; Zhou, Hong; Lv, Shuqing; Chen, Li; Wang, Jianmin

    2015-07-01

    The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft‑versus‑host disease (aGVHD). However, the role of MSCs in graft‑versus‑leukemia remains to be determined. In the present study, we co‑cultured C57BL/6 mouse bone marrow (BM)‑derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit‑8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)‑10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies.

  1. Neonatal bone marrow transplantation prevents bone pathology in a mouse model of mucopolysaccharidosis type I

    PubMed Central

    Pievani, Alice; Azario, Isabella; Antolini, Laura; Shimada, Tsutomu; Patel, Pravin; Remoli, Cristina; Rambaldi, Benedetta; Valsecchi, Maria Grazia; Riminucci, Mara; Biondi, Andrea; Tomatsu, Shunji

    2015-01-01

    Neonatal bone marrow transplantation (BMT) could offer a novel therapeutic opportunity for genetic disorders by providing sustainable levels of the missing protein at birth, thus preventing tissue damage. We tested this concept in mucopolysaccharidosis type I (MPS IH; Hurler syndrome), a lysosomal storage disorder caused by deficiency of α-l-iduronidase. MPS IH is characterized by a broad spectrum of clinical manifestations, including severe progressive skeletal abnormalities. Although BMT increases the life span of patients with MPS IH, musculoskeletal manifestations are only minimally responsive if the timing of BMT delays, suggesting already irreversible bone damage. In this study, we tested the hypothesis that transplanting normal BM into newborn MPS I mice soon after birth can prevent skeletal dysplasia. We observed that neonatal BMT was effective at restoring α-l-iduronidase activity and clearing elevated glycosaminoglycans in blood and multiple organs. At 37 weeks of age, we observed an almost complete normalization of all bone tissue parameters, using radiographic, microcomputed tomography, biochemical, and histological analyses. Overall, the magnitude of improvements correlated with the extent of hematopoietic engraftment. We conclude that BMT at a very early stage in life markedly reduces signs and symptoms of MPS I before they appear. PMID:25298037

  2. Methods and theory in bone modeling drift: comparing spatial analyses of primary bone distributions in the human humerus.

    PubMed

    Maggiano, Corey M; Maggiano, Isabel S; Tiesler, Vera G; Chi-Keb, Julio R; Stout, Sam D

    2016-01-01

    This study compares two novel methods quantifying bone shaft tissue distributions, and relates observations on human humeral growth patterns for applications in anthropological and anatomical research. Microstructural variation in compact bone occurs due to developmental and mechanically adaptive circumstances that are 'recorded' by forming bone and are important for interpretations of growth, health, physical activity, adaptation, and identity in the past and present. Those interpretations hinge on a detailed understanding of the modeling process by which bones achieve their diametric shape, diaphyseal curvature, and general position relative to other elements. Bone modeling is a complex aspect of growth, potentially causing the shaft to drift transversely through formation and resorption on opposing cortices. Unfortunately, the specifics of modeling drift are largely unknown for most skeletal elements. Moreover, bone modeling has seen little quantitative methodological development compared with secondary bone processes, such as intracortical remodeling. The techniques proposed here, starburst point-count and 45° cross-polarization hand-drawn histomorphometry, permit the statistical and populational analysis of human primary tissue distributions and provide similar results despite being suitable for different applications. This analysis of a pooled archaeological and modern skeletal sample confirms the importance of extreme asymmetry in bone modeling as a major determinant of microstructural variation in diaphyses. Specifically, humeral drift is posteromedial in the human humerus, accompanied by a significant rotational trend. In general, results encourage the usage of endocortical primary bone distributions as an indicator and summary of bone modeling drift, enabling quantitative analysis by direction and proportion in other elements and populations. © 2015 Anatomical Society.

  3. Ex vivo determination of bone tissue strains for an in vivo mouse tibial loading model

    PubMed Central

    Carriero, Alessandra; Abela, Lisa; Pitsillides, Andrew A.; Shefelbine, Sandra J.

    2014-01-01

    Previous studies introduced the digital image correlation (DIC) as a viable technique for measuring bone strain during loading. In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations on the strain contour maps measured with a DIC system. We loaded tibiae of C57BL/6 mice (12 and 18 weeks old male) up to 12 N at 8 N/min. Images of speckles on the bone surface were recorded at 1 N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps. This work provides a better understanding of parameters affecting full field strain measurements from DIC in ex vivo murine tibial loading tests. PMID:24835472

  4. Effects of experimental conditions on the release of 45calcium from prelabeled fetal mouse long bones.

    PubMed

    van Beek, E; Oostendorp-van de Ruit, M; van der Wee-Pals, L; Bloys, H; van de Bent, C; Papapoulos, S; Löwik, C

    1995-07-01

    Embryonic/neonatal bones in culture are commonly used for the study of osteoclastic resorption in vitro. For this purpose, the release of 45calcium (45Ca) from prelabeled bones is measured as an index of resorption. We studied 45Ca release from two types of long bone explants after different preparation methods: 17-day-old fetal mouse radii/ulnae with and without cartilage ends (intact radii/ulnae and shafts, respectively), and intact 18-day old metacarpals/metatarsals. In addition, we examined the effect of different culture conditions, such as cultures performed under the surface of the medium or at the interphase of medium and air, on 45Ca release and histology. When intact radii/ulnae were cultured under the surface of the medium, there was always a significant amount (10%) of net basal 45Ca release (corrected for physicochemical exchange) that was not due to osteoclastic resorption, as it could not be suppressed by inhibitors of resorption even at high concentrations. Moreover, histologically TRAcP-positive cells were almost absent after culture and the bone marrow/stromal cells in the center of the bone appeared necrotic, possibly due to a lack of oxygen. Under these culture conditions, osteoclasts could survive in shafts as well as in PTH-stimulated intact radii/ulnae, but a constant amount of 10% 45Ca, not due to resorption, was still released in the medium. When these explants were cultured at the interphase of medium and air, basal and stimulated 45Ca release originated from osteoclastic resorption. In contrast, in 18-day-old fetal mouse metacarpals/metatarsals, the experimental conditions applied did not affect 45Ca release, which was always due to resorption of the explants by osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading

    PubMed Central

    Jing, Da; Baik, Andrew D.; Lu, X. Lucas; Zhou, Bin; Lai, Xiaohan; Wang, Liyun; Luo, Erping; Guo, X. Edward

    2014-01-01

    Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca2+) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca2+ responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid-induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca2+ spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca2+ oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca2+ oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.—Jing, D., Baik, A. D., Lu, X. L., Zhou, B., Lai, X., Wang, L., Luo, E., Guo, X. E. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. PMID:24347610

  6. Phenotypic integration among trabecular and cortical bone traits establishes mechanical functionality of inbred mouse vertebrae.

    PubMed

    Tommasini, Steven M; Hu, Bin; Nadeau, Joseph H; Jepsen, Karl J

    2009-04-01

    Conventional approaches to identifying quantitative trait loci (QTLs) regulating bone mass and fragility are limited because they examine cortical and trabecular traits independently. Prior work examining long bones from young adult mice and humans indicated that skeletal traits are functionally related and that compensatory interactions among morphological and compositional traits are critical for establishing mechanical function. However, it is not known whether trait covariation (i.e., phenotypic integration) also is important for establishing mechanical function in more complex, corticocancellous structures. Covariation among trabecular, cortical, and compositional bone traits was examined in the context of mechanical functionality for L(4) vertebral bodies across a panel of 16-wk-old female AXB/BXA recombinant inbred (RI) mouse strains. The unique pattern of randomization of the A/J and C57BL/6J (B6) genome among the RI panel provides a powerful tool that can be used to measure the tendency for different traits to covary and to study the biology of complex traits. We tested the hypothesis that genetic variants affecting vertebral size and mass are buffered by changes in the relative amounts of cortical and trabecular bone and overall mineralization. Despite inheriting random sets of A/J and B6 genomes, the RI strains inherited nonrandom sets of cortical and trabecular bone traits. Path analysis, which is a multivariate analysis that shows how multiple traits covary simultaneously when confounding variables like body size are taken into consideration, showed that RI strains that tended to have smaller vertebrae relative to body size achieved mechanical functionality by increasing mineralization and the relative amounts of cortical and trabecular bone. The interdependence among corticocancellous traits in the vertebral body indicated that variation in trabecular bone traits among inbred mouse strains, which is often thought to arise from genetic factors, is also

  7. Assessment of lamellar level properties in mouse bone utilizing a novel spherical nanoindentation data analysis method

    PubMed Central

    Pathak, Siddhartha; Vachhani, Shraddha J.; Jepsen, Karl J.; Goldman, Haviva M.; Kalidindi, Surya R.

    2016-01-01

    In this work, we demonstrate the viability of using our recently developed data analysis procedures for spherical nanoindentation in conjunction with Raman spectroscopy for studying lamellar-level correlations between the local composition and local mechanical properties in mouse bone. Our methodologies allow us to convert the raw load-displacement datasets to much more meaningful indentation stress–strain curves that accurately capture the loading and unloading elastic moduli, the indentation yield points, as well as the post-yield characteristics in the tested samples. Using samples of two different inbred mouse strains, A/J and C57BL/6J (B6), we successfully demonstrate the correlations between the mechanical information obtained from spherical nanoindentation measurements to the local composition measured using Raman spectroscopy. In particular, we observe that a higher mineral-to-matrix ratio correlated well with a higher local modulus and yield strength in all samples. Thus, new bone regions exhibited lower moduli and yield strengths compared to more mature bone. The B6 mice were also found to exhibit lower modulus and yield strength values compared to the more mineralized A/J strain. PMID:22842281

  8. Targeting of primary breast cancers and metastases in a transgenic mouse model using rationally designed multifunctional SPIONs.

    PubMed

    Kievit, Forrest M; Stephen, Zachary R; Veiseh, Omid; Arami, Hamed; Wang, Tingzhong; Lai, Vy P; Park, James O; Ellenbogen, Richard G; Disis, Mary L; Zhang, Miqin

    2012-03-27

    Breast cancer remains one of the most prevalent and lethal malignancies in women. The inability to diagnose small volume metastases early has limited effective treatment of stage 4 breast cancer. Here we report the rational development and use of a multifunctional superparamagnetic iron oxide nanoparticle (SPION) for targeting metastatic breast cancer in a transgenic mouse model and imaging with magnetic resonance (MR). SPIONs coated with a copolymer of chitosan and polyethylene glycol (PEG) were labeled with a fluorescent dye for optical detection and conjugated with a monoclonal antibody against the neu receptor (NP-neu). SPIONs labeled with mouse IgG were used as a nontargeting control (NP-IgG). These SPIONs had desirable physiochemical properties for in vivo applications such as near neutral zeta potential and hydrodynamic size around 40 nm and were highly stable in serum containing medium. Only NP-neu showed high uptake in neu expressing mouse mammary carcinoma (MMC) cells which was reversed by competing free neu antibody, indicating their specificity to the neu antigen. In vivo, NP-neu was able to tag primary breast tumors and significantly, only NP-neu bound to spontaneous liver, lung, and bone marrow metastases in a transgenic mouse model of metastatic breast cancer, highlighting the necessity of targeting for delivery to metastatic disease. The SPIONs provided significant contrast enhancement in MR images of primary breast tumors; thus, they have the potential for MRI detection of micrometastases and provide an excellent platform for further development of an efficient metastatic breast cancer therapy. © 2012 American Chemical Society

  9. Differentiation of functionally active mouse T lymphocytes from functionally inactive bone marrow precursors II. Limited recovery of T-cell responses from mouse bone marrow in tissue culture.

    PubMed

    Gorczynski, M; MacRae, S

    1977-11-01

    The limited differentiation of mature T cell function from mouse bone marrow in tissue culture is described and compared with similar differentiation occuring in vivo in irradiated bone marrow protected mice. Data are presented to show that a pool of precursors, similar in size to that able to produce early (transient?) regeneration in thymectomized recipients, is responsible for the development of mitogen responsive T cells active in MLC (proliferation) and CML (development of cytotoxic cells) assays. In contrast, a helper cell population which augments antibody formation from T-depleted normal spleen cells derives from a pool of similar precursors yet does not seem to be theta positive. Similarly, larger cells (perhaps typical of those giving rise to suppressor T cells in vivo) give rise to a suppressor cell pool after 4 days of culture, though again only a fraction of this suppressor activity could be attributed to theta positive cells. It is suggested that much of the data for regenration of T lymphocytes in vitro from T-depleted sources needs to be re-interpreted in terms of this evidence for a pool of post-thymic precursors of T cells in such T-deficient cell populations.

  10. Differentiation of functionally active mouse T lymphocytes from functionally inactive bone marrow precursors II. Limited recovery of T-cell responses from mouse bone marrow in tissue culture.

    PubMed Central

    Gorczynski, M; MacRae, S

    1977-01-01

    The limited differentiation of mature T cell function from mouse bone marrow in tissue culture is described and compared with similar differentiation occuring in vivo in irradiated bone marrow protected mice. Data are presented to show that a pool of precursors, similar in size to that able to produce early (transient?) regeneration in thymectomized recipients, is responsible for the development of mitogen responsive T cells active in MLC (proliferation) and CML (development of cytotoxic cells) assays. In contrast, a helper cell population which augments antibody formation from T-depleted normal spleen cells derives from a pool of similar precursors yet does not seem to be theta positive. Similarly, larger cells (perhaps typical of those giving rise to suppressor T cells in vivo) give rise to a suppressor cell pool after 4 days of culture, though again only a fraction of this suppressor activity could be attributed to theta positive cells. It is suggested that much of the data for regenration of T lymphocytes in vitro from T-depleted sources needs to be re-interpreted in terms of this evidence for a pool of post-thymic precursors of T cells in such T-deficient cell populations. PMID:304032

  11. Establishing Biomechanical Mechanisms in Mouse Models: Practical Guidelines for Systematically Evaluating Phenotypic Changes in the Diaphyses of Long Bones

    PubMed Central

    Jepsen, Karl J; Silva, Matthew J; Vashishth, Deepak; Guo, X Edward; van der Meulen, Marjolein CH

    2016-01-01

    Mice are widely used in studies of skeletal biology, and assessment of their bones by mechanical testing is a critical step when evaluating the functional effects of an experimental perturbation. For example, a gene knockout may target a pathway important in bone formation and result in a “low bone mass” phenotype. But how well does the skeleton bear functional loads; eg, how much do bones deform during loading and how resistant are bones to fracture? By systematic evaluation of bone morphological, densitometric, and mechanical properties, investigators can establish the “biomechanical mechanisms” whereby an experimental perturbation alters whole-bone mechanical function. The goal of this review is to clarify these biomechanical mechanisms and to make recommendations for systematically evaluating phenotypic changes in mouse bones, with a focus on long-bone diaphyses and cortical bone. Further, minimum reportable standards for testing conditions and outcome variables are suggested that will improve the comparison of data across studies. Basic biomechanical principles are reviewed, followed by a description of the cross-sectional morphological properties that best inform the net cellular effects of a given experimental perturbation and are most relevant to biomechanical function. Although morphology is critical, whole-bone mechanical properties can only be determined accurately by a mechanical test. The functional importance of stiffness, maximum load, postyield displacement, and work-to-fracture are reviewed. Because bone and body size are often strongly related, strategies to adjust whole-bone properties for body mass are detailed. Finally, a comprehensive framework is presented using real data, and several examples from the literature are reviewed to illustrate how to synthesize morphological, tissue-level, and whole-bone mechanical properties of mouse long bones. PMID:25917136

  12. Preoperative Computed Tomography-Derived Bone Densities in Hounsfield Units at Implant Sites Acquired Primary Stability

    PubMed Central

    Hiasa, Kyou; Abe, Yasuhiko; Okazaki, Yohei; Nogami, Keisuke; Mizumachi, Wataru; Akagawa, Yasumasa

    2011-01-01

    The purpose of this study was to evaluate preoperative CT-derived bone densities in Hounsfield units (HU) at implant sites that acquired primary stability, and to compare these values to the optimal bone densities proposed in the literature. Fifty-one patients, 18 males (37 implant sites) and 33 females (67 implant sites) between 2003 and 2010 were assessed. CT data for different jaw sections, regions, and operating procedures were compared using the Kruskal-Wallis test and Scheffe's test for multiple comparisons (P < 0.05). The mean bone density in the maxilla was significantly lower than that in the mandible (P < 0.05); the mean bone densities in the 4 jaw regions decreased in the following order: anterior mandible > anterior maxilla > posterior mandible > posterior maxilla. The bone densities assessed by HU fell into the range of optimal bone densities associated with acquired primary implant stability proposed in the literature. PMID:22203910

  13. Near IR fluorescent conjugated poly(ethylene glycol)bisphosphonate nanoparticles for in vivo bone targeting in a young mouse model.

    PubMed

    Rudnick-Glick, S; Corem-Salkmon, E; Grinberg, I; Yehuda, R; Margel, S

    2015-11-14

    Bisphosphonate (BP) compounds are widely used in the treatment of bone disorders. This group of drugs with a high affinity to Ca(+2) ions is rapidly attracted to bone mineral, especially in areas of high resorption. We have engineered unique biodegradable BP nanoparticles (NPs) by dispersion co-polymerization of the monomers methacrylate-PEG-BP) and (3-Aminopropyl)mathacrylamide) with the crosslinker monomer tetra ethylene glycol diacrylate. These NPs possess a dual functionality: (1) covalent attachment of a dye (e.g. near IR dye) or a drug to the nanoparticles through the primary amine groups on the surface of the NPs; (2) chelation to the bone mineral hydroxyapatite through the BP on the surface of the NPs. This study describes the uptake of the unique near IR fluorescent Cy 7-conjugated BP NPs in bone of a young mouse model. Blood half-life studies revealed a relatively long half-life (approximately 5 h) due to a high concentration of PEG in the BP NPs as well as a relatively long whole body clearance (approximately 2 weeks). Body distribution studies showed a specific uptake of the BP NPs in bone. These unique engineered BP NPs are planned to be utilized in future work for diagnostic and drug delivery systems that are targeted to bone disorders.

  14. Strain measurement of a mouse bone by 3D-electronic speckle pattern interferometry (3D-ESPI)

    NASA Astrophysics Data System (ADS)

    Samala, Praveen R.; Su, Min; Liu, Sheng; Jiang, Hui H.; Yokota, Hiroki; Yang, Lianxiang

    2005-08-01

    Bone is a mechanosensitive tissue that adapts its mass, architecture and mechanical properties to mechanical loading. Appropriate mechanical loads provide an effective means to stimulate bone remodeling and prevent from bone loss. It is controversial whether in situ strain in bone is a critical determinant in enhancement of bone formation, and it is therefore important to evaluate load-driven strain in bone. Using electronic speckle pattern interferometry, we determined high-resolution three-dimensional strains on the mouse femur in response to two loading modalities: an axial loading modality (ALM) and a knee loading modality (KLM). We demonstrated that these two loading modalities induced a different pattern of strain distributions. ALM generated strain in the midshaft of cortical bone, while strains with KLM were concentrated on the distal epiphysis of the mouse femur. Since KLM is capable of enhancing bone formation in cortical bone distant from the knee, the current results indicate that in situ strain is not always necessary for load-driven bone formation.

  15. Primary bone marrow B-cell non-Hodgkin's lymphoma successfully treated with R-CHOP.

    PubMed

    Qian, Liren; Zhang, Zhi; Shen, Jianliang; Liu, Yi

    2013-01-01

    Primary isolated bone marrow disease as a presenting feature of lymphoma is very rare. We describe the case of a Chinese with isolated bone marrow small B-cell lymphoma as a first manifestation. A 55-year old woman was admitted to our hospital with fever. Her peripheral blood smear and laboratory findings were suggestive of bicytopenia. Bone marrow specimen showed diffusely distributed small-sized lymphocytes. Combined with immunophenotypic and chromosomal analysis, a diagnosis of primary bone marrow B-cell non-Hodgkin's lymphoma was made. The patient was treated with R-CHOP (rituximab and cyclophosphamide, epirubicin, vindesine, and prednisone) regimen for six cycles. She had complete remission and is still alive without relapse. We concluded that primary bone marrow mature small B-cell lymphoma is a rare but distinctive subtype of lymphoma. The prognosis for this entity is poor but rituximab-based treatment is promising for improving its outcomes.

  16. Extractable bone morphogenetic protein and correlation with induced new bone formation in an in vivo assay in the athymic mouse model.

    PubMed

    Honsawek, Sittisak; Powers, Ralph M; Wolfinbarger, Lloyd

    2005-01-01

    A correlation between extractable bone morphogenetic proteins (BMPs) in demineralized bone matrix (DBM) and osteoinduction has been suggested. Extractable BMP-4 and osteoinductivity of DBM from 40 donors were assessed using enzyme-linked immunosorbent assay (ELISA) and in vivo athymic mouse assay, respectively. Extractable BMP-4 level averaged 3.7 +/- 0.21 ng/g of DBM and correlated with osteoinductivity of the DBM in an in vivo assessment of induced newbone formation.

  17. Genetically engineered mouse models to evaluate the role of Wnt secretion in bone development and homeostasis.

    PubMed

    Williams, Bart O

    2016-03-01

    Alterations in components of the Wnt signaling pathway are associated with altered bone development and homeostasis in several human diseases. We created genetically engineered mouse models (GEMMs) that mimic the cellular defect associated with the Porcupine mutations in patients with Goltz Syndrome/Focal Dermal Hypoplasia. These GEMMs were established by utilizing mice containing a conditionally inactivatable allele of Wntless/GPR177 (a gene encoding a protein required for the transport of Porcupine-modified ligand to the plasma membrane for secretion). We crossed this strain to another which drives cre-mediated gene deletion in mature osteoblasts (Osteocalcin-cre) resulted in mice lacking the ability to secrete Wnt ligands in this cell type. These mice displayed severely reduced bone mass and provide a model to understand the effects of disrupting the ability to secrete Wnt ligands on the skeletal system. © 2016 Wiley Periodicals, Inc.

  18. Maternal beef and postweaning herring diets increase bone mineral density and strength in mouse offspring.

    PubMed

    Hussain, Aysha; Olausson, Hanna; Nilsson, Staffan; Nookaew, Intawat; Khoomrung, Sakda; Andersson, Louise; Koskela, Antti; Tuukkanen, Juha; Ohlsson, Claes; Holmäng, Agneta

    2013-12-01

    The maternal diet during gestation and lactation affects the long-term health of the offspring. We sought to determine whether maternal and postweaning crossover isocaloric diets based on fish or meat affect the geometry, mineral density, and biomechanical properties of bone in mouse offspring in adulthood. During gestation and lactation, C57BL/6 dams were fed a herring- or beef-based diet. After weaning, half of the pups in each group were fed the same diet as their dams, and half were fed the other diet. Areal bone mineral density (aBMD) and bone mineral content (BMC) of the whole body and lumbar spine were measured in the offspring by dual X-ray absorptiometry at 9 and 21 weeks of age. At 22-26 weeks, tibia bone geometry (length, cortical volumetric (v) BMD, BMC, area and thickness) was analyzed by peripheral quantitative computed tomography, and the biomechanical properties of the tibia were analyzed by the three-point bending test. Plasma insulin-like growth factor-1 was analyzed at 12 weeks. In comparison to the maternal herring diet, the maternal beef diet increased aBMD and BMC in the whole body and lumbar spine of adult offspring, as well as cortical vBMD, BMC, bone area, and thickness at the mid-diaphyseal region of the tibia and the biomechanical properties of tibia strength. In contrast, a postweaning beef diet decreased aBMD in the lumbar spine and BMC in the whole body and lumbar spine compared with a postweaning herring diet, which instead increased plasma insulin-like growth factor-1 levels. The change from a maternal beef diet before weaning to a herring diet after weaning decreased body weight and increased the cortical area, vBMD, BMC, thickness, and strength of the tibia. These significant crossover effects indicate that a preweaning maternal beef diet and a postweaning herring diet are optimal for increasing BMC and bone strength in offspring in adulthood.

  19. Chronic intermittent hypoxia preserves bone density in a mouse model of sleep apnea.

    PubMed

    Torres, Marta; Montserrat, Josep M; Pavía, Javier; Dalmases, Mireia; Ros, Domenec; Fernandez, Yolanda; Barbé, Ferran; Navajas, Daniel; Farré, Ramon

    2013-12-01

    Very recent clinical research has investigated whether obstructive sleep apnea (OSA) may modulate bone homeostasis but the few data available are conflicting. Here we report novel data obtained in a mouse study specifically designed to determine whether chronic intermittent hypoxia realistically mimicking OSA modifies bone mineral density (BMD). Normal male and female mice and orchidectomized mice (N=10 each group) were subjected to a pattern of high-frequency intermittent hypoxia (20s at 5% and 40s at 21%, 60 cycles/h) for 6h/day. Identical groups breathing room air (normoxia) were the controls. After 32 days of intermittent hypoxia/normoxia the trabecular bone mineral density (BMD) in the peripheral femora were measured by micro-CT scanning. When compared with normoxia (two-way ANOVA), intermittent hypoxia did not significantly modify BMD in the three animal groups tested. Data in this study suggest that the type of intermittent hypoxia characterizing OSA, applied as a single challenge, preserves bone homeostasis.

  20. Effect of oral calcium and calcium + fluoride treatments on mouse bone properties during suspension

    NASA Technical Reports Server (NTRS)

    Simske, S. J.; Luttges, M. W.; Allen, K. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The bone effects of oral dosages of calcium chloride with or without supplementary sodium fluoride were assessed in antiorthostatically suspended mice. Two calcium dosages were used to replace half (3.1 mM) or all(6.3 mM) of the dietary calcium lost due to reduced food intake by the suspended mice. Two groups of 6.3 mM CaCl2-treated mice were additionally treated with 0.25 or 2.5 mM NaF. The results indicate that supplementation of the mouse drinking water with calcium salts prevents bone changes induced by short-term suspension, while calcium salts in combination with fluoride are less effective as fluoride dosage increases. However, the calcium supplements change the relationship between the femur mechanical properties and the mineral composition of the bone. Because of this, it appears that oral calcium supplements are effective through a mechanism other than simple dietary supplementation and may indicate a dependence of bone consistency on systemic and local fluid conditions.

  1. Effect of oral calcium and calcium + fluoride treatments on mouse bone properties during suspension

    NASA Technical Reports Server (NTRS)

    Simske, S. J.; Luttges, M. W.; Allen, K. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The bone effects of oral dosages of calcium chloride with or without supplementary sodium fluoride were assessed in antiorthostatically suspended mice. Two calcium dosages were used to replace half (3.1 mM) or all(6.3 mM) of the dietary calcium lost due to reduced food intake by the suspended mice. Two groups of 6.3 mM CaCl2-treated mice were additionally treated with 0.25 or 2.5 mM NaF. The results indicate that supplementation of the mouse drinking water with calcium salts prevents bone changes induced by short-term suspension, while calcium salts in combination with fluoride are less effective as fluoride dosage increases. However, the calcium supplements change the relationship between the femur mechanical properties and the mineral composition of the bone. Because of this, it appears that oral calcium supplements are effective through a mechanism other than simple dietary supplementation and may indicate a dependence of bone consistency on systemic and local fluid conditions.

  2. Activated FGFR3 promotes bone formation via accelerating endochondral ossification in mouse model of distraction osteogenesis.

    PubMed

    Osawa, Yusuke; Matsushita, Masaki; Hasegawa, Sachi; Esaki, Ryusaku; Fujio, Masahito; Ohkawara, Bisei; Ishiguro, Naoki; Ohno, Kinji; Kitoh, Hiroshi

    2017-08-10

    Achondroplasia (ACH) is one of the most common short-limbed skeletal dysplasias caused by gain-of-function mutations in the fibroblast growth factor receptors 3 (FGFR3) gene. Distraction osteogenesis (DO) is a treatment option for short stature in ACH in some countries. Although the patients with ACH usually show faster healing in DO, details of the newly formed bone have not been examined. We have developed a mouse model of DO and analyzed new bone regenerates of the transgenic mice with ACH (Fgfr3(ach) mice) histologically and morphologically. We established two kinds of DO protocols, the short-DO consisted of 5days of latency period followed by 5days of distraction with a rate of 0.4mm per 24h, and the long-DO consisted of the same latency period followed by 7days of distraction with a rate of 0.3mm per 12h. The callus formation was evaluated radiologically by bone fill score and quantified by micro-CT scan in both protocols. The histomorphometric analysis was performed in the short-DO protocol by various stainings, including Villanueva Goldner, Safranin-O/Fast green, tartrate-resistant acid phosphatase, and type X collagen. Bone fill scores were significantly higher in Fgfr3(ach) mice than in wild-type mice in both protocols. The individual bone parameters, including bone volume and bone volume/tissue volume, were also significantly higher in Fgfr3(ach) mice than in wild-type mice in both protocols. The numbers of osteoblasts, as well as osteoclasts, around the trabecular bone were increased in Fgfr3(ach) mice. Cartilaginous tissues of the distraction region rapidly disappeared in Fgfr3(ach) mice compared to wild-type mice during the consolidation phase. Similarly, type X collagen-positive cells were markedly decreased in Fgfr3(ach) mice during the same period. Fgfr3(ach) mice exhibited accelerated bone regeneration after DO. Accelerated endochondral ossification could contribute to faster healing in Fgfr3(ach) mice. Copyright © 2017 Elsevier Inc. All rights

  3. Primary Hemangiopericytoma of the Parietal Bone: A Case Report

    PubMed Central

    Sipal, Sare; Demirci, Elif; Calık, Muhammet; Gundogdu, Betul; Sengul, Goksin; Gundogdu, Cemal

    2009-01-01

    Summary Hemangiopericytomas are rare hypervascular tumors arising from Zimmerman’s pericytes. They usually occur in the soft tissue, and intraosseous lesions are very rare. Surgical excision is the first choice for treatment. Many studies show that patients should be monitored for some time following treatment because of a high rate of recurrence and metastasis after radical resection. This report introduces a 56-year-old patient with a hemangiopericytoma in his parietal bone. Keywords: Parietal bone, Hemangiopericytoma PMID:25610105

  4. Characterization of benzodiazepine receptors in primary cultures of fetal mouse brain and spinal cord neurons.

    PubMed

    Huang, A; Barker, J L; Paul, S M; Moncada, V; Skolnick, P

    1980-05-26

    Primary cultures of fetal mouse brain and spinal cord were examined for the presence of binding sites for [3H]diazepam. Both brain and spinal cord cultures contain high affinity binding sites which resemble benzodiazepine receptors found in mammalian CNS with respect to both pharmacologic profile and response to exogenously applied GABA. These observations, coupled with the electrophysiologic properties of these cells suggest that primary cultures of fetal mouse brain and spinal cord may be valid models for studying the role and regulation of the benzodiazepine receptor.

  5. Isolating primary melanocyte-like cells from the mouse heart.

    PubMed

    Hwang, Hayoung; Liu, Fang; Levin, Mark D; Patel, Vickas V

    2014-09-29

    We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

  6. A novel in silico method to quantify primary stability of screws in trabecular bone.

    PubMed

    Steiner, Juri A; Christen, Patrik; Affentranger, Remo; Ferguson, Stephen J; van Lenthe, Gerrit Harry

    2017-02-27

    Insufficient primary stability of screws in bone leads to screw loosening and failure. Unlike conventional continuum finite-element models, micro-CT based finite-element analysis (micro-FE) is capable of capturing the patient-specific bone micro-architecture, providing accurate estimates of bone stiffness. However, such in silico models for screws in bone highly overestimate the apparent stiffness. We hypothesized that a more accurate prediction of primary implant stability of screws in bone is possible by considering insertion-related bone damage. We assessed two different screw types and loading scenarios in 20 trabecular bone specimens extracted from 12 cadaveric human femoral heads (N = 5 for each case). In the micro-FE model, we predicted specimen-specific Young's moduli of the peri-implant bone damage region based on morphometric parameters such that the apparent stiffness of each in silico model matched the experimentally measured stiffness of the corresponding in vitro specimen as closely as possible. The standard micro-FE models assuming perfectly intact peri-implant bone overestimated the stiffness by over 330%. The consideration of insertion related damaged peri-implant bone corrected the mean absolute percentage error down to 11.4% for both loading scenarios and screw types. Cross-validation revealed a mean absolute percentage error of 14.2%. We present the validation of a novel micro-FE modeling technique to quantify the apparent stiffness of screws in trabecular bone. While the standard micro-FE model overestimated the bone-implant stiffness, the consideration of insertion-related bone damage was crucial for an accurate stiffness prediction. This approach provides an important step toward more accurate specimen-specific micro-FE models. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  7. AN INVESTIGATION OF THE MINERAL IN DUCTILE AND BRITTLE CORTICAL MOUSE BONE

    PubMed Central

    Rodriguez-Florez, Naiara; Garcia-Tunon, Esther; Mukadam, Quresh; Saiz, Eduardo; Oldknow, Karla J.; Farquharson, Colin; Millán, José Luis; Boyde, Alan; Shefelbine, Sandra J.

    2015-01-01

    Bone is a strong and tough material composed of apatite mineral, organic matter and water. Changes in composition and organization of these building blocks affect bone’s mechanical integrity. Skeletal disorders often affect bone’s mineral phase, either by variations in the collagen or directly altering mineralization. The aim of the current study was to explore the differences in the mineral of brittle and ductile cortical bone at the mineral (nm) and tissue (µm) levels using two mouse phenotypes. Osteogenesis imperfecta murine (oim−/−) mice were used to model brittle bone; PHOSPHO1 mutants (Phospho1−/−) had ductile bone. They were compared to their respective wild-type controls. Femora were defatted and ground to powder to measure average mineral crystal size using X-ray diffraction (XRD), and to monitor the bulk mineral to matrix ratio via thermogravimetric analysis (TGA). XRD scans were run after TGA for phase identification, to assess the fractions of hydroxyapatite and β-tricalcium phosphate. Tibiae were embedded to measure elastic properties with nanoindentation and the extent of mineralization with backscattered electron microscopy (qbSEM). Interestingly, the mineral of brittle oim−/− and ductile Phospho1−/− bones had many similar characteristics. Both pathology models had smaller apatite crystals, lower mineral to matrix ratio, and showed more thermal conversion to β-tricalcium phosphate than their wild-types, indicating deviations from stoichiometric hydroxyapatite in the original mineral. The degree of mineralization of the bone matrix was different for each strain: oim−/− were hypermineralized, while Phospho1−/− were hypomineralized. However, alterations in the mineral were associated with reduced tissue elastic moduli in both pathologies. Results revealed that despite having extremely different whole bone mechanics, the mineral of oim−/− and Phospho1−/− has several similar trends at smaller length scales. This

  8. Mouse Orthotopic Xenographs of Human Prostate Primary Tumors

    DTIC Science & Technology

    2006-11-01

    prostatic hyperplasia (BPH) and 4 from cancer) were isolated from multiple samples of 4 radical prostatectomy surgical specimens, two of which belonged...pellet (12.5 mg, 90 day- release) was implanted subcutaneously in all mice. 5 PI: Loda, Massimo b) Eight primary cell cultures (4 from benign

  9. The cellular and molecular toxicity of lead in primary and clonal osteoblastic bone cells

    SciTech Connect

    Long, G.J.

    1989-01-01

    First, steady state kinetic models of lead metabolism and calcium homeostasis were developed in both primary and clonal osteoblastic bone cells. Secondly, the effect of lead on cellular calcium homeostasis was determined. Finally, the effect of lead on 1,25 (OH){sub 2}D{sub 3} induced production of osteocalcin, a protein synthesized and secreted by osteoblasts, was investigated. Lead metabolism in osteoblastic bone cells was characterized by three intracellular pools. The largest of these, S{sub 3}, included mitochondrial lead and accounted for 70 percent of total cell lead in primary osteoblastic bone cells and 85 percent of total lead in clonal osteoblastic bone cells. None of the kinetic pools were saturated at lead concentrations up to 100 {mu}M lead. Calcium homeostasis in osteoblastic bone cells was also described by a three compartment, intracellular kinetic model.

  10. Preoperative zoledronic acid therapy prevent hungry bone syndrome in patients with primary hyperparathyroidism

    PubMed Central

    Mayilvaganan, Sabaretnam; Vijaya Sarathi, H. A.; Shivaprasad, C.

    2017-01-01

    Background: Hungry bone syndrome is a common complication of surgery for primary hyperparathyroidism in India which often leads to prolonged hospitalization. There are varying reports on the use and efficacy of bisphosphonates in the prevention of hungry bone syndrome. Methods: We retrospectively analyzed the effect of preoperative bisphosphonate therapy on rates of hungry bone syndrome in our patients with primary hyperparathyroidism. A total of 19 patients underwent surgery for primary hyperparathyroidism at our institute between January 2013 and June 2015 among whom eight did not receive preoperative bisphosphonates and 11 received intravenous zoledronic acid 4 mg, 24–48 h preoperatively. Results: There was no significant difference between the two groups with respect to age, gender, duration of symptoms, preoperative serum calcium, phosphorus, parathyroid hormone, alkaline phosphatase, and the presence of radiological evidence of hyperparathyroid bone disease also did not differ between the groups. Three out of the eight patients who did not receive preoperative zoledronic acid therapy had hungry bone syndrome but none in the zoledronic acid group. The prevalence of hungry bone syndrome tended to be lower in the zoledronic acid group (P = 0.058). The need for intravenous calcium and duration of postoperative hospital stay were significantly lesser in the zoledronic acid group. Conclusion: Preoperative intravenous zoledronic acid significantly reduces the need for intravenous calcium therapy and duration of postoperative hospital stay and seems a promising option to reduce the rate of hungry bone syndrome in patients with primary hyperparathyroidism. PMID:28217502

  11. Prostaglandin E2 synthesis by human primary and metastatic bone tumors in culture.

    PubMed

    Gebhardt, M C; Lippiello, L; Bringhurst, F R; Mankin, H J

    1985-06-01

    Prostaglandin E2 (PGE2) is known to stimulate osteolysis in vitro and has been implicated in mediating bone resorption in several animal and human tumors. Little attention has yet to be directed toward local humoral control (including PGE2) of bone resorption in primary and metastatic bone tumors. For investigation of whether histologically identified areas of osteolytic or osteoblastic bone tumors differentially secrete PGE2 under in vitro conditions, culture media from explants of central and peripheral areas of tissue were sterilely collected from 13 surgical specimens of primary and metastatic bone tumors and assayed for (PGE2) radioimmunoassay. The results indicate a marked heterogeneity in the concentration of immunoreactive (I-PGE2) synthesis by tumors of different as well as similar cell type. PGE2 production was time-dependent in culture, and at 72 hours substantial increases were apparent compared to cultures of non-neoplastic fascia controls. Significantly higher levels of I-PGE2 were found in cultures derived from "bone-destructive" tumors. No difference in I-PGE2 synthesis was found between explants of peripheral versus central tissue of the same tumors. PGE2 is synthesized in culture by bone tumors characterized as destructive of bone at higher levels than "bone-forming" tumors, and this synthesis is inhibited by indomethacin.

  12. Decreasing maternal myostatin programs adult offspring bone strength in a mouse model of osteogenesis imperfecta

    PubMed Central

    Oestreich, Arin K.; Kamp, William M.; McCray, Marcus G.; Carleton, Stephanie M.; Karasseva, Natalia; Lenz, Kristin L.; Jeong, Youngjae; Daghlas, Salah A.; Yao, Xiaomei; Wang, Yong; Pfeiffer, Ferris M.; Ellersieck, Mark R.; Schulz, Laura C.; Phillips, Charlotte L.

    2016-01-01

    During fetal development, the uterine environment can have effects on offspring bone architecture and integrity that persist into adulthood; however, the biochemical and molecular mechanisms remain unknown. Myostatin is a negative regulator of muscle mass. Parental myostatin deficiency (Mstntm1Sjl/+) increases muscle mass in wild-type offspring, suggesting an intrauterine programming effect. Here, we hypothesized that Mstntm1Sjl/+ dams would also confer increased bone strength. In wild-type offspring, maternal myostatin deficiency altered fetal growth and calvarial collagen content of newborn mice and conferred a lasting impact on bone geometry and biomechanical integrity of offspring at 4 mo of age, the age of peak bone mass. Second, we sought to apply maternal myostatin deficiency to a mouse model with osteogenesis imperfecta (Col1a2oim), a heritable connective tissue disorder caused by abnormalities in the structure and/or synthesis of type I collagen. Femora of male Col1a2oim/+ offspring from natural mating of Mstntm1Sjl/+ dams to Col1a2oim/+sires had a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure compared with age, sex, and genotype-matched offspring from natural mating of Col1a2oim/+ dams to Col1a2oim/+ sires. Finally, increased bone biomechanical strength of Col1a2oim/+ offspring that had been transferred into Mstntm1Sjl/+ dams as blastocysts demonstrated that the effects of maternal myostatin deficiency were conferred by the postimplantation environment. Thus, targeting the gestational environment, and specifically prenatal myostatin pathways, provides a potential therapeutic window and an approach for treating osteogenesis imperfecta. PMID:27821779

  13. Bone marrow cell migration to the heart in a chimeric mouse model of acute chagasic disease

    PubMed Central

    Irion, Camila Iansen; Paredes, Bruno Diaz; Brasil, Guilherme Visconde; da Cunha, Sandro Torrentes; Paula, Luis Felipe; Carvalho, Alysson Roncally; de Carvalho, Antonio Carlos Campos; Carvalho, Adriana Bastos; Goldenberg, Regina Coeli dos Santos

    2017-01-01

    BACKGROUND Chagas disease is a public health problem caused by infection with the protozoan Trypanosoma cruzi. There is currently no effective therapy for Chagas disease. Although there is some evidence for the beneficial effect of bone marrow-derived cells in chagasic disease, the mechanisms underlying their effects in the heart are unknown. Reports have suggested that bone marrow cells are recruited to the chagasic heart; however, studies using chimeric mouse models of chagasic cardiomyopathy are rare. OBJECTIVES The aim of this study was to investigate the migration of bone marrow cells to the heart after T. cruzi infection in a model of chagasic disease in chimeric mice. METHODS To obtain chimerical mice, wild-type (WT) C57BL6 mice were exposed to full body irradiation (7 Gy), causing bone marrow ablation. Then, bone marrow cells from green fluorescent protein (GFP)-transgenic mice were infused into the mice. Graft effectiveness was confirmed by flow cytometry. Experimental mice were divided into four groups: (i) infected chimeric (iChim) mice; (ii) infected WT (iWT) mice, both of which received 3 × 104 trypomastigotes of the Brazil strain; (iii) non-infected chimeric (Chim) mice; and (iv) non-infected WT mice. FINDINGS At one-month post-infection, iChim and iWT mice showed first degree atrioventricular block with decreased heart rate and treadmill exercise parameters compared to those in the non-infected groups. MAIN CONCLUSIONS iChim mice showed an increase in parasitaemia, myocarditis, and the presence of amastigote nests in the heart tissue compared to iWT mice. Flow cytometry analysis did not detect haematopoietic progenitor cells in the hearts of infected mice. Furthermore, GFP+ cardiomyocytes were not detected in the tissues of chimeric mice. PMID:28767980

  14. Multifocal primary bone sarcoma in the elderly: a rare case and review of the literature.

    PubMed

    Duckworth, A D; Beggs, I; Salter, D M; Patton, J T; Porter, D E

    2012-02-01

    To our knowledge there are no reports of a primary multifocal spindle cell sarcoma affecting both long bones of the lower limb in an elderly patient. An 83-year-old man presented with a progressively painful right ankle, without a history of trauma. Radiographs demonstrated a pathological fracture through a mixed lytic and sclerotic lesion in the distal tibia. Staging investigations, including bone scintigraphy and magnetic resonance imaging, revealed a second lytic lesion in the right distal femur. A provisional diagnosis of metastatic disease was favoured and intramedullary nailing was considered. However, computed tomography of the chest, abdomen and pelvis, and relevant blood tests revealed no evidence of a primary malignancy. Open biopsy of the tibial lesion showed high-grade pleomorphic spindle cells consistent with a primary bone sarcoma. The patient was treated successfully with a right trans-femoral amputation. In conclusion, we recommend early bone biopsy to allow accurate diagnosis and appropriate skeletal management in elderly patients.

  15. Fusobacterium nucleatum and Tannerella forsythia induce synergistic alveolar bone loss in a mouse periodontitis model.

    PubMed

    Settem, Rajendra P; El-Hassan, Ahmed Taher; Honma, Kiyonobu; Stafford, Graham P; Sharma, Ashu

    2012-07-01

    Tannerella forsythia is strongly associated with chronic periodontitis, an inflammatory disease of the tooth-supporting tissues, leading to tooth loss. Fusobacterium nucleatum, an opportunistic pathogen, is thought to promote dental plaque formation by serving as a bridge bacterium between early- and late-colonizing species of the oral cavity. Previous studies have shown that F. nucleatum species synergize with T. forsythia during biofilm formation and pathogenesis. In the present study, we showed that coinfection of F. nucleatum and T. forsythia is more potent than infection with either species alone in inducing NF-κB activity and proinflammatory cytokine secretion in monocytic cells and primary murine macrophages. Moreover, in a murine model of periodontitis, mixed infection with the two species induces synergistic alveolar bone loss, characterized by bone loss which is greater than the additive alveolar bone losses induced by each species alone. Further, in comparison to the single-species infection, mixed infection caused significantly increased inflammatory cell infiltration in the gingivae and osteoclastic activity in the jaw bones. These data show that F. nucleatum subspecies and T. forsythia synergistically stimulate the host immune response and induce alveolar bone loss in a murine experimental periodontitis model.

  16. Visual impairment in an optineurin mouse model of primary open-angle glaucoma.

    PubMed

    Tseng, Henry C; Riday, Thorfinn T; McKee, Celia; Braine, Catherine E; Bomze, Howard; Barak, Ian; Marean-Reardon, Carrie; John, Simon W M; Philpot, Benjamin D; Ehlers, Michael D

    2015-06-01

    Primary open-angle glaucoma (POAG) is characterized by progressive neurodegeneration of retinal ganglion cells (RGCs). Why RGCs degenerate in low-pressure POAG remains poorly understood. To gain mechanistic insights, we developed a novel mouse model based on a mutation in human optineurin associated with hereditary, low-pressure POAG. This mouse improves the design and phenotype of currently available optineurin mice, which showed high global overexpression. Although both 18-month-old optineurin and nontransgenic control mice showed an age-related decrease in healthy axons and RGCs, the expression of mutant optineurin enhanced axonal degeneration and decreased RGC survival. Mouse visual function was determined using visual evoked potentials, which revealed specific visual impairment in contrast sensitivity. The E50K optineurin transgenic mouse described here exhibited clinical features of POAG and may be useful for mechanistic dissection of POAG and therapeutic development. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Dose and Radioadaptive Response Analysis of Micronucleus Induction in Mouse Bone Marrow

    PubMed Central

    Bannister, Laura A.; Mantha, Rebecca R.; Devantier, Yvonne; Petoukhov, Eugenia S.; Brideau, Chantal L. A.; Serran, Mandy L.; Klokov, Dmitry Y.

    2016-01-01

    Enhanced cellular DNA repair efficiency and suppression of genomic instability have been proposed as mechanisms underlying radio-adaptive responses following low-dose radiation exposures. We previously showed that low-dose γ irradiation does not generate radio-adaptation by lowering radiation-induced cytogenetic damage in mouse spleen. Since radiation may exert tissue-specific effects, we extended these results here by examining the effects of γ radiation on cytogenetic damage and proliferative index in bone marrow erythrocytes of C57BL/6 and BALB/c mice. In C57BL/6 mice, the induction of micronuclei in polychromatic erythrocytes (MN-PCE) was observed at radiation doses of 100 mGy and greater, and suppression of erythroblast maturation occurred at doses of >500 mGy. A linear dose–response relationship for MN-PCE frequencies in C57BL/6 mice was established for radiation doses between 100 mGy and 1 Gy, with departure from linearity at doses of >1 Gy. BALB/c mice exhibited increased MN-PCE frequencies above baseline following a 20 mGy radiation exposure but did not exhibit radio-sensitivity relative to C57BL/6 mice following 2 Gy exposure. Radio-adaptation of bone marrow erythrocytes was not observed in either strain of mice exposed to low-dose priming γ irradiation (single doses of 20 mGy or 100 mGy or multiple 20 mGy doses) administered at various times prior to acute 2 Gy irradiation, confirming the lack of radio-adaptive response for induction of cytogenetic damage or suppression or erythrocyte proliferation/maturation in bone marrow of these mouse strains. PMID:27649149

  18. Systemic Mesenchymal Stromal Cell Transplantation Prevents Functional Bone Loss in a Mouse Model of Age-Related Osteoporosis

    PubMed Central

    Kiernan, Jeffrey; Hu, Sally; Grynpas, Marc D.

    2016-01-01

    Age-related osteoporosis is driven by defects in the tissue-resident mesenchymal stromal cells (MSCs), a heterogeneous population of musculoskeletal progenitors that includes skeletal stem cells. MSC decline leads to reduced bone formation, causing loss of bone volume and the breakdown of bony microarchitecture crucial to trabecular strength. Furthermore, the low-turnover state precipitated by MSC loss leads to low-quality bone that is unable to perform remodeling-mediated maintenance—replacing old damaged bone with new healthy tissue. Using minimally expanded exogenous MSCs injected systemically into a mouse model of human age-related osteoporosis, we show long-term engraftment and markedly increased bone formation. This led to improved bone quality and turnover and, importantly, sustained microarchitectural competence. These data establish proof of concept that MSC transplantation may be used to prevent or treat human age-related osteoporosis. Significance This study shows that a single dose of minimally expanded mesenchymal stromal cells (MSCs) injected systemically into a mouse model of human age-related osteoporosis display long-term engraftment and prevent the decline in bone formation, bone quality, and microarchitectural competence. This work adds to a growing body of evidence suggesting that the decline of MSCs associated with age-related osteoporosis is a major transformative event in the progression of the disease. Furthermore, it establishes proof of concept that MSC transplantation may be a viable therapeutic strategy to treat or prevent human age-related osteoporosis. PMID:26987353

  19. Systemic Mesenchymal Stromal Cell Transplantation Prevents Functional Bone Loss in a Mouse Model of Age-Related Osteoporosis.

    PubMed

    Kiernan, Jeffrey; Hu, Sally; Grynpas, Marc D; Davies, John E; Stanford, William L

    2016-05-01

    Age-related osteoporosis is driven by defects in the tissue-resident mesenchymal stromal cells (MSCs), a heterogeneous population of musculoskeletal progenitors that includes skeletal stem cells. MSC decline leads to reduced bone formation, causing loss of bone volume and the breakdown of bony microarchitecture crucial to trabecular strength. Furthermore, the low-turnover state precipitated by MSC loss leads to low-quality bone that is unable to perform remodeling-mediated maintenance--replacing old damaged bone with new healthy tissue. Using minimally expanded exogenous MSCs injected systemically into a mouse model of human age-related osteoporosis, we show long-term engraftment and markedly increased bone formation. This led to improved bone quality and turnover and, importantly, sustained microarchitectural competence. These data establish proof of concept that MSC transplantation may be used to prevent or treat human age-related osteoporosis. This study shows that a single dose of minimally expanded mesenchymal stromal cells (MSCs) injected systemically into a mouse model of human age-related osteoporosis display long-term engraftment and prevent the decline in bone formation, bone quality, and microarchitectural competence. This work adds to a growing body of evidence suggesting that the decline of MSCs associated with age-related osteoporosis is a major transformative event in the progression of the disease. Furthermore, it establishes proof of concept that MSC transplantation may be a viable therapeutic strategy to treat or prevent human age-related osteoporosis. ©AlphaMed Press.

  20. Therapeutic effects of mouse bone marrow-derived clonal mesenchymal stem cells in a mouse model of inflammatory bowel disease

    PubMed Central

    Park, Jin Seok; Yi, Tac-Ghee; Park, Jong-Min; Han, Young Min; Kim, Jun-Hyung; Shin, Dong-Hee; Tak, Seon Ji; Lee, Kyuheon; Lee, Youn Sook; Jeon, Myung-Shin; Hahm, Ki-Baik; Song, Sun U; Park, Seok Hee

    2015-01-01

    Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-α, IFN-γ, IL-1β, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-κB activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy. PMID:26566304

  1. Laxative Related Primary Hyperphosphatemic Tumoral Calcinosis Identified by Bone Scintigraphy

    PubMed Central

    Asokendaran, Marcus; Lenzo, Nat Patrick

    2016-01-01

    We describe a case of a 40-year-old female patient presenting with tumor calcinosis where hypertrophic pulmonary osteoarthropathy (HPOA) was suspected given her extensive history of malignancy. Plain X-rays did not show reveal the typical periarticular calcification but did show appearances consistent with HPOA. Bone scintigraphy with 99mTc-methylene diphosphonate (MDP) is a sensitive investigation in the detection of hypertrophic osteoarthopathy but did not show findings characteristics of HPOA like bilateral symmetrical increased uptake of the radiopharmaceutical along the cortical margins of the long bones. The final diagnosis of tumor calcinosis was only made after low dose computerized tomography chest showed a moderated sized amorphous calcified cluster in the apical segment of the right upper lobe consistent. In conclusion, bone scintigraphy continues to be a useful investigation for both common and rare conditions like tumor calcinosis. The unusual three phase bone scan finding of diffuse activity throughout both lung fields, which turned to out to be tumoral calcinosis is highlighted in this case. PMID:27651742

  2. A NEW APPROACH TO PARTIALKNEE ENDOPROSTHESIS IN PRIMARY BONE SARCOMAS

    PubMed Central

    Penna, Valter; Toller, Eduardo Areas; Pinheiro, Carla; Becker, Ricardo Gehrke

    2015-01-01

    Partial knee endoprosthesis to bone sarcomas resections seems to be a good solution to treat this immature skeletal patients. The purpose of this study is to evaluate the functional score in fourteen patients, advantages and the technique indications. Methods: Retrospective analysis was done to assess in this group of patients the functional evolution and the possible complications of the procedure. 14 patients between 10 and 22 years functionally evaluated in Ennekin/ISOLS (International Society of Limb Salvage) criteria, being all of them operated in the same institution by the same surgeon. Were used distal femur and proximal tibia partial endoprosthesis. Results: General analysis demonstrated that the functional results were over than 67 percent (ISOLS criteria) in 78,6 percent of the patients, being considered excellent. 21,4 percent were considered good results, being between 50 and 66 percent. Bone storage was preserved when avoiding the adjacent segment resection. Surgery time was not prolonged in ligament reconstruction. Conclusion: Knee partial endoprosthesis are less damage to bone storage in young patients. The critics about the bad functional results are being supplied by new surgical techniques, excellent rehabilitation protocols, implants technology and the consequent learning curve. This option of treatment permits the preservation of healthy bone and provides the possibility of a revision replacement less aggressive. PMID:26998452

  3. Radioprotective effects of Dragon's blood and its extract against gamma irradiation in mouse bone marrow cells.

    PubMed

    Ran, Yuanyuan; Wang, Ran; Lin, Fankai; Hasan, Murtaza; Jia, Qiutian; Tang, Bo; Xia, Yu; Shan, Shuangquan; Wang, Xiao; Li, Qiang; Deng, Yulin; Qing, Hong

    2014-06-01

    The radioprotective effects of Dragon's blood (DB) and its extracts (DBE) were investigated using the chromosomal aberrant test, micronucleus and oxidative stress assay for anti-clastogenic and anti-oxidative activity. Adult BALB/C mice were exposed to the whole body irradiation with 4 Gy (60)Co γ-rays. DB and DBE were administered orally once a day from 5 days prior to irradiation treatment to 1 day after irradiation. The mice were sacrificed on 24 h after irradiation. The cells of bone marrow were measured by counting different types of chromosomal aberrations and the frequency of micronuclei. Oxidative stress response was carried out by analysis of serum from blood. DB and DBE significantly decreased the number of bone marrow cells with chromosome aberrations after irradiation with respect to irradiated alone group. The administration of DB and DBE also significantly reduced the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE). In addition, DB and DBE markedly increased the activity of antioxidant enzymes and the level of antioxidant molecular. Malondialdehyde (MDA) and nitric oxide (NO) levels in serum were significantly reduced by DB and DBE treatment. Our data suggested that DB and DBE have potential radioprotective properties in mouse bone marrow after (60)Co γ-ray exposure, which support their candidature as a potential radioprotective agent. Copyright © 2013 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

  4. Survival benefit with radium-223 dichloride in a mouse model of breast cancer bone metastasis.

    PubMed

    Suominen, Mari I; Rissanen, Jukka P; Käkönen, Rami; Fagerlund, Katja M; Alhoniemi, Esa; Mumberg, Dominik; Ziegelbauer, Karl; Halleen, Jussi M; Käkönen, Sanna-Maria; Scholz, Arne

    2013-06-19

    Bone metastases are associated with increased morbidity and poor prognosis in breast cancer patients. Radium-223 dichloride is a calcium mimetic that localizes to bone, providing targeted therapy for skeletal metastasis. We investigated the mode of action of radium-223 dichloride using breast cancer cell, osteoclast, and osteoblast cultures as well as a mouse model of breast cancer bone metastasis. A single dose of radium-223 dichloride was used in three different settings mimicking the prevention or treatment of bone metastasis. Disease progression was monitored using fluorescence and radiographic imaging and histological analyses. The effect of radium-223 dichloride alone and in combination with doxorubicin or zoledronic acid on survival of mice was analyzed by Kaplan-Meier methods. All statistical tests used were two-sided. Radium-223 dichloride incorporated into bone matrix and inhibited proliferation of breast cancer cells and differentiation of osteoblasts and osteoclasts (all P values < .001) in vitro. In an established bone metastasis setting, radium-223 dichloride prevented tumor-induced cachexia (0/14 vs 7/14 control mice) and decreased osteolysis by 56% and tumor growth by 43% (all P values < .05). Radium-223 dichloride induced double-strand DNA breaks in cancer cells in vivo. Finally, radium-223 dichloride extended survival as a monotherapy (29.2 days, 95% confidence interval [CI] = 26.6 to 31.8 days, P = .039) and in combination with zoledronic acid (31.4 days, 95% CI = 28.8 to 34.0 days, P = .004) or doxorubicin (31.5 days, 95% CI = 29.5 to 33.5 days, P < .001) compared to the vehicle group (24.9 days, 95% CI = 23.4 to 26.4 days). Similar but even more pronounced effects were observed when radium-223 dichloride was administered in a preventive or micrometastatic setting. Our findings strongly support the development of radium-223 dichloride for the treatment of breast cancer patients with or at high risk of developing bone metastases.

  5. Mouse newborn cells allow highly productive mouse cytomegalovirus replication, constituting a novel convenient primary cell culture system.

    PubMed

    Le-Trilling, Vu Thuy Khanh; Trilling, Mirko

    2017-01-01

    Mammalian cell culture is indispensable for most aspects of current biomedical research. Immortalized cell lines are very convenient, but transforming principles (e.g. oncogenic viruses or their oncogenes) can heavily influence the experimental outcome. Primary cells do not share this apparent disadvantage but are more laborious to generate. Certain viruses (e.g. mouse cytomegalovirus) do not replicate efficiently in most transformed cell lines. In the past, such viruses have been routinely propagated on primary mouse embryonic fibroblasts (MEF) established around day 17 (d17) of gestation. According to new regulations of the European Union, experiments using gravid mammals and/or their embryos in the last trimester (>d14 in the case of mice) of gestation do require explicit permission of the local authorities responsible for animal care and use. Applying for such permission is time-consuming and often inflexible. Embryonic fibroblasts could also be produced at earlier time points of pregnancy from younger and smaller embryos. Obviously, this approach consumes more pregnant mice and embryos. Newborn mice are larger thus yielding more cells per sacrificed animal and the new Directive (2010/63/EU) excludes the killing of animals solely for the use of their organs or tissues. We established a convenient protocol to generate adherent mouse newborn cells (MNC). A direct comparison of MNC with MEF revealed that MNC fully recapitulate all tested aspects of a broad panel of virological parameters (plaque size, final titers, viral replication kinetics, viral gene expression, drug and interferon susceptibility as well as species specificity). The herein described approach allows researchers the legal use of primary cells and contributes to the 3R (replace, reduce, refine) guiding principles-especially the 'reduce' aspect-for the use of animals in scientific research. Additionally, it offers the option to directly compare in vitro and in vivo experiments when MNC are

  6. Mouse newborn cells allow highly productive mouse cytomegalovirus replication, constituting a novel convenient primary cell culture system

    PubMed Central

    Le-Trilling, Vu Thuy Khanh

    2017-01-01

    Mammalian cell culture is indispensable for most aspects of current biomedical research. Immortalized cell lines are very convenient, but transforming principles (e.g. oncogenic viruses or their oncogenes) can heavily influence the experimental outcome. Primary cells do not share this apparent disadvantage but are more laborious to generate. Certain viruses (e.g. mouse cytomegalovirus) do not replicate efficiently in most transformed cell lines. In the past, such viruses have been routinely propagated on primary mouse embryonic fibroblasts (MEF) established around day 17 (d17) of gestation. According to new regulations of the European Union, experiments using gravid mammals and/or their embryos in the last trimester (>d14 in the case of mice) of gestation do require explicit permission of the local authorities responsible for animal care and use. Applying for such permission is time-consuming and often inflexible. Embryonic fibroblasts could also be produced at earlier time points of pregnancy from younger and smaller embryos. Obviously, this approach consumes more pregnant mice and embryos. Newborn mice are larger thus yielding more cells per sacrificed animal and the new Directive (2010/63/EU) excludes the killing of animals solely for the use of their organs or tissues. We established a convenient protocol to generate adherent mouse newborn cells (MNC). A direct comparison of MNC with MEF revealed that MNC fully recapitulate all tested aspects of a broad panel of virological parameters (plaque size, final titers, viral replication kinetics, viral gene expression, drug and interferon susceptibility as well as species specificity). The herein described approach allows researchers the legal use of primary cells and contributes to the 3R (replace, reduce, refine) guiding principles—especially the ‘reduce’ aspect—for the use of animals in scientific research. Additionally, it offers the option to directly compare in vitro and in vivo experiments when MNC are

  7. An RNA-seq Protocol to Identify mRNA Expression Changes in Mouse Diaphyseal Bone: Applications in Mice with Bone Property Altering Lrp5 Mutations

    PubMed Central

    Ayturk, Ugur M.; Jacobsen, Christina M.; Christodoulou, Danos C.; Gorham, Joshua; Seidman, Jonathan G.; Seidman, Christine E.; Robling, Alexander G.; Warman, Matthew L.

    2013-01-01

    Loss-of-function and certain missense mutations in the Wnt co-receptor LRP5 significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knockin mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5+/+), knockout (Lrp5−/−), and high bone mass (HBM)-causing (Lrp5p.A214V/+) alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating non-skeletal tissue (i.e., blood, bone marrow, and skeletal muscle) in our data. These methods included pre-digestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal’s right and left tibiae and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 “sufficient” (i.e., Lrp5+/+ and Lrp5p.A214V/+) and “insufficient” (Lrp5−/−) diaphyseal bone, and far fewer differentially expressed genes between Lrp5p.A214V/+ and Lrp5+/+ diaphyseal bone. PMID:23553928

  8. Primary pericranial Ewing's sarcoma on the temporal bone: A case report

    PubMed Central

    Kawano, Hiroto; Nitta, Naoki; Ishida, Mitsuaki; Fukami, Tadateru; Nozaki, Kazuhiko

    2016-01-01

    Background: Primary Ewing's sarcoma originating in the pericranium is an extremely rare disease entity. Case Description: A 9-year-old female patient was admitted to our department due to a left temporal subcutaneous mass. The mass was localized under the left temporal muscle and attached to the surface of the temporal bone. Head computed tomography revealed a mass with bony spicule formation on the temporal bone, however, it did not show bone destruction or intracranial invasion. F-18 fluorodeoxyglucose positron emission tomography showed no lesions other than the mass on the temporal bone. Magnetic resonance imaging showed that the mass was located between the temporal bone and the pericranium. The mass was completely resected with the underlying temporal bone and the overlying deep layer of temporal muscle, and was diagnosed as primary Ewing's sarcoma. Because the tumor was located in the subpericranium, we created a new classification, “pericranial Ewing's sarcoma,” and diagnosed the present tumor as pericranial Ewing's sarcoma. Conclusion: We herein present an extremely rare case of primary pericranial Ewing's sarcoma that developed on the temporal bone. PMID:27308095

  9. [Hematopoietic growth factors in primary and therapy-related bone marrow insufficiency].

    PubMed

    Hansen, B; Hippe, E; Jacobsen, G K; Johnsen, H E

    1992-06-08

    This investigation is retrospective and comprises 20 patients with bone-marrow insufficiency. During the period 1.4.1988-1.3.1991, these patients were treated with erythropoietin (Epo), the granulocyte-macrophage-colony-stimulating factor (GM-CSF) or the granulocyte-colony-stimulating factor (G-CSF). Thirteen patients had primary bone-marrow insufficiency: six had the myelodysplastic syndrome, three had primary myelofibrosis, two aplastic anemia and two myelomatosis. On account of dominating symptoms of anemia, five patients received Epo while eight received GM-CSF as part of an extensive clinical trial of this preparation. Seven patients with relapse of the haematological malignant disease had bone-marrow insufficiency and pancytopenia secondary to intensive chemotherapy/irradiation: four of these patients received GM-CSF and two received G-CSF with the object of increasing bone-marrow regeneration and to render further chemotherapy possible. One patient received GM-CSF with the object of improving bone-marrow function after autologous bone-marrow transplantation. Treatment with Epo for ten months combined with treatment with interferon for six months resulted in normalization of the haemoglobin concentration in one patient with bone-marrow insufficiency on account of primary myelofibrosis. Treatment with Epo for briefer periods in lower doses was without effect in four other patients with primary bone-marrow insufficiency. Treatment with GM-CSF and G-CSF resulted in neutrophil leukocytosis in 12 out of 15 patients (80%) and, in six out of 14 patients (43%), increased marrow cellularity was demonstrated by means of histological examination of the bone-marrow. One patient showed normal haemoglobin levels during treatment with GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Tumor-targeting Salmonella typhimurium A1-R inhibits human prostate cancer experimental bone metastasis in mouse models.

    PubMed

    Toneri, Makoto; Miwa, Shinji; Zhang, Yong; Hu, Cameron; Yano, Shuya; Matsumoto, Yasunori; Bouvet, Michael; Nakanishi, Hayao; Hoffman, Robert M; Zhao, Ming

    2015-10-13

    Bone metastasis is a frequent occurrence in prostate cancer patients and often is lethal. Zoledronic acid (ZOL) is often used for bone metastasis with limited efficacy. More effective models and treatment methods are required to improve the outcome of prostate cancer patients. In the present study, the effects of tumor-targeting Salmonella typhimurium A1-R were analyzed in vitro and in vivo on prostate cancer cells and experimental bone metastasis. Both ZOL and S. typhimurium A1-R inhibited the growth of PC-3 cells expressing red fluorescent protien in vitro. To investigate the efficacy of S. typhimurium A1-R on prostate cancer experimental bone metastasis, we established models of both early and advanced stage bone metastasis. The mice were treated with ZOL, S. typhimurium A1-R, and combination therapy of both ZOL and S. typhimurium A1-R. ZOL and S. typhimurium A1-R inhibited the growth of solitary bone metastases. S. typhimurium A1-R treatment significantly decreased bone metastasis and delayed the appearance of PC-3 bone metastases of multiple mouse models. Additionally, S. typhimurium A1-R treatment significantly improved the overall survival of the mice with multiple bone metastases. The results of the present study indicate that S. typhimurium A1-R is useful to prevent and inhibit prostate cancer bone metastasis and has potential for future clinical use in the adjuvant setting.

  11. Modelling of optical aberrations caused by light propagation in mouse cranial bone using second harmonic generation imaging

    NASA Astrophysics Data System (ADS)

    Tehrani, Kayvan; Kner, Peter; Mortensen, Luke J.

    2017-02-01

    Multiphoton imaging through the bone to image into the bone marrow or the brain is an emerging need in the scientific community. Due to the highly scattering nature of bone, bone thinning or removal is typically required to enhance the resolution and signal intensity at the imaging plane. The optical aberrations and scattering in the bone significantly affect the resolution and signal to noise ratio of deep tissue microscopy. Multiphoton microscopy uses long wavelength (nearinfrared and infrared) excitation light to reduce the effects of scattering. However, it is still susceptible to optical aberrations and scattering since the light propagates through several layers of media with inhomogeneous indices of refraction. Mechanical removal of bone is highly invasive, laborious, and cannot be applied in experiments where imaging inside of the bone is desired. Adaptive optics technology can compensate for these optical aberrations and potentially restore the diffraction limited point spread function of the system even in deep tissue. To design an adaptive optics system, a priori knowledge of the sample structure assists selection of the proper correction element and sensing methods. In this work we present the characterization of optical aberrations caused by mouse cranial bone, using second harmonic generation imaging of bone collagen. We simulate light propagation through the bone, calculate aberrations and determine the correction that can be achieved using a deformable mirror.

  12. Tumor-targeting Salmonella typhimurium A1-R inhibits human prostate cancer experimental bone metastasis in mouse models

    PubMed Central

    Toneri, Makoto; Miwa, Shinji; Zhang, Yong; Hu, Cameron; Yano, Shuya; Matsumoto, Yasunori; Bouvet, Michael; Nakanishi, Hayao; Hoffman, Robert M.; Zhao, Ming

    2015-01-01

    Bone metastasis is a frequent occurrence in prostate cancer patients and often is lethal. Zoledronic acid (ZOL) is often used for bone metastasis with limited efficacy. More effective models and treatment methods are required to improve the outcome of prostate cancer patients. In the present study, the effects of tumor-targeting Salmonella typhimurium A1-R were analyzed in vitro and in vivo on prostate cancer cells and experimental bone metastasis. Both ZOL and S. typhimurium A1-R inhibited the growth of PC-3 cells expressing red fluorescent protien in vitro. To investigate the efficacy of S. typhimurium A1-R on prostate cancer experimental bone metastasis, we established models of both early and advanced stage bone metastasis. The mice were treated with ZOL, S. typhimurium A1-R, and combination therapy of both ZOL and S. typhimurium A1-R. ZOL and S. typhimurium A1-R inhibited the growth of solitary bone metastases. S. typhimurium A1-R treatment significantly decreased bone metastasis and delayed the appearance of PC-3 bone metastases of multiple mouse models. Additionally, S. typhimurium A1-R treatment significantly improved the overall survival of the mice with multiple bone metastases. The results of the present study indicate that S. typhimurium A1-R is useful to prevent and inhibit prostate cancer bone metastasis and has potential for future clinical use in the adjuvant setting. PMID:26431498

  13. Inhibition of RANKL blocks skeletal tumor progression and improves survival in a mouse model of breast cancer bone metastasis.

    PubMed

    Canon, Jude R; Roudier, Martine; Bryant, Rebecca; Morony, Sean; Stolina, Marina; Kostenuik, Paul J; Dougall, William C

    2008-01-01

    Bone metastases cause severe skeletal morbidity including fractures and hypercalcemia. Tumor cells in bone induce activation of osteoclasts, which mediate bone resorption and release of growth factors from bone matrix, resulting in a "vicious cycle" of bone breakdown and tumor proliferation. Receptor activator of NF-kappaB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival, and is blocked by a soluble decoy receptor, osteoprotegerin (OPG). In human malignancies that metastasize to bone, dysregulation of the RANK/RANKL/OPG pathway can increase the RANKL:OPG ratio, a condition which favors excessive osteolysis. In a mouse model of bone metastasis, RANKL protein levels in MDA-MB-231 (MDA-231) tumor-bearing bones were significantly higher than tumor-free bones. The resulting tumor-induced osteoclastogenesis and osteolysis was dose-dependently inhibited by recombinant OPG-Fc treatment, supporting the essential role for RANKL in this process. Using bioluminescence imaging in a mouse model of metastasis, we monitored the anti-tumor efficacy of RANKL inhibition on MDA-231 human breast cancer cells in a temporal manner. Treatment with OPG-Fc in vivo inhibited growth of MDA-231 tumor cells in bony sites when given both as a preventative (dosed day 0) and as a therapeutic agent for established bone metastases (dosed day 7). One mechanism by which RANKL inhibition reduced tumor burden appears to be indirect through inhibition of the "vicious cycle" and involved an increase in tumor cell apoptosis, as measured by active caspase-3. Here, we demonstrate for the first time that OPG-Fc treatment of mice with established bone metastases resulted in an overall improvement in survival.

  14. Growth of Mycobacterium lepraemurium in nonstimulated and stimulated mouse peritoneal-derived and bone marrrow-derived macrophages in vitro.

    PubMed Central

    Alexander, J; Smith, C C

    1978-01-01

    Mycobacterium lepraemurium cells were found to multiply in normal mouse peritoneal-derived and bone marrow-derived macrophages in vitro. Whereas activated peritoneal-derived macrophages demonstrated marked bacteriostasis for M. lepraemurium, significant bactericidal activity was exhibited by activated bone marrow-derived macrophages. However, only a small proportion of the bacterial were killed by activated bone marrow-derived macrophages with subsequent and enhanced bacteria growth. It is suggested that a rapid turnover of monocytes in active lesions is required to control mycobacterial infections in vivo. These results would suggest that careful consideration be given to the choice of the host cell in studies involving obligate intracellular parasites. PMID:365762

  15. Lack of prolidase causes a bone phenotype both in human and in mouse.

    PubMed

    Besio, Roberta; Maruelli, Silvia; Gioia, Roberta; Villa, Isabella; Grabowski, Peter; Gallagher, Orla; Bishop, Nicholas J; Foster, Sarah; De Lorenzi, Ersilia; Colombo, Raffaella; Diaz, Josè Luis Dapena; Moore-Barton, Haether; Deshpande, Charu; Aydin, Halil Ibrahim; Tokatli, Aysegul; Kwiek, Bartlomiej; Kasapkara, Cigdem Seher; Adisen, Esra Ozsoy; Gurer, Mehmet Ali; Di Rocco, Maja; Phang, James M; Gunn, Teresa M; Tenni, Ruggero; Rossi, Antonio; Forlino, Antonella

    2015-03-01

    The degradation of the main fibrillar collagens, collagens I and II, is a crucial process for skeletal development. The most abundant dipeptides generated from the catabolism of collagens contain proline and hydroxyproline. In humans, prolidase is the only enzyme able to hydrolyze dipeptides containing these amino acids at their C-terminal end, thus being a key player in collagen synthesis and turnover. Mutations in the prolidase gene cause prolidase deficiency (PD), a rare recessive disorder. Here we describe 12 PD patients, 9 of whom were molecularly characterized in this study. Following a retrospective analysis of all of them a skeletal phenotype associated with short stature, hypertelorism, nose abnormalities, microcephaly, osteopenia and genu valgum, independent of both the type of mutation and the presence of the mutant protein was identified. In order to understand the molecular basis of the bone phenotype associated with PD, we analyzed a recently identified mouse model for the disease, the dark-like (dal) mutant. The dal/dal mice showed a short snout, they were smaller than controls, their femurs were significantly shorter and pQCT and μCT analyses of long bones revealed compromised bone properties at the cortical and at the trabecular level in both male and female animals. The differences were more pronounce at 1 month being the most parameters normalized by 2 months of age. A delay in the formation of the second ossification center was evident at postnatal day 10. Our work reveals that reduced bone growth was due to impaired chondrocyte proliferation and increased apoptosis rate in the proliferative zone associated with reduced hyperthrophic zone height. These data suggest that lack of prolidase, a cytosolic enzyme involved in the final stage of protein catabolism, is required for normal skeletogenesis especially at early age when the requirement for collagen synthesis and degradation is the highest.

  16. Cortical and trabecular bone adaptation to incremental load magnitudes using the mouse tibial axial compression loading model.

    PubMed

    Weatherholt, Alyssa M; Fuchs, Robyn K; Warden, Stuart J

    2013-01-01

    The mouse tibial axial compression loading model has recently been described to allow simultaneous exploration of cortical and trabecular bone adaptation within the same loaded element. However, the model frequently induces cortical woven bone formation and has produced inconsistent results with regards to trabecular bone adaptation. The aim of this study was to investigate bone adaptation to incremental load magnitudes using the mouse tibial axial compression loading model, with the ultimate goal of revealing a load that simultaneously induced lamellar cortical and trabecular bone adaptation. Adult (16 weeks old) female C57BL/6 mice were randomly divided into three load magnitude groups (5, 7 and 9N), and had their right tibia axially loaded using a continuous 2-Hz haversine waveform for 360 cycles/day, 3 days/week for 4 consecutive weeks. In vivo peripheral quantitative computed tomography was used to longitudinally assess midshaft tibia cortical bone adaptation, while ex vivo micro-computed tomography and histomorphometry were used to assess both midshaft tibia cortical and proximal tibia trabecular bone adaptation. A dose response to loading magnitude was observed within cortical bone, with increasing load magnitude inducing increasing levels of lamellar cortical bone adaptation within the upper two thirds of the tibial diaphysis. Greatest cortical bone adaptation was observed at the midshaft where there was a 42% increase in estimated mechanical properties (polar moment of inertia) in the highest (9N) load group. A dose response to load magnitude was not clearly evident within trabecular bone, with only the highest load (9N) being able to induce measureable adaptation (31% increase in trabecular bone volume fraction at the proximal tibia). The ultimate finding was that a load of 9N (engendering a tensile strain of 1833 με on medial surface of the midshaft tibia) was able to simultaneously induce measurable lamellar cortical and trabecular bone adaptation

  17. A Mouse Model for Studying Nutritional Programming: Effects of Early Life Exposure to Soy Isoflavones on Bone and Reproductive Health.

    PubMed

    Ward, Wendy E; Kaludjerovic, Jovana; Dinsdale, Elsa C

    2016-05-11

    Over the past decade, our research group has characterized and used a mouse model to demonstrate that "nutritional programming" of bone development occurs when mice receive soy isoflavones (ISO) during the first days of life. Nutritional programming of bone development can be defined as the ability for diet during early life to set a trajectory for better or compromised bone health at adulthood. We have shown that CD-1 mice exposed to soy ISO during early neonatal life have higher bone mineral density (BMD) and greater trabecular inter-connectivity in long bones and lumbar spine at young adulthood. These skeletal sites also withstand greater forces before fracture. Because the chemical structure of ISO resembles that of 17-β-estradiol and can bind to estrogen receptors in reproductive tissues, it was prudent to expand analyses to include measures of reproductive health. This review highlights aspects of our studies in CD-1 mice to understand the early life programming effects of soy ISO on bone and reproductive health. Preclinical mouse models can provide useful data to help develop and guide the design of studies in human cohorts, which may, depending on findings and considerations of safety, lead to dietary interventions that optimize bone health.

  18. A Mouse Model for Studying Nutritional Programming: Effects of Early Life Exposure to Soy Isoflavones on Bone and Reproductive Health

    PubMed Central

    Ward, Wendy E.; Kaludjerovic, Jovana; Dinsdale, Elsa C.

    2016-01-01

    Over the past decade, our research group has characterized and used a mouse model to demonstrate that “nutritional programming” of bone development occurs when mice receive soy isoflavones (ISO) during the first days of life. Nutritional programming of bone development can be defined as the ability for diet during early life to set a trajectory for better or compromised bone health at adulthood. We have shown that CD-1 mice exposed to soy ISO during early neonatal life have higher bone mineral density (BMD) and greater trabecular inter-connectivity in long bones and lumbar spine at young adulthood. These skeletal sites also withstand greater forces before fracture. Because the chemical structure of ISO resembles that of 17-β-estradiol and can bind to estrogen receptors in reproductive tissues, it was prudent to expand analyses to include measures of reproductive health. This review highlights aspects of our studies in CD-1 mice to understand the early life programming effects of soy ISO on bone and reproductive health. Preclinical mouse models can provide useful data to help develop and guide the design of studies in human cohorts, which may, depending on findings and considerations of safety, lead to dietary interventions that optimize bone health. PMID:27187422

  19. Effects of implant neck design on primary stability and overload in a type IV mandibular bone.

    PubMed

    Chou, I-Chiang; Lee, Shyh-Yuan; Jiang, Cho-Pei

    2014-11-01

    This study investigates the effect of implant neck design on primary stability and overload using 3D finite element analysis. Four commercial dental implants and mandibular segments are created. Various parameters including the osseointegration condition (non-osseointegration and full osseointegration), force direction (vertical and horizontal), and cortical bone thickness (Tc = 0.3, 0.5, and 1 mm) are considered. The vertical and horizontal forces, 500 N and 250 N, are statically applied at the top of the platform, respectively. Micromotion and von Mises stress are employed to evaluate the risk of osseointegration and bone fatigue before osseointegration condition. After osseointegration, the principal stress is used to analyze the bone overload. Maximal von Mises stress and micromotion of the peri-implant bone decreased as cortical bone thickness increased. Horizontal force induces stress concentration in the bone around the implant neck easier than that of vertical force, and it may result in crestal bone loss. Thinner cortical bone should avoid dental implantation because it causes a noteworthy larger micromotion and stress concentration in cortical bone in particular Tc less than 0.3 mm. Copyright © 2014 John Wiley & Sons, Ltd.

  20. Culturing primary human osteoblasts on electrospun poly(lactic-co-glycolic acid) and poly(lactic-co-glycolic acid)/nanohydroxyapatite scaffolds for bone tissue engineering.

    PubMed

    Li, Mengmeng; Liu, Wenwen; Sun, Jiashu; Xianyu, Yunlei; Wang, Jidong; Zhang, Wei; Zheng, Wenfu; Huang, Deyong; Di, Shiyu; Long, Yun-Ze; Jiang, Xingyu

    2013-07-10

    In this work, we fabricated polymeric fibrous scaffolds for bone tissue engineering using primary human osteoblasts (HOB) as the model cell. By employing one simple approach, electrospinning, we produced poly(lactic-co-glycolic acid) (PLGA) scaffolds with different topographies including microspheres, beaded fibers, and uniform fibers, as well as the PLGA/nanohydroxyapatite (nano-HA) composite scaffold. The bone-bonding ability of electrospun scaffolds was investigated by using simulated body fluid (SBF) solution, and the nano-HA in PLGA/nano-HA composite scaffold can significantly enhance the formation of the bonelike apatites. Furthermore, we carried out in vitro experiments to test the performance of electrospun scaffolds by utilizing both mouse preosteoblast cell line (MC 3T3 E1) and HOB. Results including cell viability, alkaline phosphatase (ALP) activity, and osteocalcin concentration demonstrated that the PLGA/nano-HA fibers can promote the proliferation of HOB efficiently, indicating that it is a promising scaffold for human bone repair.

  1. Responsiveness of human prostate carcinoma bone tumors to interleukin-2 therapy in a mouse xenograft tumor model.

    PubMed

    Kocheril, S V; Grignon, D J; Wang, C Y; Maughan, R L; Montecillo, E J; Talati, B; Tekyi-Mensah, S; Pontes, J e; Hillman, G G

    1999-01-01

    We have tested an immunotherapy approach for the treatment of metastatic prostate carcinoma using a bone tumor model. Human PC-3 prostate carcinoma tumor cells were heterotransplanted into the femur cavity of athymic Balb/c nude mice. Tumor cells replaced marrow cells in the bone cavity, invaded adjacent bone and muscle tissues, and formed a palpable tumor at the hip joint. PC-3/IF cell lines, generated from bone tumors by serial in vivo passages, grew with faster kinetics in the femur and metastasized to inguinal lymph nodes. Established tumors were treated with systemic interleukin-2 (IL-2) injections. IL-2 significantly inhibited the formation of palpable tumors and prolonged mouse survival at nontoxic low doses. Histologically IL-2 caused vascular damage and infiltration of polymorphonuclear cells and lymphocytes in the tumor as well as necrotic areas with apoptotic cells. These findings suggest destruction of tumor cells by systemic IL-2 therapy and IL-2 responsiveness of prostate carcinoma bone tumors.

  2. Radiation sensitivity and cycling status of mouse bone marrow prothymocytes and day 8 colony forming units spleen (CFUs)

    SciTech Connect

    Boersma, W.J.

    1983-11-01

    Mouse bone marrow prothymocytes as determined in an in vivo thymus regeneration assay have an in vitro gamma radiation sensitivity which is different from that of spleen colony forming cells (CFUs). Determination of Do according to in vivo irradiation revealed similar but insignificant differences. Prothymocytes in normal bone marrow maintain a low but slightly different proliferative state as compared to CFUs, according to determinations using the /sup 3/H-TdR suicide technique. In regenerating bone marrow prothymocytes were found to be sensitive to an inhibitory effect of in vitro incubation with cold thymidine. CFUs and normal bone marrow prothymocytes were not affected by cold thymidine. Taking into account the cold thymidine effect it can be concluded that prothymocytes and CFUs in regenerating bone marrow are fully in cycle. These results are best explained when prothymocytes and CFUs are considered to be different cells.

  3. Guidelines for histopathological specimen examination and diagnostic reporting of primary bone tumours

    PubMed Central

    2011-01-01

    This review is intended to provide histopathologists with guidelines for clinical assessment, specimen handling and diagnostic reporting of benign and malignant primary bone tumours. Information from radiology, surgical, oncology and other clinical colleagues involved in the diagnosis and treatment of primary bone tumours should be properly assessed before undertaking a structured approach to specimen handling and histological reporting. This ensures that the information needed for planning appropriate treatment of these complex tumours is provided. Consistency in diagnostic evaluation with respect to both terminology and report content facilitates liaison at multidisciplinary bone tumour meetings and collaboration between cancer units and networks, as well as providing a common database for audit of the clinical, radiological and pathological aspects of bone tumours. PMID:22613930

  4. Effects of Loading Duration and Short Rest Insertion on Cancellous and Cortical Bone Adaptation in the Mouse Tibia

    PubMed Central

    Yang, Haisheng; Embry, Rachel E.; Main, Russell P.

    2017-01-01

    The skeleton’s osteogenic response to mechanical loading can be affected by loading duration and rest insertion during a series of loading events. Prior animal loading studies have shown that the cortical bone response saturates quickly and short rest insertions between load cycles can enhance cortical bone formation. However, it remains unknown how loading duration and short rest insertion affect load-induced osteogenesis in the mouse tibial compressive loading model, and particularly in cancellous bone. To address this issue, we applied cyclic loading (-9 N peak load; 4 Hz) to the tibiae of three groups of 16 week-old female C57BL/6 mice for two weeks, with a different number of continuous load cycles applied daily to each group (36, 216 and 1200). A fourth group was loaded under 216 daily load cycles with a 10 s rest insertion after every fourth cycle. We found that as few as 36 load cycles per day were able to induce osteogenic responses in both cancellous and cortical bone. Furthermore, while cortical bone area and thickness continued to increase through 1200 cycles, the incremental increase in the osteogenic response decreased as load number increased, indicating a reduced benefit of the increasing number of load cycles. In the proximal metaphyseal cancellous bone, trabecular thickness increased with load up to 216 cycles. We also found that insertion of a 10 s rest between load cycles did not improve the osteogenic response of the cortical or cancellous tissues compared to continuous loading in this model given the age and sex of the mice and the loading parameters used here. These results suggest that relatively few load cycles (e.g. 36) are sufficient to induce osteogenic responses in both cortical and cancellous bone in the mouse tibial loading model. Mechanistic studies using the mouse tibial loading model to examine bone formation and skeletal mechanobiology could be accomplished with relatively few load cycles. PMID:28076363

  5. Effects of Loading Duration and Short Rest Insertion on Cancellous and Cortical Bone Adaptation in the Mouse Tibia.

    PubMed

    Yang, Haisheng; Embry, Rachel E; Main, Russell P

    2017-01-01

    The skeleton's osteogenic response to mechanical loading can be affected by loading duration and rest insertion during a series of loading events. Prior animal loading studies have shown that the cortical bone response saturates quickly and short rest insertions between load cycles can enhance cortical bone formation. However, it remains unknown how loading duration and short rest insertion affect load-induced osteogenesis in the mouse tibial compressive loading model, and particularly in cancellous bone. To address this issue, we applied cyclic loading (-9 N peak load; 4 Hz) to the tibiae of three groups of 16 week-old female C57BL/6 mice for two weeks, with a different number of continuous load cycles applied daily to each group (36, 216 and 1200). A fourth group was loaded under 216 daily load cycles with a 10 s rest insertion after every fourth cycle. We found that as few as 36 load cycles per day were able to induce osteogenic responses in both cancellous and cortical bone. Furthermore, while cortical bone area and thickness continued to increase through 1200 cycles, the incremental increase in the osteogenic response decreased as load number increased, indicating a reduced benefit of the increasing number of load cycles. In the proximal metaphyseal cancellous bone, trabecular thickness increased with load up to 216 cycles. We also found that insertion of a 10 s rest between load cycles did not improve the osteogenic response of the cortical or cancellous tissues compared to continuous loading in this model given the age and sex of the mice and the loading parameters used here. These results suggest that relatively few load cycles (e.g. 36) are sufficient to induce osteogenic responses in both cortical and cancellous bone in the mouse tibial loading model. Mechanistic studies using the mouse tibial loading model to examine bone formation and skeletal mechanobiology could be accomplished with relatively few load cycles.

  6. Mechanical Unloading of Mouse Bone in Microgravity Significantly Alters Cell Cycle Gene Set Expression

    NASA Astrophysics Data System (ADS)

    Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Kaplan, Warren; Burns, Brnedan

    2012-07-01

    unloading in spaceflight, we conducted genome wide microarray analysis of total RNA isolated from the mouse pelvis. Specifically, 16 week old mice were subjected to 15 days spaceflight onboard NASA's STS-131 space shuttle mission. The pelvis of the mice was dissected, the bone marrow was flushed and the bones were briefly stored in RNAlater. The pelvii were then homogenized, and RNA was isolated using TRIzol. RNA concentration and quality was measured using a Nanodrop spectrometer, and 0.8% agarose gel electrophoresis. Samples of cDNA were analyzed using an Affymetrix GeneChip\\S Gene 1.0 ST (Sense Target) Array System for Mouse and GenePattern Software. We normalized the ST gene arrays using Robust Multichip Average (RMA) normalization, which summarizes perfectly matched spots on the array through the median polish algorithm, rather than normalizing according to mismatched spots. We also used Limma for statistical analysis, using the BioConductor Limma Library by Gordon Smyth, and differential expression analysis to identify genes with significant changes in expression between the two experimental conditions. Finally we used GSEApreRanked for Gene Set Enrichment Analysis (GSEA), with Kolmogorov-Smirnov style statistics to identify groups of genes that are regulated together using the t-statistics derived from Limma. Preliminary results show that 6,603 genes expressed in pelvic bone had statistically significant alterations in spaceflight compared to ground controls. These prominently included cell cycle arrest molecules p21, and p18, cell survival molecule Crbp1, and cell cycle molecules cyclin D1, and Cdk1. Additionally, GSEA results indicated alterations in molecular targets of cyclin D1 and Cdk4, senescence pathways resulting from abnormal laminin maturation, cell-cell contacts via E-cadherin, and several pathways relating to protein translation and metabolism. In total 111 gene sets out of 2,488, about 4%, showed statistically significant set alterations. These

  7. Skoog Primary Periosteoplasty versus Secondary Alveolar Bone Grafting in Unilateral Cleft Lip and Alveolus: Long-Term Effects on Alveolar Bone Formation and Maxillary Growth.

    PubMed

    Jabbari, Fatima; Hakelius, Malin M; Thor, Andreas L I; Reiser, Erika A; Skoog, Valdemar T; Nowinski, Daniel J

    2017-01-01

    Clefts involving the alveolus are treated using one of two strategies: primary periosteoplasty at the time of lip repair or secondary alveolar bone grafting at mixed dentition. Most teams favor secondary alveolar bone grafting because of its high success rate, and concerns have been raised that primary periosteoplasty may interfere with maxillary growth. However, primary periosteoplasty may obviate the need for future bone grafting and is still practiced in some centers. Few studies compare the long-term outcomes of these two strategies. Fifty-seven consecutive patients born with unilateral cleft lip and alveolus were studied retrospectively. All patients underwent primary lip repair using Skoog's method; 28 patients underwent primary periosteoplasty at the time of lip repair and the remaining 29 underwent secondary alveolar bone grafting at mixed dentition. Occlusal radiographs obtained at ages 10 and 16 years were analyzed for alveolar bone height. Cephalometric analysis assessed growth at ages 5, 10, and 18 years. Seventeen of 28 patients treated using primary periosteoplasty required later secondary bone grafting, and the bone height at age 16 years was lower in the primary periosteoplasty group (p < 0.0001). There was a more pronounced decrease in maxillary protrusion from ages 5 to 10 years in the primary periosteoplasty group (p < 0.03). However, at age 18 there was no significant difference in maxillary growth between the two groups. Primary periosteoplasty did not seem to inhibit long-term maxillary growth but was ineffective as a method of reconstructing the alveolar cleft. Therapeutic, III.

  8. Primary aneurysmal bone cyst of the petrous temporal bone: A case report and review of literature

    PubMed Central

    Sharma, Mayur; Velho, Vernon; Kharosekar, Hrushikesh

    2016-01-01

    Aneurysmal bone cyst (ABC) arising in the petrous portion of the temporal bone is a rare entity with only five such reported cases in the literature. We report the case of a 28-year-old man who presented with a tender swelling in the right preauricular region with right ear discharge and conductive hearing loss of 4 years' duration. Computed tomography and Magnetic Resonance imaging showed a destructive lesion in the right petrous bone with cavitation consistent with the diagnosis of ABC. Gross total resection of the lesion was achieved and diagnosis was confirmed histologically. The patient had no recurrence at 12 months of follow-up. This report presents the unusual location of an uncommon bony tumor with a review of its clinical, radiological, and histopathological features as well as the treatment modalities available. PMID:27695554

  9. Differential sensitivity of a mouse myeloid leukemia cell line and normal mouse bone marrow cells to X-ray-induced chromosome aberrations

    SciTech Connect

    Aardema, M.J.; Au, W.W.; Hand, R.E. Jr.; Preston, R.J.

    1985-11-01

    Cell line ML-1 was established from a myelogenous leukemia of an RFM mouse. The ML-1 cells and in vitro normal mouse bone marrow cells were analyzed to determine if there was a differential sensitivity to X-ray-induced chromosome aberrations in G1 cells and/or differences in postirradiation cell cycle progression. Cells identified as being in G1 at the time of irradiation by their staining pattern after replication in 5-bromodeoxyuridine were analyzed for all types of chromosomal aberrations following X-ray doses of 0.5, 1.0, 1.5, and 2.0 Gy. ML-1 cells showed a greater sensitivity to the induction of both chromosome-type aberrations and chromatid-type aberrations compared to normal mouse bone marrow cells, which only contained chromosome-type aberrations. The presence of chromatid-type aberrations in the ML-1 cells and not normal bone marrow cells suggested a differential progression through the cell cycle for the two cell types after irradiation. Mitotic index and flow cytometric analyses were performed and showed that both cell types have a delay in progression from G2 into mitosis, but only the normal mouse bone marrow cells have a delay in progression from G1 into S, as well as delayed progression through the S phase following X-irradiation. These results indicate that the ML-1 leukemia cells have an increased radiosensitivity. These same characteristics have been observed in ataxia telangiectasia cells and may well represent a general feature of cells with increased radiosensitivity.

  10. Cancer Cell Expression of Autotaxin Controls Bone Metastasis Formation in Mouse through Lysophosphatidic Acid-Dependent Activation of Osteoclasts

    PubMed Central

    David, Marion; Wannecq, Estelle; Descotes, Françoise; Jansen, Silvia; Deux, Blandine; Ribeiro, Johnny; Serre, Claire-Marie; Grès, Sandra; Bendriss-Vermare, Nathalie; Bollen, Mathieu; Saez, Simone; Aoki, Junken; Saulnier-Blache, Jean-Sébastien; Clézardin, Philippe; Peyruchaud, Olivier

    2010-01-01

    Background Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorbtive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models. Methodology/Principal Findings Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to inmmunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis. Conclusion/Significance Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates

  11. Cancer cell expression of autotaxin controls bone metastasis formation in mouse through lysophosphatidic acid-dependent activation of osteoclasts.

    PubMed

    David, Marion; Wannecq, Estelle; Descotes, Françoise; Jansen, Silvia; Deux, Blandine; Ribeiro, Johnny; Serre, Claire-Marie; Grès, Sandra; Bendriss-Vermare, Nathalie; Bollen, Mathieu; Saez, Simone; Aoki, Junken; Saulnier-Blache, Jean-Sébastien; Clézardin, Philippe; Peyruchaud, Olivier

    2010-03-17

    Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorptive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models. Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to immunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis. Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and osteoclast

  12. An in vivo mouse model of primary dysmenorrhea

    PubMed Central

    YANG, Lu; CAO, Zhengyu; YU, Boyang; CHAI, Chengzhi

    2015-01-01

    Primary dysmenorrhea (PD) is a common gynecological disorder. Hitherto, animal models which recapitulate clinical features of PD have not been fully established. We aimed to examine whether a pain model in mice could mimic the clinic features of PD. After pretreated with estradiol benzoate (1 mg/kg/day) intraperitoneally (i.p.) for 3 consecutive days, non-pregnant female Imprinting Control Region mice (6–8 weeks old) was injected with 0.4 U of oxytocin to induce the stretching or writhing response which was recorded for a time period of 30 min. During the writhing period, the uterine artery blood flow alterations were examined by Doppler ultrasound detection. After writhing test, the uterine morphological changes were observed by hematoxylin and eosin (H&E) staining histopathology. In addition, enzyme-linked immunosorbent assay kit was used to measure the levels of prostaglandins F2α/prostaglandins E2 (PGF2α/PGE2) and TXB2 (a metabolite of TXA2)/6-keto-PGF1α (a metabolite of PGI2) in the uterine tissue homogenates and plasma, respectively. Western blot analyses were performed to determine the expressions of oxytocin receptor (OTR), beta2-adrenergic receptor (beta2-AR), and cyclooxygenase-2 (COX-2) in uterine, which are responsible for the uterine contraction. The writhing response only occurred in the estrogen pretreated female mice. The area of uterine myometrium significantly decreased along with the increased thickness in the oxytocin-induced estrogen pretreated mice model. The uterine artery blood flow velocity dropped, while the pulsatility index and resistance index slightly increased after the injection of oxytocin. The PGF2α/PGE2 level significantly increased and the plasma TXB2/6-keto-PGF1α level significantly enhanced. Compared with the control group, the uterine histopathology demonstrated moderate to severe edema of endometrium lamina propria. In consistent with the uterine morphological changes, a significant reduction of beta2-AR and a

  13. An in vivo mouse model of primary dysmenorrhea.

    PubMed

    Yang, Lu; Cao, Zhengyu; Yu, Boyang; Chai, Chengzhi

    2015-01-01

    Primary dysmenorrhea (PD) is a common gynecological disorder. Hitherto, animal models which recapitulate clinical features of PD have not been fully established. We aimed to examine whether a pain model in mice could mimic the clinic features of PD. After pretreated with estradiol benzoate (1 mg/kg/day) intraperitoneally (i.p.) for 3 consecutive days, non-pregnant female Imprinting Control Region mice (6-8 weeks old) was injected with 0.4 U of oxytocin to induce the stretching or writhing response which was recorded for a time period of 30 min. During the writhing period, the uterine artery blood flow alterations were examined by Doppler ultrasound detection. After writhing test, the uterine morphological changes were observed by hematoxylin and eosin (H&E) staining histopathology. In addition, enzyme-linked immunosorbent assay kit was used to measure the levels of prostaglandins F2α/prostaglandins E2 (PGF2α/PGE2) and TXB2 (a metabolite of TXA2)/6-keto-PGF1α (a metabolite of PGI2) in the uterine tissue homogenates and plasma, respectively. Western blot analyses were performed to determine the expressions of oxytocin receptor (OTR), beta2-adrenergic receptor (beta2-AR), and cyclooxygenase-2 (COX-2) in uterine, which are responsible for the uterine contraction. The writhing response only occurred in the estrogen pretreated female mice. The area of uterine myometrium significantly decreased along with the increased thickness in the oxytocin-induced estrogen pretreated mice model. The uterine artery blood flow velocity dropped, while the pulsatility index and resistance index slightly increased after the injection of oxytocin. The PGF2α/PGE2 level significantly increased and the plasma TXB2/6-keto-PGF1α level significantly enhanced. Compared with the control group, the uterine histopathology demonstrated moderate to severe edema of endometrium lamina propria. In consistent with the uterine morphological changes, a significant reduction of beta2-AR and a

  14. PULSED FOCUSED ULTRASOUND TREATMENT OF MUSCLE MITIGATES PARALYSIS-INDUCED BONE LOSS IN THE ADJACENT BONE: A STUDY IN A MOUSE MODEL

    PubMed Central

    Poliachik, Sandra L.; Khokhlova, Tatiana D.; Wang, Yak-Nam; Simon, Julianna C.; Bailey, Michael R.

    2015-01-01

    Bone loss can result from bed rest, space flight, spinal cord injury or age-related hormonal changes. Current bone loss mitigation techniques include pharmaceutical interventions, exercise, pulsed ultrasound targeted to bone and whole body vibration. In this study, we attempted to mitigate paralysis-induced bone loss by applying focused ultrasound to the midbelly of a paralyzed muscle. We employed a mouse model of disuse that uses onabotulinumtoxinA-induced paralysis, which causes rapid bone loss in 5 d. A focused 2 MHz transducer applied pulsed exposures with pulse repetition frequency mimicking that of motor neuron firing during walking (80 Hz), standing (20 Hz), or the standard pulsed ultrasound frequency used in fracture healing (1 kHz). Exposures were applied daily to calf muscle for 4 consecutive d. Trabecular bone changes were characterized using micro-computed tomography. Our results indicated that application of certain focused pulsed ultrasound parameters was able to mitigate some of the paralysis-induced bone loss. PMID:24857416

  15. Knockdown of toll-like receptor 4 signaling pathways ameliorate bone graft rejection in a mouse model of allograft transplantation

    PubMed Central

    Hsieh, Jeng-Long; Shen, Po-Chuan; Wu, Po-Ting; Jou, I-Ming; Wu, Chao-Liang; Shiau, Ai-Li; Wang, Chrong-Reen; Chong, Hao-Earn; Chuang, Shu-Han; Peng, Jia-Shiou; Chen, Shih-Yao

    2017-01-01

    Non-union occurring in structural bone grafting is a major problem in allograft transplantation because of impaired interaction between the host and graft tissue. Activated toll-like receptor (TLR) induces inflammatory cytokines and chemokines and triggers cell-mediated immune responses. The TLR-mediated signal pathway is important for mediating allograft rejection. We evaluated the effects of local knockdown of the TLR4 signaling pathway in a mouse segmental femoral graft model. Allografts were coated with freeze-dried lentiviral vectors that encoded TLR4 and myeloid differentiation primary response gene 88 (MyD88) short-hairpin RNA (shRNA), which were individually transplanted into the mice. They were assessed morphologically, radiographically, and histologically for tissue remodeling. Union occurred in autografted but not in allografted mice at the graft and host junctions after 4 weeks. TLR4 and MyD88 expression was up-regulated in allografted mice. TLR4 and MyD88 shRNAs inhibited TLR4 and MyD88 expression, which led to better union in the grafted sites. More regulatory T-cells in the draining lymph nodes suggested inflammation suppression. Local inhibition of TLR4 and MyD88 might reduce immune responses and ameliorate allograft rejection. PMID:28393847

  16. Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan

    PubMed Central

    Cuong, Dang Van; Kim, Hyoung Kyu; Marquez, Jubert; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo

    2016-01-01

    Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 µg/ml BG, 100 µg/ml peptidoglycan (PGN), or 10 µM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation. PMID:26937218

  17. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    SciTech Connect

    Kawahara, Takeshi

    2012-11-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed {alpha}, {beta}, and {gamma} subunits of high-affinity immunoglobulin E (IgE) receptor (Fc{epsilon}RI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-{alpha}, and cyclooxygenase 2, and production of prostaglandin D{sub 2} and leukotriene C{sub 4} in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-{alpha} expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on Fc{epsilon}RI- and TLR-mediated effector functions of mast cells.

  18. Cranial irradiation induces bone marrow-derived microglia in adult mouse brain tissue.

    PubMed

    Okonogi, Noriyuki; Nakamura, Kazuhiro; Suzuki, Yoshiyuki; Suto, Nana; Suzue, Kazutomo; Kaminuma, Takuya; Nakano, Takashi; Hirai, Hirokazu

    2014-07-01

    Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV-GFP mice, aged 8-12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

  19. Ewing's sarcoma of bone tumor cells produces MCSF that stimulates monocyte proliferation in a novel mouse model of Ewing's sarcoma of bone.

    PubMed

    Margulies, B S; DeBoyace, S D; Damron, T A; Allen, M J

    2015-10-01

    Ewing's sarcoma of bone is a primary childhood malignancy of bone that is treated with X-radiation therapy in combination with surgical excision and chemotherapy. To better study Ewing's sarcoma of bone we developed a novel model of primary Ewing's sarcoma of bone and then treated animals with X-radiation therapy. We identified that uncontrolled tumor resulted in lytic bone destruction while X-radiation therapy decreased lytic bone destruction and increased limb-length asymmetry, a common, crippling complication of X-radiation therapy. Osteoclasts were indentified adjacent to the tumor, however, we were unable to detect RANK-ligand in the Ewing's tumor cells in vitro, which lead us to investigate alternate mechanisms for osteoclast formation. Ewing's sarcoma tumor cells and archival Ewing's sarcoma of bone tumor biopsy samples were shown to express MCSF, which could promote osteoclast formation. Increased monocyte numbers were detected in peripheral blood and spleen in animals with untreated Ewing's sarcoma tumor while monocyte number in animals treated with x-radiation had normal numbers of monocytes. Our data suggest that our Ewing's sarcoma of bone model will be useful in the study Ewing's sarcoma tumor progression in parallel with the effects of chemotherapy and X-radiation therapy.

  20. Ewing's Sarcoma of Bone Tumor Cells Produce MCSF that Stimulates Monocyte Proliferation in a Novel Mouse Model of Ewing's Sarcoma of Bone

    PubMed Central

    Margulies, BS; DeBoyace, SD; Damron, TA; Allen, MJ

    2015-01-01

    Ewing's sarcoma of bone is a primary childhood malignancy of bone that is treated with X-radiation therapy in combination with surgical excision and chemotherapy. To better study Ewing's sarcoma of bone we developed a novel model of primary Ewing's sarcoma of bone and then treated animals with X-radiation therapy. We identified that uncontrolled tumor resulted in lytic bone destruction while X-radiation therapy decreased lytic bone destruction and increased limb-length asymmetry, a common, crippling complication of X-radiation therapy. Osteoclasts were indentified adjacent to the tumor, however, we were unable to detect RANK-ligand in the Ewing's tumor cells in vitro, which lead us to investigate alternate mechanisms for osteoclast formation. Ewing's sarcoma tumor cells and archival Ewing's sarcoma of bone tumor biopsy samples were shown to express MCSF, which could promote osteoclast formation. Increased monocyte numbers were detected in peripheral blood and spleen in animals with untreated Ewing's sarcoma tumor while monocyte number in animals treated with x-radiation had normal numbers of monocytes. Our data suggest that our Ewing's sarcoma of bone model will be useful in the study Ewing's sarcoma tumor progression in parallel with the effects of chemotherapy and X-radiation therapy. PMID:26051470

  1. Hypomorphic mutation in mouse Nppc gene causes retarded bone growth due to impaired endochondral ossification

    SciTech Connect

    Tsuji, Takehito Kondo, Eri; Yasoda, Akihiro; Inamoto, Masataka; Kiyosu, Chiyo; Nakao, Kazuwa; Kunieda, Tetsuo

    2008-11-07

    Long bone abnormality (lbab/lbab) is a spontaneous mutant mouse characterized by dwarfism with shorter long bones. A missense mutation was reported in the Nppc gene, which encodes C-type natriuretic peptide (CNP), but it has not been confirmed whether this mutation is responsible for the dwarf phenotype. To verify that the mutation causes the dwarfism of lbab/lbab mice, we first investigated the effect of CNP in lbab/lbab mice. By transgenic rescue with chondrocyte-specific expression of CNP, the dwarf phenotype in lbab/lbab mice was completely compensated. Next, we revealed that CNP derived from the lbab allele retained only slight activity to induce cGMP production through its receptor. Histological analysis showed that both proliferative and hypertrophic zones of chondrocytes in the growth plate of lbab/lbab mice were markedly reduced. Our results demonstrate that lbab/lbab mice have a hypomorphic mutation in the Nppc gene that is responsible for dwarfism caused by impaired endochondral ossification.

  2. Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

    PubMed Central

    Hosogane, Naobumi; Huang, Zhiping; Rawlins, Bernard A.; Liu, Xia; Boachie-Adjei, Oheneba; Boskey, Adele L.; Zhu, Wei

    2010-01-01

    Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations. PMID:20362069

  3. Tooth-bone morphogenesis during postnatal stages of mouse first molar development.

    PubMed

    Lungová, Vlasta; Radlanski, Ralf J; Tucker, Abigail S; Renz, Herbert; Míšek, Ivan; Matalová, Eva

    2011-06-01

    The first mouse molar (M1) is the most common model for odontogenesis, with research particularly focused on prenatal development. However, the functional dentition forms postnatally, when the histogenesis and morphogenesis of the tooth is completed, the roots form and the tooth physically anchors into the jaw. In this work, M1 was studied from birth to eruption, assessing morphogenesis, proliferation and apoptosis, and correlating these with remodeling of the surrounding bony tissue. The M1 completed crown formation between postnatal (P) days 0-2, and the development of the tooth root was initiated at P4. From P2 until P12, cell proliferation in the dental epithelium reduced and shifted downward to the apical region of the forming root. In contrast, proliferation was maintained or increased in the mesenchymal cells of the dental follicle. At later stages, before tooth eruption (P20), cell proliferation suddenly ceased. This withdrawal from the cell cycle correlated with tooth mineralization and mesenchymal differentiation. Apoptosis was observed during all stages of M1 postnatal morphogenesis, playing a role in the removal of cells such as osteoblasts in the mandibular region and working together with osteoclasts to remodel the bone around the developing tooth. At more advanced developmental stages, apoptotic cells and bodies accumulated in the cell layers above the tooth cusps, in the path of eruption. Three-dimensional reconstruction of the developing postnatal tooth and bone indicates that the alveolar crypts form by resorption underneath the primordia, whereas the ridges form by active bone growth between the teeth and roots to form a functional complex.

  4. Tooth-bone morphogenesis during postnatal stages of mouse first molar development

    PubMed Central

    Lungová, Vlasta; Radlanski, Ralf J; Tucker, Abigail S; Renz, Herbert; Míšek, Ivan; Matalová, Eva

    2011-01-01

    The first mouse molar (M1) is the most common model for odontogenesis, with research particularly focused on prenatal development. However, the functional dentition forms postnatally, when the histogenesis and morphogenesis of the tooth is completed, the roots form and the tooth physically anchors into the jaw. In this work, M1 was studied from birth to eruption, assessing morphogenesis, proliferation and apoptosis, and correlating these with remodeling of the surrounding bony tissue. The M1 completed crown formation between postnatal (P) days 0–2, and the development of the tooth root was initiated at P4. From P2 until P12, cell proliferation in the dental epithelium reduced and shifted downward to the apical region of the forming root. In contrast, proliferation was maintained or increased in the mesenchymal cells of the dental follicle. At later stages, before tooth eruption (P20), cell proliferation suddenly ceased. This withdrawal from the cell cycle correlated with tooth mineralization and mesenchymal differentiation. Apoptosis was observed during all stages of M1 postnatal morphogenesis, playing a role in the removal of cells such as osteoblasts in the mandibular region and working together with osteoclasts to remodel the bone around the developing tooth. At more advanced developmental stages, apoptotic cells and bodies accumulated in the cell layers above the tooth cusps, in the path of eruption. Three-dimensional reconstruction of the developing postnatal tooth and bone indicates that the alveolar crypts form by resorption underneath the primordia, whereas the ridges form by active bone growth between the teeth and roots to form a functional complex. PMID:21418206

  5. Raman spectroscopy detects deterioration in biomechanical properties of bone in a glucocorticoid-treated mouse model of rheumatoid arthritis

    NASA Astrophysics Data System (ADS)

    Maher, Jason R.; Takahata, Masahiko; Awad, Hani A.; Berger, Andrew J.

    2011-08-01

    Although glucocorticoids are frequently prescribed for the symptomatic management of inflammatory disorders such as rheumatoid arthritis, extended glucocorticoid exposure is the leading cause of physician-induced osteoporosis and leaves patients at a high risk of fracture. To study the biochemical effects of glucocorticoid exposure and how they might affect biomechanical properties of the bone, Raman spectra were acquired from ex vivo tibiae of glucocorticoid- and placebo-treated wild-type mice and a transgenic mouse model of rheumatoid arthritis. Statistically significant spectral differences were observed due to both treatment regimen and mouse genotype. These differences are attributed to changes in the overall bone mineral composition, as well as the degree of phosphate mineralization in tibial cortical bone. In addition, partial least squares regression was used to generate a Raman-based prediction of each tibia's biomechanical strength as quantified by a torsion test. The Raman-based predictions were as accurate as those produced by microcomputed tomography derived parameters, and more accurate than the clinically-used parameter of bone mineral density. These results suggest that Raman spectroscopy could be a valuable tool for monitoring bone biochemistry in studies of bone diseases such as osteoporosis, including tests of drugs being developed to combat these diseases.

  6. Primary hyperparathyroidism presenting with acute pancreatitis and asymptomatic bone involvement

    PubMed Central

    Saif, Aasem

    2015-01-01

    Summary A 15-year-old female patient presented to the emergency room with vomiting and abdominal pain. She had two similar attacks in the previous three months both of them were diagnosed as pancreatitis in two different hospitals. On admission, her serum calcium and parathyroid hormone levels were very high. CT scan revealed left inferior parathyroid adenoma. Investigations to rule out possible multiple endocrine neoplasia were all negative. The patient was managed by intravenous fluids and furosemide to lower her serum calcium level. Then, left inferior parathyroidectomy was done. Postoperatively, the patient had hungry bone syndrome with severe hypocalcaemia and was managed by intravenous calcium infusion for five days in the intensive care unit. Later, she was kept on oral calcium and vitamin D supplementation. She became symptom-free and her serum calcium improved gradually. PMID:26604950

  7. Primary bone microanatomy records developmental aspects of life history in catarrhine primates.

    PubMed

    McFarlin, Shannon C; Terranova, Carl J; Zihlman, Adrienne L; Bromage, Timothy G

    2016-03-01

    A central challenge in human origins research is to understand how evolution has shaped modern human life history. As fossilized remains of our ancestors provide the only direct evidence for life history evolution, efforts to reconstruct life history in paleontological contexts have focused on hard tissues, particularly on dental development. However, among investigators of other vertebrate groups, there is a long tradition of examining primary bone microstructure to decipher growth rates and maturational timing, based on an empirical relationship between the microanatomy of primary bone and the rate at which it is deposited. We examined ontogenetic variation in primary bone microstructure at the midshaft femur of Chlorocebus aethiops, Hylobates lar, and Pan troglodytes to test whether tissue type proportions vary in accordance with predictions based on body mass growth patterns described previously. In all taxa, younger age classes were characterized by significantly higher percent areas of fibro-lamellar and/or parallel-fibered tissues, while older age classes showed significantly higher proportions of lamellar bone. In prior experimental studies, fibro-lamellar and parallel-fibered tissue types have been associated with faster depositional rates than lamellar bone. Principal components analysis revealed differences among taxa in the timing of this transition, and in the particular tissue types observed among individuals of similar dental emergence status. Among M1 and M2 age classes, higher proportions of parallel-fibered and fibro-lamellar tissues were observed in those taxa characterized by reportedly faster body mass growth rates. Further, persistence of fibro-lamellar tissue throughout DECID, M1 and M2 age classes in chimpanzees contrasts with the pattern reported previously for modern humans. Despite the necessary limitations of our cross-sectional study design and the secondary remodeling of bone in primates, large areas of primary bone remain intact and

  8. Implant osseointegration in the absence of primary bone anchorage: a clinical report.

    PubMed

    Villa, Roberto; Polimeni, Giuseppe; Wikesjö, Ulf M E

    2010-11-01

    The authors identified no report describing implant primary stability obtained by external fixation as a means to achieve osseointegration in craniofacial settings. This article describes a situation in which an implant was placed without direct contact with the resident bone; primary stability was provided by an external device. An edentulous patient was restored with 5 endosseous titanium implants to support a mandibular fixed prosthesis. An implant placed in the right central incisor position was removed after 48 hours and replaced with a shorter and narrower implant without contact with resident bone. Thus, primary stability for the implant was provided by rigid fixation to the prosthesis rather than by bone anchorage. At recall examinations after 6 and 27 months, all implants, including the implant in the right central incisor position, showed clinical and radiographic signs of osseointegration. Resonance frequency analysis indicated acceptable stability and osseointegration for all implants. Observations of this patient suggest that implant osseointegration can be achieved by providing primary stability using a fixed complete denture. Primary bone anchorage/contact does not appear to be critical to the osseointegration process. Copyright © 2010 The Editorial Council of the Journal of Prosthetic Dentistry. Published by Mosby, Inc. All rights reserved.

  9. Resonance in the Mouse Tibia as a Predictor of Frequencies and Locations of Loading-Induced Bone Formation

    PubMed Central

    Zhao, Liming; Dodge, Todd; Nemani, Arun; Yokota, Hiroki

    2013-01-01

    To enhance new bone formation for the treating of patients with osteopenia and osteoporosis, various mechanical loading regimens have been developed. Although a wide spectrum of loading frequencies is proposed in those regimens, a potential linkage between loading frequencies and locations of loading-induced bone formation is not well understood. In this study, we addressed a question: Does mechanical resonance play a role in frequency dependent bone formation? If so, can the locations of enhanced bone formation be predicted through the modes of vibration? Our hypothesis is that mechanical loads applied at a frequency near the resonant frequencies enhance bone formation, specifically in areas that experience high principal strains. To test the hypothesis, we conducted axial tibia loading using low, medium, or high frequency to the mouse tibia, as well as finite element analysis. The experimental data demonstrated dependence of the maximum bone formation on location and frequency of loading. Samples loaded with the low frequency waveform exhibited peak enhancement of bone formation in the proximal tibia, while the high frequency waveform offered the greatest enhancement in the midshaft and distal sections. Furthermore, the observed dependence on loading frequencies was correlated to the principal strains in the first five resonance modes at 8.0 to 42.9 Hz. Collectively, the results suggest that resonance is a contributor to the frequencies and locations of maximum bone formation. Further investigation of the observed effects of resonance may lead to the prescribing of personalized mechanical loading treatments. PMID:23575747

  10. Role of aquaporin 9 in cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes

    SciTech Connect

    Shinkai, Yasuhiro; Sumi, Daigo; Toyama, Takashi; Kaji, Toshiyuki; Kumagai, Yoshito

    2009-06-01

    Aquaporin (AQP) 9 is a member of the aquaglyceroporin subfamily of AQPs in the transfer of water and small solutes such as glycerol and arsenite. It is well recognized that arsenic toxicity is associated with intracellular accumulation of this metalloid. In the present study, we examined the contribution of AQP9 to the uptake of inorganic arsenite, thereby increasing arsenic-induced cytotoxicity in primary mouse hepatocytes. Pretreatment with sorbitol as a competitive inhibitor of AQP9 and siRNA-mediated knockdown of AQP9 resulted in a significant decrease of arsenite uptake in the cell and its cytotoxicity. Furthermore, overexpression of AQP9 in HEK293 cells led to the enhancement of intracellular arsenic concentration, resulting in enhanced cytotoxicity after arsenite exposure. These results suggest that AQP9 is a channel to define arsenite sensitivity in primary mouse hepatocytes.

  11. Cholera toxin-stimulated bone resorption in cultured mouse calvarial bones not inhibited by calcitonin: a possible interaction at the stimulatory G protein.

    PubMed

    Ransjö, M; Lerner, U H; Ljunggren, O

    1994-12-01

    We examined the effect of calcitonin in cultured mouse calvarial bones after prestimulation with different activators of adenylyl cyclase. Calcitonin (100 ng/ml), added after 48 h of culture, inhibited bone resorption (assessed as release of 45Ca from prelabeled bones cultured for 96-144 h) stimulated with parathyroid hormone (PTH, 10 nM; 0-144 h) or the adenylyl cyclase stimulator forskolin (2 microM; 0-144 h). However, no effect of calcitonin was demonstrated when bone resorption was prestimulated with the adenylyl cyclase stimulator cholera toxin, at and above 1 ng/ml, at any time point studied. In contrast, two other types of inhibitors of bone resorption in vitro, the carbonic anhydrase inhibitor acetazolamide (10 microM) and the aminobisphosphonate AHPrBP (10 microM), significantly inhibited cholera toxin-stimulated bone resorption. No cyclic AMP response to calcitonin was seen after preculture for 48 h with cholera toxin (0.1-100 ng/ml), although bones precultured in basic medium, in the absence or presence of forskolin, were still able to respond to calcitonin with elevation of cyclic AMP. Binding studies with [125I]calcitonin demonstrated that the preculture with cholera toxin did not affect the binding of calcitonin to the receptor. In summary, our data show that cholera toxin pretreatment makes calvarial bones insensitive to calcitonin-induced inhibition of bone resorption as a result of an interaction with cholera toxin at the level of calcitonin receptor-linked signal transduction. We suggest that the interaction, distal to the calcitonin receptor, is caused by the irreversible activation of Gs produced by cholera toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Measurement of mitochondrial oxygen consumption rates in mouse primary neurons and astrocytes.

    PubMed

    Ribeiro, Sofia M; Giménez-Cassina, Alfredo; Danial, Nika N

    2015-01-01

    The introduction of microplate-based assays that measure extracellular fluxes in intact, living cells has revolutionized the field of cellular bioenergetics. Here, we describe a method for real time assessment of mitochondrial oxygen consumption rates in primary mouse cortical neurons and astrocytes. This method requires the Extracellular Flux Analyzer Instrument (XF24, Seahorse Biosciences), which uses fluorescent oxygen sensors in a microplate assay format.

  13. Defective Endochondral Ossification-Derived Matrix and Bone Cells Alter the Lymphopoietic Niche in Collagen X Mouse Models

    PubMed Central

    Sweeney, Elizabeth; Roberts, Douglas; Lin, Angela; Guldberg, Robert

    2013-01-01

    Despite the appreciated interdependence of skeletal and hematopoietic development, the cell and matrix components of the hematopoietic niche remain to be fully defined. Utilizing mice with disrupted function of collagen X (ColX), a major hypertrophic cartilage matrix protein associated with endochondral ossification, our data identified a cytokine defect in trabecular bone cells at the chondro-osseous hematopoietic niche as a cause for aberrant B lymphopoiesis in these mice. Specifically, analysis of ColX transgenic and null mouse chondro-osseous regions via micro-computed tomography revealed an altered trabecular bone environment. Additionally, cocultures with hematopoietic and chondro-osseous cell types highlighted impaired hematopoietic support by ColX transgenic and null mouse derived trabecular bone cells. Further, cytokine arrays with conditioned media from the trabecular osteoblast cocultures suggested an aberrant hematopoietic cytokine milieu within the chondro-osseous niche of the ColX deficient mice. Accordingly, B lymphopoiesis was rescued in the ColX mouse derived trabecular osteoblast cocultures with interlukin-7, stem cell factor, and stromal derived factor-1 supplementation. Moreover, B cell development was restored in vivo after injections of interlukin-7. These data support our hypothesis that endrochondrally-derived trabecular bone cells and matrix constituents provide cytokine-rich niches for hematopoiesis. Furthermore, this study contributes to the emerging concept that niche defects may underlie certain immuno-osseous and hematopoietic disorders. PMID:23656481

  14. Haematopoietic progenitor cells from adult bone marrow differentiate into cells that express oligodendroglial antigens in the neonatal mouse brain.

    PubMed

    Bonilla, Sonia; Alarcón, Pedro; Villaverde, Ramón; Aparicio, Pedro; Silva, Augusto; Martínez, Salvador

    2002-02-01

    Stem cells are self-renewable, pluripotent cells that, in adult life, proliferate by a characteristic asymmetric division in which one daughter cell is committed to differentiation whereas the other remains a stem cell. These cells are also characterized by their ability to differentiate into various cell types under heterotopic environmental influences. In the present study, we have explored the potential of adult haematopoietic bone marrow cells to differentiate into cells of oligodendroglial lineage under physiological, active myelinating conditions. We present evidence of generation of cells expressing oligodendroglial specific markers from a bone marrow subpopulation enriched on adult haematopoietic progenitor cells (CD117+) in vivo after intracerebral transplantation into the neonatal mouse brain. Our results suggest that adult bone marrow cells have the capacity to undergo differentiation from haematopoietic to oligodendroglial cells and add support the validity of bone marrow transplants as an alternative treatment for demyelinating diseases of the CNS including Multiple Sclerosis.

  15. Rare case study of a primary carcinoma of the petrous bone and a brief literature review.

    PubMed

    Atallah, Ihab; Karkas, Alexandre; Righini, Christian Adrien; Lantuejoul, Sylvie; Schmerber, Sébastien

    2015-04-01

    Temporal bone carcinoma is an aggressive tumor with multiple unconfirmed risk factors. Herein, we present a rare case of a primary petrous bone carcinoma in a female patient (65 years old) with an irrelevant medical history. She presented a postauricular swelling that revealed a multilocular osteolytic cystic lesion of the mastoid portion of the temporal bone on the CT scan. The patient underwent resection of the lesion and pathological analysis revealed moderately to well-differentiated squamous cell carcinoma. Postoperative radiotherapy was carried out. Until the present time, the patient shows complete remission on regular clinical and radiological follow-up. Although no widely accepted strategy for managing temporal bone tumors exists, a review of the literature showed that surgery with or without radiotherapy is the treatment of choice. Preoperative assessment and accurate staging are vital in ensuring that the treatment is adequate for each disease stage. © 2014 Wiley Periodicals, Inc.

  16. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    SciTech Connect

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    2009-10-30

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  17. Cancellous impaction bone grafting of acetabular defects in complex primary and revision total hip arthroplasty.

    PubMed

    Patil, Nilesh; Hwang, Katherine; Goodman, Stuart B

    2012-03-07

    The reconstruction of major acetabular bone defects during revision, conversion, and primary total hip arthroplasties (THAs) is challenging. We reviewed a consecutive series of 168 THAs (108 revisions, 8 conversions, and 52 primary THAs) performed by 1 surgeon (S.B.G.) between 1997 and 2008 using impaction bone grafting for acetabular reconstruction. Autograft, cancellous allograft croutons, and demineralized bone matrix were used to fill bone defects as needed. The acetabular bone deficiency was classified according to the American Academy of Orthopaedic Surgeons: type I, segmental deficiency with significant rim defect; type II, cavitary defects medially or posteriorly; type III, combined cavitary and segmental deficiency; type IV, pelvic discontinuity; and type V, arthrodesis. According to this method, 56 hips had type I, 31 hips had type II, 48 hips had type III, and 27 hips had type IV deficiencies. Of the 168 patients, 19 subsequently died of causes unrelated to the THA, and 11 were lost to follow-up. All patients had at least 2 years of follow-up. Average Harris Hip Score improved from 45.5±17.9 preoperatively to 81.1±16.5 postoperatively (P<.05) for revision THAs, from 40.0±11.3 preoperatively to 85.0±12.8 postoperatively (P<.05) for conversion THAs, and from 42.3±14.9 preoperatively to 85.0±12.0 postoperatively (P<.05) for primary THAs. All impaction grafted bone (allograft, autograft, or a combination) incorporated radiographically, thus restoring bone stock. Complications included 1 early infection, which was managed successfully with debridement and liner exchange, and 2 late infections that were managed successfully with staged revision. Two revisions required subsequent re-revision for late loosening. Two hip dislocations occurred, 1 of which required surgical treatment to place a constrained liner. Copyright 2012, SLACK Incorporated.

  18. Vaccination with DKK1-derived peptides promotes bone formation and bone mass in an aged mouse osteoporosis model.

    PubMed

    Wu, Qiong; Li, Rui-Shu; Zhao, Yue; Wang, Zhi-Xia; Tang, Yan-Chun; Zhang, Jing; Liu, Jian-Ning; Tan, Xiang-Yang

    2014-08-01

    The investigation of agents for the treatment of osteoporosis has been a long-standing effort. The Wnt pathway plays an important role in bone formation and regeneration, and expression of Wnt pathway inhibitors, Dickkopf-1 (DKK1), appears to be associated with changes in bone mass. Inactivation of DKK1 leads to substantially increased bone mass in genetically manipulated animals. DKK1-derived peptides (DDPs) were added to BMP2-stimulated MC3T3-E1 preosteoblastic cells in vitro to evaluate inhibitory activity of DDPs in MC3T3-E1 cell differentiation. Study was extended in vivo on old female mice to show whether or not inhibition of endogenous DKK1 biological activity using DDPs vaccination approach leads to increase of bone formation, bone density, and improvement of bone microstructure. We reported that synthetic DDPs were able to reduce alkaline phosphatase activity, prevent mineralization and inhibit the differentiation of MC3T3-E1 cells in vitro. Furthermore, vaccination with these DDPs in aged female mice 4 times for a total period of 22 weeks promoted bone mass and bone microstructure. 3D microCT and histomorphometric analysis showed that there were significant increase in bone mineral densities, improvement of bone microstructure and promotion of bone formation in the vaccinated mice, especially in the mice vaccinated with DDP-A and DDP-C. Histological and scanning electron microscopy image analysis also indicated that vaccination increased trabecular bone mass and significantly decreased fragmentation of bone fibers. Taken together, these preclinical results suggest that vaccination with DDPs represents a promising new therapeutic approach for the treatment of bone-related disorders, such as osteoporosis.

  19. β-Caryophyllene promotes osteoblastic mineralization, and suppresses osteoclastogenesis and adipogenesis in mouse bone marrow cultures in vitro

    PubMed Central

    Yamaguchi, Masayoshi; Levy, Robert M.

    2016-01-01

    Osteoporosis is induced by the reduction in bone mass through decreased osteoblastic osteogenesis and increased osteoclastic bone resorption, and it is associated with obesity and diabetes. Osteoblasts and adipocytes are derived from bone marrow mesenchymal stem cells. The prevention of osteoporosis is an important public health concern in aging populations. β-caryophyllene, a component of various essential oils, is a selective agonist of the cannabinoid receptor type 2 and exerts cannabimimetic anti-inflammatory effects in animals. The present study aimed to identify the effect of β-caryophyllene on adipogenesis, osteoblastic mineralization and osteoclastogenesis in mouse bone marrow cell cultures in vitro. Bone marrow cells obtained from mouse femoral tissues were cultured in the presence of β-caryophyllene (0.1–100 µM) in vitro. The results revealed that β-caryophyllene stimulated osteoblastic mineralization, and suppressed adipogenesis and osteoclastogenesis. Thus, β-caryophyllene may be used as a therapeutic agent for the prevention and treatment of osteoporosis. PMID:28105093

  20. Sandwich-type PLLA-nanosheets loaded with BMP-2 induce bone regeneration in critical-sized mouse calvarial defects.

    PubMed

    Huang, Kuo-Chin; Yano, Fumiko; Murahashi, Yasutaka; Takano, Shuta; Kitaura, Yoshiaki; Chang, Song Ho; Soma, Kazuhito; Ueng, Steve W N; Tanaka, Sakae; Ishihara, Kazuhiko; Okamura, Yosuke; Moro, Toru; Saito, Taku

    2017-09-01

    To overcome serious clinical problems caused by large bone defects, various approaches to bone regeneration have been researched, including tissue engineering, biomaterials, stem cells and drug screening. Previously, we developed a free-standing biodegradable polymer nanosheet composed of poly(L-lactic acid) (PLLA) using a simple fabrication process consisting of spin-coating and peeling techniques. Here, we loaded recombinant human bone morphogenetic protein-2 (rhBMP-2) between two 60-nm-thick PLLA nanosheets, and investigated these sandwich-type nanosheets in bone regeneration applications. The PLLA nanosheets displayed constant and sustained release of the loaded rhBMP-2 for over 2months in vitro. Moreover, we implanted the sandwich-type nanosheets with or without rhBMP-2 into critical-sized defects in mouse calvariae. Bone regeneration was evident 4weeks after implantation, and the size and robustness of the regenerated bone had increased by 8weeks after implantation in mice implanted with the rhBMP-2-loaded nanosheets, whereas no significant bone formation occurred over a period of 20weeks in mice implanted with blank nanosheets. The PLLA nanosheets loaded with rhBMP-2 may be useful in bone regenerative medicine; furthermore, the sandwich-type PLLA nanosheet structure may potentially be applied as a potent prolonged sustained-release carrier of other molecules or drugs. Here we describe sandwich-type poly(L-lactic acid) (PLLA) nanosheets loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) as a novel method for bone regeneration. Biodegradable 60-nm-thick PLLA nanosheets display strong adhesion without any adhesive agent. The sandwich-type PLLA nanosheets displayed constant and sustained release of the loaded rhBMP-2 for over 2months in vitro. The nanosheets with rhBMP-2 markedly enhanced bone regeneration when they were implanted into critical-sized defects in mouse calvariae. In addition to their application for bone regeneration, PLLA

  1. Imaging primary mouse sarcomas after radiation therapy using cathepsin-activatable fluorescent imaging agents

    PubMed Central

    Cuneo, Kyle C.; Mito, Jeffrey K.; Javid, Melodi P.; Ferrer, Jorge M.; Kim, Yongbaek; Lee, W. David; Bawendi, Moungi G.; Brigman, Brian E.; Kirsch, David G.

    2014-01-01

    Purpose Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS), and other tumors, undergo radiation therapy (RT) prior to surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes and various tissues, including the tumor, were imaged using a handheld imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b positive tumor associated immune cells. Conclusions In this primary mouse model of STS, RT does not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes label tumor cells and tumor associated macrophages. Our results support including patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes. PMID:23391816

  2. Prolonged bioluminescence monitoring in mouse ex vivo bone culture revealed persistent circadian rhythms in articular cartilages and growth plates.

    PubMed

    Okubo, Naoki; Minami, Yoichi; Fujiwara, Hiroyoshi; Umemura, Yasuhiro; Tsuchiya, Yoshiki; Shirai, Toshiharu; Oda, Ryo; Inokawa, Hitoshi; Kubo, Toshikazu; Yagita, Kazuhiro

    2013-01-01

    The bone is a metabolically active organ which undergoes repeated remodeling cycles of bone resorption and formation. In this study, we revealed a robust and extremely long-lasting circadian rhythm in ex vivo culture maintained for over six months from the femoral bone of a PERIOD2(Luciferase) mouse. Furthermore, we also identified robust circadian clocks in flat bones. High- or low-magnification real-time bioluminescence microscopic imaging revealed that the robust circadian rhythms emanated from the articular cartilage and the epiphyseal cartilage within the growth plate of juvenile animals. Stimulation by forskolin or dexamethasone treatment caused type 0 phase resetting, indicating canonical entraining properties of the bone clock. Together, our findings from long-term ex vivo culture revealed that "tissue-autonomous" circadian rhythm in the articular cartilage and the growth plate of femoral bone functions for several months even in an organ culture condition, and provided a useful in vitro assay system investigating the role of the biological clock in bone formation or development.

  3. The function of CCR3 on mouse bone marrow-derived mast cells in vitro.

    PubMed

    Collington, Sarah J; Westwick, John; Williams, Timothy J; Weller, Charlotte L

    2010-01-01

    The mechanisms governing the population of tissues by mast cells are not fully understood, but several studies using human mast cells have suggested that expression of the chemokine receptor CCR3 and migration to its ligands may be important. In CCR3-deficient mice, a change in mast cell tissue distribution in the airways following allergen challenge was reported compared with wild-type mice. In addition, there is evidence that CCR3 is important in mast cell maturation in mouse. In this study, bone marrow-derived mast cells (BMMCs) were cultured and CCR3 expression and the migratory response to CCR3 ligands were characterized. In addition, BMMCs were cultured from wild-type and CCR3-deficient mice and their phenotype and migratory responses were compared. CCR3 messenger RNA was detectable in BMMCs, but this was not significantly increased after activation by immunoglobulin E (IgE). CCR3 protein was not detected on BMMCs during maturation and expression could not be enhanced after IgE activation. Resting and IgE-activated immature and mature BMMCs did not migrate in response to the CCR3 ligands eotaxin- 1 and eotaxin-2. Comparing wild-type and CCR3-deficient BMMCs, there were no differences in mast cell phenotype or ability to migrate to the mast cell chemoattractants leukotriene B4 and stem cell factor. The results of this study show that CCR3 may not mediate mast cell migration in mouse BMMCs in vitro. These observations need to be considered in relation to the findings of CCR3 deficiency on mast cells in vivo.

  4. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.

    PubMed

    Marquardt, D L; Walker, L L; Heinemann, S

    1994-05-01

    Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

  5. [Brown bone tumor as the first manifestation of primary hyperparathyroidism].

    PubMed

    Marcos García, M; Pino Rivero, V; Keituqwa Yáñez, T; Alcaraz Fuentes, M; Trinidad Ruiz, G; Blasco Huelva, A

    2003-01-01

    We report a clinical case of a 26 years old female who had a 2 years evolution chin tumour with hypercalcemia (11.8 mg/dl) and PTH (paratohormone) of 761 pg/ml. She underwent a CT scan and MRI of the mandible, as well as a biopsy followed by excision of the tumour by the maxilofacial surgeons. Our ENT Department asked for a Scintigraphy (Tc99s-mibi) and thoracic-cervical CT, which showed a lesion that turned out to be an adenoma of the lower right parathyroid gland after surgery and pathological examination. The patient suffered a Primary hyperparathyroidism that was the main stimulus for the Brown Tumour made up by macrophagos and multinuclear giant cells, being this the first manifestation of the metabolic disorder. This form of hyperparathyroidism is very rare in the clinic. We do a literature review to establish the differential diagnosis for such pathology.

  6. Inflammation-driven bone formation in a mouse model of ankylosing spondylitis: sequential not parallel processes.

    PubMed

    Tseng, Hsu-Wen; Pitt, Miranda E; Glant, Tibor T; McRae, Allan F; Kenna, Tony J; Brown, Matthew A; Pettit, Allison R; Thomas, Gethin P

    2016-01-29

    Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly

  7. Bone Marrow Mesenchymal Stem Cells Attenuate Mitochondria Damage Induced by Hypoxia in Mouse Trophoblasts

    PubMed Central

    Wang, Lingjuan; Xu, Xiaoyan; Kang, Lina

    2016-01-01

    Objective We aimed to observe the change of mitochondrial function and structure as well as the cell function induced by hypoxia in mouse trophoblasts, and moreover, to validate the restoration of these changes after co-culture with bone marrow mesenchymal stem cells (hereinafter referred to as “MSCs”). Further, we explored the mechanism of MSCs attenuating the functional damage of trophoblasts caused by hypoxia. Methods Cells were divided into two groups, trophoblasts and MSCs+trophoblasts respectively, and the two groups of cells were incubated with normoxia or hypoxia. Chemiluminescence was used to assay the β-HCG and progesterone in cell culture supernatants quantitatively. Western blotting and PCR were applied to detect the expression of Mfn2, MMP-2, MMP-9 and integrin β1 in the two groups. The mitochondrial membrane potential of each group of cells was detected with JC-1 dye and the ATP content was measured by the phosphomolybdic acid colorimetric method. We utilized transmission electron microscopy for observing the ultrastructure of mitochondria in trophoblasts. Finally, we assessed the cell apoptosis with flow cytometry (FCM) and analyzed the expression of the apoptosis related genes—Bcl-2, Bax, Caspase3 and Caspase9 by western blotting. Results The results showed that the Mfn2 expression was reduced after 4 h in hypoxia compared with that in normoxia, but increased in the co-culture group when compared with that in the separated-culture group (p<0.05). In addition, compared with the separated-culture group, theβ-HCG and progesterone levels in the co-culture group were significantly enhanced (p<0.05), and so were the expressions of MMP-2, MMP-9 and integrin β1 (p<0.05). Moreover, it exhibited significantly higher in ATP levels and intensified about the mitochondrial membrane potential in the co-culture group. TEM revealed disorders of the mitochondrial cristae and presented short rod-like structure and spheroids in hypoxia, however, in the co

  8. Analgesic effects of adenylyl cyclase inhibitor NB001 on bone cancer pain in a mouse model

    PubMed Central

    Kang, Wen-bo; Yang, Qi; Guo, Yan-yan; Wang, Lu; Wang, Dong-sheng; Cheng, Qiang; Li, Xiao-ming; Tang, Jun; Zhao, Jian-ning; Liu, Gang; Zhuo, Min

    2016-01-01

    Background Cancer pain, especially the one caused by metastasis in bones, is a severe type of pain. Pain becomes chronic unless its causes and consequences are resolved. With improvements in cancer detection and survival among patients, pain has been considered as a great challenge because traditional therapies are partially effective in terms of providing relief. Cancer pain mechanisms are more poorly understood than neuropathic and inflammatory pain states. Chronic inflammatory pain and neuropathic pain are influenced by NB001, an adenylyl cyclase 1 (AC1)-specific inhibitor with analgesic effects. In this study, the analgesic effects of NB001 on cancer pain were evaluated. Results Pain was induced by injecting osteolytic murine sarcoma cell NCTC 2472 into the intramedullary cavity of the femur of mice. The mice injected with sarcoma cells for four weeks exhibited significant spontaneous pain behavior and mechanical allodynia. The continuous systemic application of NB001 (30 mg/kg, intraperitoneally, twice daily for three days) markedly decreased the number of spontaneous lifting but increased the mechanical paw withdrawal threshold. NB001 decreased the concentrations of cAMP and the levels of GluN2A, GluN2B, p-GluA1 (831), and p-GluA1 (845) in the anterior cingulate cortex, and inhibited the frequency of presynaptic neurotransmitter release in the anterior cingulate cortex of the mouse models. Conclusions NB001 may serve as a novel analgesic to treat bone cancer pain. Its analgesic effect is at least partially due to the inhibition of AC1 in anterior cingulate cortex. PMID:27612915

  9. Lamin B1 protein is required for dendrite development in primary mouse cortical neurons.

    PubMed

    Giacomini, Caterina; Mahajani, Sameehan; Ruffilli, Roberta; Marotta, Roberto; Gasparini, Laura

    2016-01-01

    Lamin B1, a key component of the nuclear lamina, plays an important role in brain development and function. A duplication of the human lamin B1 (LMNB1) gene has been linked to adult-onset autosomal dominant leukodystrophy, and mouse and human loss-of-function mutations in lamin B1 are susceptibility factors for neural tube defects. In the mouse, experimental ablation of endogenous lamin B1 (Lmnb1) severely impairs embryonic corticogenesis. Here we report that in primary mouse cortical neurons, LMNB1 overexpression reduces axonal outgrowth, whereas deficiency of endogenous Lmnb1 results in aberrant dendritic development. In the absence of Lmnb1, both the length and complexity of dendrites are reduced, and their growth is unresponsive to KCl stimulation. This defective dendritic outgrowth stems from impaired ERK signaling. In Lmnb1-null neurons, ERK is correctly phosphorylated, but phospho-ERK fails to translocate to the nucleus, possibly due to delocalization of nuclear pore complexes (NPCs) at the nuclear envelope. Taken together, these data highlight a previously unrecognized role of lamin B1 in dendrite development of mouse cortical neurons through regulation of nuclear shuttling of specific signaling molecules and NPC distribution.

  10. Lamin B1 protein is required for dendrite development in primary mouse cortical neurons

    PubMed Central

    Giacomini, Caterina; Mahajani, Sameehan; Ruffilli, Roberta; Marotta, Roberto; Gasparini, Laura

    2016-01-01

    Lamin B1, a key component of the nuclear lamina, plays an important role in brain development and function. A duplication of the human lamin B1 (LMNB1) gene has been linked to adult-onset autosomal dominant leukodystrophy, and mouse and human loss-of-function mutations in lamin B1 are susceptibility factors for neural tube defects. In the mouse, experimental ablation of endogenous lamin B1 (Lmnb1) severely impairs embryonic corticogenesis. Here we report that in primary mouse cortical neurons, LMNB1 overexpression reduces axonal outgrowth, whereas deficiency of endogenous Lmnb1 results in aberrant dendritic development. In the absence of Lmnb1, both the length and complexity of dendrites are reduced, and their growth is unresponsive to KCl stimulation. This defective dendritic outgrowth stems from impaired ERK signaling. In Lmnb1-null neurons, ERK is correctly phosphorylated, but phospho-ERK fails to translocate to the nucleus, possibly due to delocalization of nuclear pore complexes (NPCs) at the nuclear envelope. Taken together, these data highlight a previously unrecognized role of lamin B1 in dendrite development of mouse cortical neurons through regulation of nuclear shuttling of specific signaling molecules and NPC distribution. PMID:26510501

  11. Sulfuretin promotes osteoblastic differentiation in primary cultured osteoblasts and in vivo bone healing

    PubMed Central

    Yun, Hyung-Mun; Lim, Hyun-Chang; Kim, Ga-Hyun; Lee, Dong-Sung; Kim, Youn-Chul; Oh, Hyuncheol; Kim, Eun-Cheol

    2016-01-01

    Although sulfuretin, the major flavonoid of Rhus verniciflua Stokes, has a variety of biological actions, its in vitro and in vivo effects on osteogenic potential remain poorly understood. The objective of the present study was to investigate the effects of sulfuretin on in vitro osteoblastic differentiation and the underlying signal pathway mechanisms in primary cultured osteoblasts and on in vivo bone formation using critical-sized calvarial defects in mice. Sulfuretin promoted osteogenic differentiation of primary osteoblasts, with increased ALP activity and mineralization, and upregulated differentiation markers, including ALP, osteocalcin, and osteopontin, in a concentration-dependent manner. The expression levels of Runx2, BMP-2, and phospho-Smad1/5/8 were upregulated by sulfuretin. Moreover, sulfuretin increased phosphorylation of Akt, mTOR, ERK, and JNK. Furthermore, sulfuretin treatment increased mRNA expression of Wnt ligands, phosphorylation of GSK3, and nuclear β-catenin protein expression. In vivo studies with calvarial bone defects revealed that sulfuretin significantly enhanced new bone formation by micro-computed tomography and histologic analysis. Collectively, these data suggest that sulfuretin acts through the activation of BMP, mTOR, Wnt/β-catenin, and Runx2 signaling to promote in vitro osteoblast differentiation and facilitate in vivo bone regeneration, and might be have therapeutic benefits in bone disease and regeneration. PMID:27713171

  12. Automatic detection and quantitative analysis of cells in the mouse primary motor cortex

    NASA Astrophysics Data System (ADS)

    Meng, Yunlong; He, Yong; Wu, Jingpeng; Chen, Shangbin; Li, Anan; Gong, Hui

    2014-09-01

    Neuronal cells play very important role on metabolism regulation and mechanism control, so cell number is a fundamental determinant of brain function. Combined suitable cell-labeling approaches with recently proposed three-dimensional optical imaging techniques, whole mouse brain coronal sections can be acquired with 1-μm voxel resolution. We have developed a completely automatic pipeline to perform cell centroids detection, and provided three-dimensional quantitative information of cells in the primary motor cortex of C57BL/6 mouse. It involves four principal steps: i) preprocessing; ii) image binarization; iii) cell centroids extraction and contour segmentation; iv) laminar density estimation. Investigations on the presented method reveal promising detection accuracy in terms of recall and precision, with average recall rate 92.1% and average precision rate 86.2%. We also analyze laminar density distribution of cells from pial surface to corpus callosum from the output vectorizations of detected cell centroids in mouse primary motor cortex, and find significant cellular density distribution variations in different layers. This automatic cell centroids detection approach will be beneficial for fast cell-counting and accurate density estimation, as time-consuming and error-prone manual identification is avoided.

  13. Hepatocellular Carcinoma with Cervical Spine and Pelvic Bone Metastases Presenting as Unknown Primary Neoplasm.

    PubMed

    Hwang, Sea Won; Lee, Ji Eun; Lee, Jung Min; Hong, Sook Hee; Lee, Myung Ah; Chun, Hoo Geun; Chun, Ho Jong; Lee, Sung Hak; Jung, Eun Sun

    2015-07-01

    The occurrence of hepatocellular carcinoma (HCC) is closely associated with viral hepatitis or alcoholic hepatitis. Although active surveillance is ongoing in Korea, advanced or metastatic HCC is found at initial presentation in many patients. Metastatic HCC presents with a hypervascular intrahepatic tumor and extrahepatic lesions such as lung or lymph node metastases. Cases of HCC presenting as carcinoma of unknown primary have been rarely reported. The authors experienced a case of metastatic HCC in a patient who presented with a metastatic bone lesion but no primary intrahepatic tumor. This case suggests that HCC should be considered as a differential diagnosis when evaluating the primary origin of metastatic carcinoma.

  14. In vivo longitudinal visualization of bone marrow engraftment process in mouse calvaria using two-photon microscopy.

    PubMed

    Le, Viet-Hoan; Lee, Seunghun; Lee, Seungwon; Wang, Taejun; Hyuk Jang, Won; Yoon, Yeoreum; Kwon, Soonjae; Kim, Hyekang; Lee, Seung-Woo; Hean Kim, Ki

    2017-03-09

    Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level. However, the current method used to access the mouse calvaria allows for only a few imaging times in the same mouse because of scar formation and inflammation induced by multiple surgeries. Longitudinal imaging of the BM may help better understand its microenvironment. In this study, a mouse calvarial window model was developed for longitudinal imaging that involves attaching a cover glass window onto the mouse calvaria and sealing the surrounding exposed area with cyanoacrylate glue and dental cement. The model was used for the longitudinal two-photon microscopy (TPM) imaging of the BM engraftment process. The same BM cavity sites were imaged multiple times over 4 weeks after BM transplantation (BMT). Temporal changes in the BM microenvironment, such as the reconstitution of transplanted BM cells and the recovery of vasculature, were observed and analysed qualitatively and quantitatively. Longitudinal intravital microscopy using the mouse calvarial window model was successfully demonstrated and may be useful for further BM studies.

  15. In vivo longitudinal visualization of bone marrow engraftment process in mouse calvaria using two-photon microscopy

    PubMed Central

    Le, Viet-Hoan; Lee, Seunghun; Lee, Seungwon; Wang, Taejun; Hyuk Jang, Won; Yoon, Yeoreum; Kwon, Soonjae; Kim, Hyekang; Lee, Seung-Woo; Hean Kim, Ki

    2017-01-01

    Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level. However, the current method used to access the mouse calvaria allows for only a few imaging times in the same mouse because of scar formation and inflammation induced by multiple surgeries. Longitudinal imaging of the BM may help better understand its microenvironment. In this study, a mouse calvarial window model was developed for longitudinal imaging that involves attaching a cover glass window onto the mouse calvaria and sealing the surrounding exposed area with cyanoacrylate glue and dental cement. The model was used for the longitudinal two-photon microscopy (TPM) imaging of the BM engraftment process. The same BM cavity sites were imaged multiple times over 4 weeks after BM transplantation (BMT). Temporal changes in the BM microenvironment, such as the reconstitution of transplanted BM cells and the recovery of vasculature, were observed and analysed qualitatively and quantitatively. Longitudinal intravital microscopy using the mouse calvarial window model was successfully demonstrated and may be useful for further BM studies. PMID:28276477

  16. Restricted replication of mouse hepatitis virus A59 in primary mouse brain astrocytes correlates with reduced pathogenicity.

    PubMed Central

    van Berlo, M F; Wolswijk, G; Calafat, J; Koolen, M J; Horzinek, M C; van der Zeijst, B A

    1986-01-01

    Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) are drastically attenuated in their pathogenic properties. Intracerebral inoculation of mice with 10(5) PFU of mutant ts342 results in prolonged infection of the central nervous system, whereas 100 PFU of wild-type virus are lethal (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). In the Sac(-) cell line ts342 grows as well at 37 degrees C (the body temperature of mice) as at 31 degrees C (the permissive temperature). There is, however, a difference in primary cultures of mouse brain astrocytes. After infection with ts342, astrocytes produced low levels of infectious virus (5.2 +/- 3.7%) compared with virus yields after infection with wild-type virus. The fraction of wild-type virus- and ts342-infected cells was similar. Electron microscopy showed in wild-type virus-infected cells abundant virions in smooth vesicles usually closely associated with a well-developed Golgi apparatus. In mutant-infected cells no mature ts342 virus particles were found. There was no difference between ts342 and wild-type virus regarding the intracellular virus-specific RNAs. In ts342-infected cells the viral glycoproteins E2 and E1 were not detectable or were barely detectable. Either the mRNAs for the glycoproteins are not translated or the proteins are rapidly broken down. Revertants of ts342 were isolated. They grew as well as wild-type virus in astrocytes, indicating that they apparently produced sufficient amounts of E2 and E1, the ts defect itself rather than a second site mutation is responsible for the defect in replication, and the ts defect acts in unison with host-cell factors. The revertants also regained the lethal properties of wild-type virus. Images PMID:3009857

  17. Periprosthetic bone turnover after primary total hip arthroplasty measured by single-photon emission computed tomography.

    PubMed

    Venesmaa, P; Vanninen, E; Miettinen, H; Kröger, H

    2012-01-01

    Alterations in periprosthetic bone are common sequlae of prosthetic implants.This prospective 3-year study was performed to follow regional periprosthetic bone turnover after uncomplicated total hip arthroplasty (THA) using single-photon emission computed tomography (SPECT). Eighteen patients (nine men, nine women: mean age 61 years, range from 50 to 73 years) with primary hip osteoarthritis underwent either uncemented or cemented THA. The SPECT measurements were taken 6, 12, and 36 months after THA. Bone mineral density (BMD) measurements were performed on the patients during follow-up. The mean SPECT uptake ratios decreased significantly in the regions of interest (ROIs) during follow-up compared to baseline value, in the trochanter major (p = 0.006), the trochanter minor (p = 0.009) and the total area (p = 0.018). Despite these decreases the uptake ratios in the medial cortex (p = 0.014), tip (p = 0.002) and total area (p = 0.016) remained significantly higher in the operated side than in the contralateral side (non-operated) 3 years after THA. Changes in bone turnover in the vicinity of the implant did not correlate with changes in periprosthetic BMD. Local periprosthetic bone turnover decreased slowly after THA and did not recover to the level typical of non-operated hips. This led us to suggest that bone turnover around the femoral implants remains increased more than 3 years after THA even if there are no signs of failure of the prosthesis.

  18. Osseous and dental outcomes of primary gingivoperiosteoplasty with iliac bone graft: A radiological evaluation.

    PubMed

    Touzet-Roumazeille, Sandrine; Vi-Fane, Brigitte; Kadlub, Natacha; Genin, Michaël; Dissaux, Caroline; Raoul, Gwenaël; Ferri, Joël; Vazquez, Marie-Paule; Picard, Arnaud

    2015-07-01

    Primary alveolar cleft repair has two main purposes: to restore normal morphology and normal function. Gingivoperiosteoplasty with bone grafting in mixed dentition has been a well-established procedure. We hypothesized that 1) performance of this surgery in deciduous dentition would provide favorable bone graft osseointegration, and 2) would improve the support of incisor teeth eruption, thereby avoiding maxillary growth disturbances. We conducted a retrospective study of clinical and tridimensional radiological data for 73 patients with alveolar clefts (with or without lip and palate clefts) who underwent gingivoperiosteoplasty with iliac bone graft in deciduous dentition. Pre- and post-operative Cone Beam Computed Tomography (CBCT) comparison allowed evaluation of the ratio between bone graft volume and initial cleft volume (BGV/ICV ratio), and measurement of central incisor teeth movements. This series of 73 patients included 44 males and 29 females, with a mean age of 5.5 years. Few complications were observed. Post-operative CBCT was performed at 7.4 months. The mean BGV/ICV ratio was 0.62. Axial rotation was significantly improved post-operatively (p = 0.004). Gingivoperiosteoplasty with iliac bone graft is safe when performed in deciduous dentition and results in a sufficient bone graft volume to support lateral incisor eruption and upper central incisor tooth position improvement. Copyright © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  19. Study on bone density at various skeletal sites for the diagnosis of primary osteoporosis.

    PubMed

    Jiang, E; Wang, Zizheng; Meng, Qingle; Li, Shaohua; Wang, Feng; Shao, Guoqiang; Zhang, Lele

    2012-09-01

    The objective of this study was to evaluate the diagnostic value of bone density changes in lumbar vertebrae and femoral necks in patients with primary osteoporosis (OP) at various ages. Dual-energy X-ray absorptiometry (DXA) scans were performed on patients who had their primary visits between March 2008 and February 2009. The bone mineral density (BMD) of the lumbar vertebrae 1-4 (L1-L4) in anteroposterior projection and the proximal femoral neck in lateral projection were measured. If the BMD values (T score) of any site is -2.5 or less (T ≤ -2.5), the patients were diagnosed as primary OP, and the T scores were statistically analyzed. The 81 patients who had lumbar vertebrae with a T ≤ -2.5 led to a positive rate of 80.1 % in the diagnosis of primary OP; the 47 patients who had femoral neck with a T ≤ -2.5 gave a positive rate of 47.0 %. The patients with type I or type II primary OP were divided into two age groups of ≤70 and ≥71 years old. The comparison of lumbar spine T score values did not show significant statistical difference (P > 0.05) between the age groups, while the result of the femoral necks revealed significant difference between the two groups (P < 0.001). In diagnosis of primary OP, anteroposterior lumbar spine offers a significantly higher detection rate than that of the femoral neck, but to the patients older than 70, the measurement of femoral neck may generate higher detection rate. It is more sensitive to measure lumbar trabecular bone, especially to the patients in early postmenopausal period. BMD in elderly patients may falsely increase with age, attention should be paid to the determination of the hip bone mass.

  20. Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats.

    PubMed

    Orriss, Isabel R; Hajjawi, Mark O R; Huesa, Carmen; MacRae, Vicky E; Arnett, Timothy R

    2014-11-01

    The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of bone formation. The widespread use of transgenic mice has created a particular need for a reliable, simple method that allows the differentiation and bone‑forming activity of murine osteoblasts to be studied. In the present study, we established such a method and identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells isolated from neonatal rodent calvariae by collagenase digestion were cultured for 14‑28 days before staining for tissue non-specific alkaline phosphatase (TNAP) and bone mineralisation (alizarin red). The reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of mineralised 'trabecular‑shaped' bone nodules, occurred only following culture in α minimum essential medium (αMEM) and took 21‑28 days. Dexamethasone (10 nM) inhibited bone mineralisation in the mouse osteoblasts. By contrast, TNAP expression and bone formation by rat osteoblasts were observed following culture in both αMEM and Dulbecco's modified Eagle's medium (DMEM) after approximately 14 days (although ~3‑fold more effectively in αMEM) and was strongly dependent on dexamethasone. Both the mouse and rat osteoblasts required ascorbate (50 µg/ml) for osteogenic differentiation and β‑glycerophosphate (2 mM) for mineralisation. The rat and mouse osteoblasts showed similar sensitivity to the well‑established inhibitors of mineralisation, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP; 1‑100 µM). The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former. These findings offer a reliable technique for inducing mouse osteoblasts to form bone in vitro and a more effective method for culturing bone‑forming rat osteoblasts.

  1. Oxidized and Original article degraded mitochondrial polynucleotides (DeMPs), especially RNA, are potent immunogenic regulators in primary mouse macrophages.

    PubMed

    Saxena, Abhinav R; Gao, Linda Y; Srivatsa, Shachi; Bobersky, Elizabeth Z; Periasamy, Sivakumar; Hunt, Danielle T; Altman, Kyle E; Crawford, Dana R

    2017-03-01

    Certain mitochondrial components can act as damage-associated molecular patterns (DAMPs) or danger signals, triggering a proinflammatory response in target (usually immune) cells. We previously reported the selective degradation of mitochondrial DNA and RNA in response to cellular oxidative stress, and the immunogenic effect of this DNA in primary mouse astrocytes. Here, we extend these studies to assess the immunogenic role of both mitochondrial DNA and RNA isolated from hydrogen peroxide (HP) treated HA1 cells (designated "DeMPs" for degraded mitochondrial polynucleotides) using mouse bone marrow derived macrophages (BMDMs), a conventional immune cell type. DeMPs and control mitochondrial DNA (cont mtDNA) and RNA (cont mtRNA) were transfected into BMDMs and cell-free media analyzed for the presence of proinflammatory cytokines (IL-6, MCP-1, and TNFα) and Type I interferon (IFN-α and IFN-β). Cont mtDNA induced IL-6 and MCP-1 production, and this effect was even greater with DeMP DNA. A similar response was observed for Type I interferons. An even stronger induction of proinflammatory cytokine and type 1 interferons was observed for cont mtRNA. However, contrary to DeMP DNA, DeMP RNA attenuated rather than potentiated the cont mtRNA cytokine inductions. This attenuation effect was not accompanied by an IL-10 or TGFβ anti-inflammatory response. All DeMP effects were observed at multiple oxidant concentrations. Finally, DeMP production and immunogenicity overlaps with cellular adaptive response and so may contribute to cellular oxidant protection. These results provide new insight into the immunogenicity of mitochondrial polynucleotides, and identify new roles and selective consequences of cellular oxidation.

  2. Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages.

    PubMed

    Singh, Mahavir; Kapoor, Aniruddh; McCracken, James; Hill, Bradford; Bhatnagar, Aruni

    2017-03-01

    Macrophages are critical drivers of the immune response during infection and inflammation. The pathogenesis of several inflammatory conditions, such as diabetes, cancer and sepsis has been linked with aldose reductase (AR), a member of the aldo-keto reductase (AKR) superfamily. However, the role of AR in the early stages of innate immunity such as phagocytosis remains unclear. In this study, we examined the role of AR in regulating the growth and the phagocytic activity of bone marrow-derived mouse macrophages (BMMs) from AR-null and wild-type (WT) mice. We found that macrophages derived from AR-null mice were larger in size and had a slower growth rate than those derived from WT mice. The AR-null macrophages also displayed higher basal, and lipopolysaccharide (LPS) stimulated phagocytic activity than WT macrophages. Moreover, absence of AR led to a marked increase in cellular levels of both ATP and NADPH. These data suggest that metabolic pathways involving AR suppress macrophage energy production, and that inhibition of AR could induce a favorable metabolic state that promotes macrophage phagocytosis. Hence, modulation of macrophage metabolism by inhibition of AR might represent a novel strategy to modulate host defense responses and to modify metabolism to promote macrophage hypertrophy and phagocytosis under inflammatory conditions.

  3. Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development

    NASA Technical Reports Server (NTRS)

    Simske, Steven J.; Bateman, Ted A.; Smith, Erin E.; Ferguson, Virginia L.; Chapes, Stephen K.

    2002-01-01

    We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls.

  4. Transplantation of Bone Marrow-Derived Mesenchymal Stem Cells into the Developing Mouse Eye

    PubMed Central

    Lee, Eun-Shil; Yu, Song-Hee; Jang, Yu-Jin; Hwang, Dong-Youn; Jeon, Chang-Jin

    2011-01-01

    Mesenchymal stem cells (MSCs) have been studied widely for their potential to differentiate into various lineage cells including neural cells in vitro and in vivo. To investigate the influence of the developing host environment on the integration and morphological and molecular differentiation of MSCs, human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the developing mouse retina. Enhanced green fluorescent protein (GFP)-expressing BM-MSCs were transplanted by intraocular injections into mice, ranging in ages from 1 day postnatal (PN) to 10 days PN. The survival dates ranged from 7 days post-transplantation (DPT) to 28DPT, at which time an immunohistochemical analysis was performed on the eyes. The transplanted BM-MSCs survived and showed morphological differentiation into neural cells and some processes within the host retina. Some transplanted cells expressed microtubule associated protein 2 (MAP2ab, marker for mature neural cells) or glial fibrillary acid protein (GFAP, marker for glial cells) at 5PN 7DPT. In addition, some transplanted cells integrated into the developing retina. The morphological and molecular differentiation and integration within the 5PN 7DPT eye was greater than those of other-aged host eye. The present findings suggest that the age of the host environment can strongly influence the differentiation and integration of BM-MSCs. PMID:22096261

  5. Antigenotoxicity protection of Carapa guianensis oil against mitomycin C and cyclophosphamide in mouse bone marrow.

    PubMed

    Lemes, Susy R; Chaves, Dwight A; Silva, Nelson J DA; Carneiro, Cristiene C; Chen-Chen, Lee; Almeida, Luciane M DE; Gonçalves, Pablo J; Melo-Reis, Paulo R DE

    2017-06-29

    The aim of this study was to evaluate the possible protective of C. guianensis oil against MMC and CP, which are direct- and indirect-acting chemical mutagens, using the micronucleus test. Three experiments were performed. First the C. guianensis oil was co-administered to mice at doses of 250, 500 and 1000 mg/kg bw with 4 mg/kg bw MMC or 50 mg/kg bw CP. Second, the mutagenic drug (CP) was administered ip 50 mg/kg bw and after 6 and 12 hours 250 and 500 mg/kg bw of C. guianensis oil were administered. In the last, C. guianensis oil was administrated (250 and 500 mg/kg bw) during five days and after it was administered ip 50 mg/kg bw CP. The results obtained showed that the C. guianensis oil is not cytotoxic neither genotoxic to mouse bone marrow. Regarding the antimutagenic effect, all doses of C. guianensis oil were significantly (p < 0.05) effective in reducing the frequency of micronucleated polychromatic erythrocytes, when compared with MMC or CP alone. Based on these results, our results suggest that the C. guianensis oil shows medicinal potential as an antimutagenic agent, modulating the mutagenicity caused by both direct- and indirect-acting chemical mutagens, in a mammalian model.

  6. Influences of reduced expression of maternal bone morphogenetic protein 2 on mouse embryonic development.

    PubMed

    Singh, A P; Castranio, T; Scott, G; Guo, D; Harris, M A; Ray, M; Harris, S E; Mishina, Y

    2008-01-01

    Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. In the course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3' untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal mouse tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/-) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. The number of embryos exhibiting these abnormalities was increased when, due to different genotypes, expression levels of Bmp2 in maternal tissues were lower. These results suggest that the expression levels of Bmp2 in both embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds.

  7. Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development

    NASA Technical Reports Server (NTRS)

    Simske, Steven J.; Bateman, Ted A.; Smith, Erin E.; Ferguson, Virginia L.; Chapes, Stephen K.

    2002-01-01

    We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls.

  8. Prognostic factors for elderly patients with primary malignant bone and soft tissue tumors

    PubMed Central

    IWAI, TADASHI; HOSHI, MANABU; TAKADA, JUN; OEBISU, NAOTO; AONO, MASANARI; TAKAMI, MASATSUGU; IEGUCHI, MAKOTO; NAKAMURA, HIROAKI

    2015-01-01

    The number of patients with primary malignant bone and soft tissue tumors in Japan is increasing in line with the increasing size of the elderly population. The aim of the present study was to determine the prognostic factors of primary malignant bone or soft tissue tumors in elderly patients. Clinical data was obtained from 90 patients, aged ≥65 years, with primary malignant bone or soft tissue tumors (bone, 20 cases; and soft tissue, 70 cases), treated at the Osaka City University Hospital between 1993 and 2013. Clinical information prior to treatment and tumor type, location, size, depth, grade and American Society of Anesthesiologists-Physical Status (ASA-PS) score were evaluated in order to identify prognostic factors using the Cox proportional hazards regression model. In addition, 5-year survival rates were evaluated using the Kaplan-Meier method. The average follow-up period was 44.8 months and the 5-year overall survival rate was 77.5%. In the multivariate analysis, ASA-PS score and high-grade sarcoma were found to be associated with a poorer overall survival. No significant differences were observed between the patient group aged 65–74 years and that aged ≥75 years. In general, aging is associated with physically reduced function and an increased prevalence of comorbidities. It was therefore expected that increasing age may be a predictive factor for poor prognosis. However, the results of the present study suggested that ASA-PS score and tumor grade were significant factors associated with poor prognosis, whereas increasing age was not. Therefore, the treatment of elderly patients with primary bone and soft tissue tumors should not be based on age. PMID:26622753

  9. Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

    PubMed

    Stalvey, Michael S; Clines, Katrina L; Havasi, Viktoria; McKibbin, Christopher R; Dunn, Lauren K; Chung, W Joon; Clines, Gregory A

    2013-01-01

    Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/-) mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling

  10. Properties of the primary somatosensory cortex projection to the primary motor cortex in the mouse.

    PubMed

    Petrof, Iraklis; Viaene, Angela N; Sherman, S Murray

    2015-04-01

    The primary somatosensory (S1) and primary motor (M1) cortices are reciprocally connected, and their interaction has long been hypothesized to contribute to coordinated motor output. Very little is known, however, about the nature and synaptic properties of the S1 input to M1. Here we wanted to take advantage of a previously developed sensorimotor slice preparation that preserves much of the S1-to-M1 connectivity (Rocco MM, Brumberg JC. J Neurosci Methods 162: 139-147, 2007), as well as available optogenetic methodologies, in order to investigate the synaptic profile of this projection. Our data show that S1 input to pyramidal cells of M1 is highly homogeneous, possesses many features of a "driver" pathway, such as paired-pulse depression and lack of metabotropic glutamate receptor activation, and is mediated through axons that terminate in both small and large synaptic boutons. Our data suggest that S1 provides M1 with afferents that possess synaptic and anatomical characteristics ideal for the delivery of strong inputs that can "drive" postsynaptic M1 cells, thereby potentially affecting their output.

  11. Cytokine response in mouse bone marrow derived macrophages after infection with pathogenic and non-pathogenic Rift Valley fever virus.

    PubMed

    Roberts, Kimberly K; Hill, Terence E; Davis, Melissa N; Holbrook, Michael R; Freiberg, Alexander N

    2015-07-01

    Rift Valley fever virus (RVFV) is the most pathogenic member of the genus Phlebovirus within the family Bunyaviridae, and can cause severe disease in humans and livestock. Until recently, limited information has been published on the cellular host response elicited by RVFV, particularly in macrophages and dendritic cells, which play critical roles in stimulating adaptive and innate immune responses to viral infection. In an effort to define the initial response of host immunomodulatory cells to infection, primary mouse bone marrow derived macrophages (BMDM) were infected with the pathogenic RVFV strain ZH501, or attenuated strains MP-12 or MP-12 based Clone13 type (rMP12-C13 type), and cytokine secretion profiles examined. The secretion of T helper (Th)1-associated antiviral cytokines, chemokines and various interleukins increased rapidly after infection with the attenuated rMP12-C13 type RVFV, which lacks a functional NSs virulence gene. In comparison, infection with live-attenuated MP-12 encoding a functional NSs gene appeared to cause a delayed immune response, while pathogenic ZH501 ablates the immune response almost entirely. These data demonstrate that NSs can inhibit components of the BMDM antiviral response and supports previous work indicating that NSs can specifically regulate the type I interferon response in macrophages. Furthermore, our data demonstrate that genetic differences between ZH501 and MP-12 reduce the ability of MP-12 to inhibit antiviral signalling and subsequently reduce virulence in BMDM, demonstrating that viral components other than NSs play a critical role in regulating the host response to RVFV infection.

  12. Cytokine response in mouse bone marrow derived macrophages after infection with pathogenic and non-pathogenic Rift Valley fever virus

    PubMed Central

    Roberts, Kimberly K.; Hill, Terence E.; Davis, Melissa N.; Holbrook, Michael R.

    2015-01-01

    Rift Valley fever virus (RVFV) is the most pathogenic member of the genus Phlebovirus within the family Bunyaviridae, and can cause severe disease in humans and livestock. Until recently, limited information has been published on the cellular host response elicited by RVFV, particularly in macrophages and dendritic cells, which play critical roles in stimulating adaptive and innate immune responses to viral infection. In an effort to define the initial response of host immunomodulatory cells to infection, primary mouse bone marrow derived macrophages (BMDM) were infected with the pathogenic RVFV strain ZH501, or attenuated strains MP-12 or MP-12 based Clone13 type (rMP12-C13 type), and cytokine secretion profiles examined. The secretion of T helper (Th)1-associated antiviral cytokines, chemokines and various interleukins increased rapidly after infection with the attenuated rMP12-C13 type RVFV, which lacks a functional NSs virulence gene. In comparison, infection with live-attenuated MP-12 encoding a functional NSs gene appeared to cause a delayed immune response, while pathogenic ZH501 ablates the immune response almost entirely. These data demonstrate that NSs can inhibit components of the BMDM antiviral response and supports previous work indicating that NSs can specifically regulate the type I interferon response in macrophages. Furthermore, our data demonstrate that genetic differences between ZH501 and MP-12 reduce the ability of MP-12 to inhibit antiviral signalling and subsequently reduce virulence in BMDM, demonstrating that viral components other than NSs play a critical role in regulating the host response to RVFV infection. PMID:25759029

  13. Minor histocompatibility antigens on transfused leukoreduced units of red blood cells induce bone marrow transplant rejection in a mouse model.

    PubMed

    Desmarets, Maxime; Cadwell, Chantel M; Peterson, Kenneth R; Neades, Renee; Zimring, James C

    2009-09-10

    When successful, human leukocyte antigen (HLA)-matched bone marrow transplantation with reduced-intensity conditioning is a cure for several nonmalignant hematologic disorders that require chronic transfusion, such as sickle cell disease and aplastic anemia. However, there are unusually high bone marrow transplant (BMT) rejection rates in these patients. Rejection correlates with the number of transfusions before bone marrow transplantation, and it has been hypothesized that preimmunization to antigens on transfused blood may prime BMT rejection. Using a novel mouse model of red blood cell (RBC) transfusion and major histocompatibility complex-matched bone marrow transplantation, we report that transfusion of RBC products induced BMT rejection across minor histocompatibility antigen (mHA) barriers. It has been proposed that contaminating leukocytes are responsible for transfusion-induced BMT rejection; however, filter leukoreduction did not prevent rejection in the current studies. Moreover, we generated a novel transgenic mouse with RBC-specific expression of a model mHA and demonstrated that transfusion of RBCs induced a CD8(+) T-cell response. Together, these data suggest that mHAs on RBCs themselves are capable of inducing BMT rejection. Cellular immunization to mHAs is neither monitored nor managed by current transfusion medicine practice; however, the current data suggest that mHAs on RBCs may represent an unappreciated and significant consequence of RBC transfusion.

  14. Osteoprotegerin inhibits osteolysis and decreases skeletal tumor burden in syngeneic and nude mouse models of experimental bone metastasis.

    PubMed

    Morony, S; Capparelli, C; Sarosi, I; Lacey, D L; Dunstan, C R; Kostenuik, P J

    2001-06-01

    Certain malignancies, including breast cancer, frequently metastasize to bone, where the tumor cells induce osteoclasts to locally destroy bone. Osteoprotegerin (OPG), a member of the tumor necrosis factor receptor family, is a negative regulator of osteoclast differentiation, activation, and survival. We tested the ability of recombinant OPG to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in two animal models. In a syngeneic model, mouse colon adenocarcinoma (Colon-26) cells were injected into the left ventricle of mice. Treatment with OPG dose-dependently decreased the number and area of radiographically evident lytic bone lesions, which, at the highest dose, were undetectable. Histologically, OPG also decreased skeletal tumor burden and tumor-associated osteoclasts. In a nude mouse model, OPG treatment completely prevented radiographic osteolytic lesions caused by human MDA-MB-231 breast cancer cells. Histologically, OPG decreased skeletal tumor burden by 75% and completely eradicated MDA tumor-associated osteoclasts. In both models, OPG had no effect on metastatic tumor burden in a panel of soft tissue organs. These data indicate that OPG may be an effective therapy for preventing osteolysis and decreasing skeletal tumor burden in patients with bone metastasis.

  15. Vital staining for cell death identifies Atg9a-dependent necrosis in developmental bone formation in mouse

    PubMed Central

    Imagawa, Yusuke; Saitoh, Tatsuya; Tsujimoto, Yoshihide

    2016-01-01

    Programmed cell death has a crucial role in various biological events, including developmental morphogenesis. Recent evidence indicates that necrosis contributes to programmed cell death in addition to apoptosis, but it is unclear whether necrosis acts as a compensatory mechanism for failure of apoptosis or has an intrinsic role during development. In contrast to apoptosis, there have been no techniques for imaging physiological necrosis in vivo. Here we employ vital staining using propidium iodide to identify cells with plasma membrane disruption (necrotic cells) in mouse embryos. We discover a form of necrosis at the bone surface, which does not occur in embryos with deficiency of the autophagy-related gene Atg9a, although it is unaffected by Atg5 knockout. We also find abnormalities of the bone surface in Atg9a knockout mice, suggesting an important role of Atg9a-dependent necrosis in bone surface formation. These findings suggest that necrosis has an active role in developmental morphogenesis. PMID:27811852

  16. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    SciTech Connect

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. )

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  17. Co-regulation of Primary Mouse Hepatocyte Viability and Function by Oxygen and Matrix

    PubMed Central

    Buck, Lorenna D.; Inman, S. Walker; Rusyn, Ivan; Griffith, Linda G.

    2014-01-01

    Although oxygen and extracellular matrix cues both influence differentiation state and metabolic function of primary rat and human hepatocytes, relatively little is known about how these factors together regulate behaviors of primary mouse hepatocytes in culture. To determine the effects of pericellular oxygen tension on hepatocellular function, we employed 2 methods of altering oxygen concentration in the local cellular microenvironment of cells cultured in the presence or absence of an extracellular matrix (Matrigel) supplement. By systematically altering medium depth and gas phase oxygen tension, we created multiple oxygen regimes (hypoxic, normoxic, and hyperoxic) and measured the local oxygen concentrations in the pericellular environment using custom-designed oxygen microprobes. From these measurements of oxygen concentrations, we derived values of oxygen consumption rates under a spectrum of environmental contexts, thus providing the first reported estimates of these values for primary mouse hepatocytes. Oxygen tension and matrix microenvironment were found to synergistically regulate hepatocellular survival and function as assessed using quantitative image analysis for cells stained with vital dyes, and assessment of secretion of albumin. Hepatocellular viability was affected only at strongly hypoxic conditions. Surprisingly, albumin secretion rates were greatest at a moderately supra-physiological oxygen concentration, and this effect was mitigated at still greater supra-physiological concentrations. Matrigel enhanced the effects of oxygen on retention of function. This study underscores the importance of carefully controlling cell density, medium depth and gas phase oxygen, as the effects of these parameters on local pericellular oxygen tension and subsequent hepatocellular function are profound. PMID:24222008

  18. Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.

    PubMed

    Kim, Yohan; Kang, Kyojin; Jeong, Jaemin; Paik, Seung Sam; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Park, Jisun; Choi, Dongho

    2017-02-01

    The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6-8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10(7) hepatocytes with 80%-90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

  19. Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure

    PubMed Central

    Kim, Yohan; Kang, Kyojin; Jeong, Jaemin; Paik, Seung Sam; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Park, Jisun

    2017-01-01

    Purpose The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. Methods To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 107 hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Results Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Conclusion Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers. PMID:28203553

  20. Immunodepletion and Hypoxia Preconditioning of Mouse Compact Bone Cells as a Novel Protocol to Isolate Highly Immunosuppressive Mesenchymal Stem Cells.

    PubMed

    Sivanathan, Kisha Nandini; Gronthos, Stan; Grey, Shane T; Rojas-Canales, Darling; Coates, Patrick T

    2017-04-01

    Compact bones (CB) are major reservoirs of mouse mesenchymal stem cells (mMSC). Here, we established a protocol to isolate MSC from CB and tested their immunosuppressive potential. Collagenase type II digestion of BM-flushed CB from C57B/6 mice was performed to liberate mMSC precursors from bone surfaces to establish nondepleted mMSC. CB cells were also immunodepleted based on the expression of CD45 (leukocytes) and TER119 (erythroid cells) to eliminate hematopoietic cells. CD45(-)TER119(-) CB cells were subsequently used to generate depleted mMSC. CB nondepleted and depleted mMSC progenitors were cultured under hypoxic conditions to establish primary mMSC cultures. CB depleted mMSC compared to nondepleted mMSC showed greater cell numbers at subculturing and had increased functional ability to differentiate into adipocytes and osteoblasts. CB depleted mMSC had high purity and expressed key mMSC markers (>85% Sca-1, CD29, CD90) with no mature hematopoietic contaminating cells (<5% CD45, CD11b) when subcultured to passage 5 (P5). Nondepleted mMSC cultures, however, were less pure and heterogenous with <72% Sca-1(+), CD29(+), and CD90(+) cells at early passages (P1 or P2), along with high percentages of contaminating CD11b(+) (35.6%) and CD45(+) (39.2%) cells that persisted in culture long term. Depleted and nondepleted mMSC nevertheless exhibited similar potency to suppress total (CD3(+)), CD4(+) and CD8(+) T cell proliferation, in a dendritic cell allostimulatory one-way mixed lymphocyte reaction. CB depleted mMSC, pretreated with proinflammatory cytokines IFN-γ, TNF-α, and IL-17A, showed superior suppression of CD8(+) T cell, but not CD4(+) T cell proliferation, relative to untreated-mMSC. In conclusion, CB depleted mMSC established under hypoxic conditions and treated with selective cytokines represent a novel source of potent immunosuppressive MSC. As these cells have enhanced immune modulatory function, they may represent a superior product for use in clinical

  1. The relief of bone pain in primary biliary cirrhosis with calcium infusions

    PubMed Central

    Ajdukiewicz, A. B.; Agnew, J. E.; Byers, P. D.; Wills, M. R.; Sherlock, Sheila

    1974-01-01

    Intravenous calcium infusions produced subjective relief of bone pain in 14 patients with primary biliary cirrhosis. The bone pain had developed despite long-term parenteral vitamin D therapy. The pain returned after two to three months, but a subsequent course of infusions again brought relief. Before treatment satisfactory iliac crest bone biopsies were obtained in 11 of the patients and were normal in seven; two patients had biopsies indicating osteomalacia and two osteoporosis. After treatment a repeat biopsy in one of the patients with osteomalacia showed marked reduction in osteoid. The infusion treatment produced no change in plasma calcium concentration, serum phosphate, or serum alkaline phosphatase. Absorption of oral calcium was also unchanged. PMID:4279816

  2. Case Report Diffuse large B-cell lymphoma in the primary bone marrow.

    PubMed

    Hu, Y; Chen, S L; Huang, Z X; Gao, W; An, N

    2015-06-11

    This study aimed to improve understanding of the diagnosis, treatment, and prognosis of primary bone marrow (PBM) diffuse large B-cell lymphoma (DLBCL), a rare illness. We report a 56-year-old man with pancytopenia and hyperbilirubinemia but without lymphadenopathy, hepatomegaly, or splenomegaly. Bone marrow aspiration, flow cytometry, biopsy, and immunohistochemistry confirmed DLBCL. Two cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone were administered. Blood cell numbers and hyperbilirubinemia improved. Although the patient did not completely recover, he survived for at least 3 years after chemotherapy and receiving blood transfusions. PBM DLBCL is a distinct, aggressive lymphoma characterized by lymphoma cells only in the bone marrow and effectively treated via chemotherapy. Prognoses for PBM DLBCL vary.

  3. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    SciTech Connect

    Cuneo, Kyle C.; Mito, Jeffrey K.; Javid, Melodi P.; Ferrer, Jorge M.; Kim, Yongbaek; Lee, W. David; Bawendi, Moungi G.; Brigman, Brian E.; Kirsch, David G.

    2013-05-01

    Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

  4. Dysregulated TGF-β signaling alters bone microstructure in a mouse model of Loeys-Dietz syndrome.

    PubMed

    Dewan, Ashvin K; Tomlinson, Ryan E; Mitchell, Stuart; Goh, Brian C; Yung, Rachel M; Kumar, Sarvesh; Tan, Eric W; Faugere, Marie-Claude; Dietz, Harry C; Clemens, Thomas L; Sponseller, Paul D

    2015-10-01

    Loeys-Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular and skeletal abnormalities resembling Marfan syndrome, including a predisposition for pathologic fracture. LDS is caused by heterozygous mutations in the genes encoding transforming growth factor-β (TGF-β) type 1 and type 2 receptors. In this study, we characterized the skeletal phenotype of mice carrying a mutation in the TGF-β type 2 receptor associated with severe LDS in humans. Cortical bone in LDS mice showed significantly reduced tissue area, bone area, and cortical thickness with increased eccentricity. However, no significant differences in trabecular bone volume were observed. Dynamic histomorphometry performed in calcein-labeled mice showed decreased mineral apposition rates in cortical and trabecular bone with normal numbers of osteoblasts and osteoclasts. Mechanical testing of femurs by three-point bending revealed reduced femoral strength and fracture resistance. In vitro, osteoblasts from LDS mice demonstrated increased mineralization with enhanced expression of osteoblast differentiation markers compared with control cells. These changes were associated with impaired TGF-β1-induced Smad2 and Erk1/2 phosphorylation and upregulated TGF-β1 ligand mRNA expression, compatible with G357W as a loss-of-function mutation in the TGF-β type 2 receptor. Paradoxically, phosphorylated Smad2/3 in cortical osteocytes measured by immunohistochemistry was increased relative to controls, possibly suggesting the cross-activation of TGF-β-related receptors. The skeletal phenotype observed in the LDS mouse closely resembles the principal structural features of bone in humans with LDS and establishes this mouse as a valid in vivo model for further investigation of TGF-β receptor signaling in bone. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  5. Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting

    PubMed Central

    Lowell, Clifford A.

    2017-01-01

    In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 × 106 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured ex vivo to understand their roles in various biological processes. PMID:28180137

  6. In vitro comparison of primary stability of two implant designs in D3 bone

    PubMed Central

    Ortega-Aranegui, Ricardo; López-Quiles, Juan

    2017-01-01

    Background Primary stability (PS) is a key factor for implant survival rate and depends on implant design or bone quality. The aim of this study was to compare different thread designs implants, evaluating PS with periotest values (PV) and implant stability quotient (ISQ) values through resonance frequency analysis (RFA). Material and Methods A total of 60 implants (Radhex®, Inmet-Garnick S.A., Guadalajara, Spain) were placed in freshly bovine ribs in vitro. Two designs were used: 30 tapered body with single thread design (PHI) and 30 tapered body with double thread design implants (PHIA). Both designs were 4mm wide and 12mm long. Implants were placed according to manufacturer’s guidelines. Osstell™ and Periotest® devices were used to evaluate PS by a blinded independent observer. Computed tomographies (CTs) of the ribs were made (BrightSpeed Series CT systems, GE Healthcare, Milwaukee, WI, USA) and bone quality surrounding each implant was evaluated in Hounsfield Units (HU) using Ez3D Plus software (Vatech Co., Korea). Bone quality was classified according to Misch and Kircos in D1, D2, D3 or D4. Results All implants were mechanically stable. Only implants placed in D3 bone (350-850 HU) were selected for the study: 28 PHI and 26 PHIA. The one way ANOVA showed significant difference (p<0.005) among two implants designs in ISQ values (61,55 ± 6,67 in PHI and 68,94 ± 5,82 in PHIA). No significant difference (p = 0,171) was shown in PV between two designs (-4,47 ± 1,39 in PHI and -4,77 ± 0,87 in PHIA). Conclusions Higher PS was found using Osstell™ device in implants with double thread design (PHIA) in comparison to implants with single thread design (PHI) in D3 bone. Key words:Dental implant, primary stability, Osstell, Periotest, bone quality, implant design. PMID:28578374

  7. Bone Quality determined by Fourier Transform Infrared Imaging Analysis in Mild Primary Hyperparathyroidism

    PubMed Central

    Zoehrer, Ruth; Dempster, David W.; Bilezikian, John P.; Zhou, Hua; Silverberg, Shonni J.; Shane, Elizabeth; Roschger, Paul; Paschalis, Eleftherios P.; Klaushofer, Klaus

    2008-01-01

    Context: Mild primary hyperparathyroidism (PHPT) is characterized by asymptomatic hypercalcemia, most commonly in the absence of classical signs and symptoms. Hence, there is need to characterize this disorder with particular attention to the skeleton. Design: We determined the ratio of pyridinium and dehydrodihydroxylysinonorleucine collagen cross-links in 46 iliac crest bone biopsies from patients with PHPT (14 men, aged 28–68 yr; 32 women, aged 26–74 yr) by Fourier transform infrared imaging. The results were compared with previously reported collagen cross-links ratio determined in iliac crest biopsies from normal subjects. Results: PHPT patients exhibited significantly lower pyridinium to dehydrodihydroxylysinonorleucine collagen cross-links ratio, compared with normal controls. Parathyroidectomy restored values to those comparable with normal controls. Moreover, the differences among PHPT subjects were gender dependent, with female PHPT patients having a statistically significant lower ratio, compared with either male PHPT patients or normal controls. Comparison of the obtained outcomes with histomorphometry showed that the collagen cross-link ratio was strongly correlated with rate of bone formation, and mineralizing surface, in individual patients. This ratio was also correlated with bone mineralization density distribution parameters obtained in the same patients. The strongest correlations were with bone mineralization density distribution variables reflecting heterogeneity of mineralization and primary mineralization parameters. Conclusions: The results are consistent with the high turnover state manifested in PHPT patients. Reduced collagen cross-link ratio in patients with PHPT would be expected to reduce the stiffness of bone tissue. These observations provide a more complete assessment of bone material properties in this disorder. PMID:18593769

  8. Primary lymphoma of bone in the pediatric and young adult population.

    PubMed

    Chisholm, Karen M; Ohgami, Robert S; Tan, Brent; Hasserjian, Robert P; Weinberg, Olga K

    2017-02-01

    Primary lymphoma of bone (PLB) accounts for 3% to 7% of primary neoplasms of bone and must be distinguished from more common bone tumors in the pediatric population such as osteosarcoma, Ewing sarcoma, and other small round blue cell tumors. In this study, pathology databases from 4 institutions were queried for PLB in individuals 1 to 21 years old. A total of 54 cases of PLB were identified, including 41 diffuse large B-cell lymphomas (DLBCL, 76%), 8 B-lymphoblastic lymphomas (BLL, 15%), 3 anaplastic large cell lymphomas (ALCL, 6%), and 2 low-grade follicular lymphomas (4%). The male/female ratio was 1.8:1 and median age was 16 years (range, 2-21). Patients with DLBCL were significantly older (P<.001), and patients with ALCL and BLL were significantly younger (P=.050 and P=.008, respectively) when compared with the other patients. Due to necrosis, crush artifact, and/or insufficient material, 30% of cases required multiple biopsies for diagnosis. The femur, tibia, pelvic bones, humerus, and vertebrae were most commonly involved. DLBCL patients had significantly more solitary bone involvement (P=.001), whereas BLL had significantly more polyostotic involvement (P<.001). Of the 37 patients with outcome data, all had no evidence of disease on last follow-up. This largest pediatric series of PLB identifies DLBCL as the most frequent subtype and documents rarer occurrences of BLL, ALCL, and follicular lymphomas. The differential diagnosis of bone neoplasms in pediatric patients, including those with necrosis, should include PLB. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. A rare clinical case of the isolated primary frontal bone osteoma.

    PubMed

    Pohranychna, Kh

    2016-09-01

    To study a rare clinical case of isolated primary osteoma localized in the frontal bone, provide its detailed clinical and pathomorphological analysis, and evaluate statistical indicators of occurrence frequency and localization of facial skeleton osteomas during 2006-2015. The retrospective case records analysis of patients undergoing treatment in the ma-xillofacial department of Lviv Regional Clinical Hospital and suffering from facial skeleton osteomas was conducted. The clinical examination was carried out in accordance with the inspection protocol required for the examination of patients diagnosed with tumors and tumor-like neoplasms in a particular clinical case. Frontal and lateral views were used in the course of X-ray imaging. Histological studies included macroscopy and microscopy. According to the retrospective case record analysis made over the last 10 years 346 cases of facial bone osteoma were revealed and proved histologically comprising 3.7% of all benign osteogeneous maxillofacial tumors. For the first time in 10 years osteoma localized in the frontal bone had been revealed and thoroughly studied. Macroscopically - a semi-spherical and immovable neoformation 7 cm in diameter of hard consistency, splaying out at 4 cm and being adherent with the frontal bone. Roentgenologically - homogeneous intense shadowing of a round form with relatively distinct outlines complying with clinical sizes. According to the histological data, the tumor consists mostly of the solid osseous tissue and the spongy osseous tissue with the evident thickened trabeculas of the bone located in different directions. The analysis of historical data demonstrated extremely rare localization of primary osteoma of the frontal bone, and indicated the uniqueness of the case and significant clinical importance of its detailed study.

  10. The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

    PubMed

    Tsujigiwa, Hidetsugu; Hirata, Yasuhisa; Katase, Naoki; Buery, Rosario Rivera; Tamamura, Ryo; Ito, Satoshi; Takagi, Shin; Iida, Seiji; Nagatsuka, Hitoshi

    2013-03-01

    Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) were transplanted into C57BL/6 J wild mice. After transplantation, bone injury was created using a 1.0-mm drill. Bone healing was histologically assessed at 3, 7, 14, and 28 postoperative days. Immunohistochemistry for GFP; double-fluorescent immunohistochemistry for GFP-F4/80, GFP-CD34, and GFP-osteocalcin; and double-staining for GFP and tartrate-resistant acid phosphatase were performed. Bone marrow transplantation successfully replaced the hematopoietic cells into GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts or osteocytes in the repair stage were GFP-negative, whereas osteoclasts in the repair and remodeling stages and hematopoietic cells were GFP-positive. The results indicated that bone marrow-derived cells might not differentiate into osteoblasts. The role of bone marrow-derived cells might be limited to adjustment of the microenvironment by differentiating into inflammatory cells, osteoclasts, or endothelial cells in immature blood vessels.

  11. Microarchitecture, but Not Bone Mechanical Properties, Is Rescued with Growth Hormone Treatment in a Mouse Model of Growth Hormone Deficiency

    PubMed Central

    Kristensen, Erika; Hallgrímsson, Benedikt; Morck, Douglas W.; Boyd, Steven K.

    2012-01-01

    Growth hormone (GH) deficiency is related to an increased fracture risk although it is not clear if this is due to compromised bone quality or a small bone size. We investigated the relationship between bone macrostructure, microarchitecture and mechanical properties in a GH-deficient (GHD) mouse model undergoing GH treatment commencing at an early (prepubertal) or late (postpubertal) time point. Microcomputed tomography images of the femur and L4 vertebra were obtained to quantify macrostructure and vertebral trabecular microarchitecture, and mechanical properties were determined using finite element analyses. In the GHD animals, bone macrostructure was 25 to 43% smaller as compared to the GH-sufficient (GHS) controls (P < 0.001). GHD animals had 20% and 19% reductions in bone volume ratio (BV/TV) and trabecular thickness (Tb.Th), respectively. Whole bone mechanical properties of the GHD mice were lower at the femur and vertebra (67% and 45% resp.) than the GHS controls (P < 0.001). Both early and late GH treatment partially recovered the bone macrostructure (15 to 32 % smaller than GHS controls) and the whole bone mechanical properties (24 to 43% larger than GHD animals) although there remained a sustained 27–52% net deficit compared to normal mice (P < 0.05). Importantly, early treatment with GH led to a recovery of BV/TV and Tb.Th with a concomitant improvement of trabecular mechanical properties. Therefore, the results suggest that GH treatment should start early, and that measurements of microarchitecture should be considered in the management of GHD. PMID:22505889

  12. The effect of dipeptidyl peptidase-IV inhibition on bone in a mouse model of type 2 diabetes

    PubMed Central

    Gallagher, Emily Jane; Sun, Hui; Kornhauser, Caroline; Tobin-Hess, Aviva; Epstein, Sol; Yakar, Shoshana; LeRoith, Derek

    2017-01-01

    Background Individuals with type 2 diabetes (T2D) are at greater risk of bone fractures than those without diabetes. Certain oral diabetic medications may further increase the risk of fracture. Dipeptidyl peptidase-IV (DPP-IV) inhibitors are incretin-based therapies that are being increasingly used for the management of T2D. It has been hypothesized that these agents may reduce fracture risk in those with T2D. In this study, we used a mouse model of T2D to examine the effects of the DPP-IV inhibitor, MK-0626, on bone. Methods Male wild type (WT) and diabetic muscle-lysine-arginine (MKR) mice were treated with MK-0626, pioglitazone, alendronate or vehicle. The effects of treatment with MK-0626 on bone microarchitecture and turnover were compared with treatment with pioglitazone, alendronate and vehicle. Osteoblast differentiation was determined by alkaline phosphatase staining of bone marrow cells from WT and MKR mice after treatment with pioglitazone, MK-0626 or phosphate buffered saline. Results We found that MK-0626 had neutral effects on cortical and trabecular bone in diabetic mice. Pioglitazone had detrimental effects on the trabecular bone of WT but not of diabetic mice. Alendronate caused improvements in cortical and trabecular bone architecture in diabetic and WT mice. MK-0626 did not alter osteoblast differentiation, but pioglitazone impaired osteoblast differentiation in vitro. Conclusions Overall, the DPP-IV inhibitor, MK-0626, had no adverse effects on bone in an animal model of T2D or directly on osteoblasts in culture. These findings are reassuring as DPP-IV inhibitors are being widely used to treat patients with T2D who are already at an increased risk of fractures. PMID:24023014

  13. Examination of ERα signaling pathways in bone of mutant mouse models reveals the importance of ERE-dependent signaling.

    PubMed

    Chokalingam, Kumar; Roforth, Matthew M; Nicks, Kristy M; McGregor, Ulrike; Fraser, Daniel; Khosla, Sundeep; Monroe, David G

    2012-11-01

    The mechanisms of estrogen receptor (ER)-α activity can be categorized into those involving direct (classical) or indirect (nonclassical) DNA binding. Although various mouse models have demonstrated the importance of ERα in bone, the specific gene expression patterns affected by these modes of ERα action are unknown. In this report, the gene expression patterns of ERα-deficient (ERKO) mice and nonclassical ER knock-in (NERKI) mice, which can function only by nonclassical means, were analyzed. Three-month-old mice were ovariectomized and implanted with estrogen pellets for 1 month to normalize estrogen levels. Microarray analysis of flushed cortical bone revealed 28% (210 of 763) of the genes differentially expressed in ERKO mice were altered in NERKI mice, suggesting estrogen response element-dependent regulation of these genes in bone. Pathway analysis revealed alterations in genes involved in focal adhesion and extracellular matrix interactions. However, the majority of genes regulated in ERKO mice (72%) were unique (i.e. not altered in NERKI mice), suggesting these are regulated by nonclassical mechanisms. To further explore the pathways affected in ERKO mice, we performed focused quantitative PCR arrays for genes involved in various aspects of bone physiology. Genes involved in bone formation, senescence, apoptosis, and autophagy were significantly regulated. Overall, the majority of the genes regulated by ERα in bone are via nonclassical pathways. However, because NERKI mice display an osteoporotic phenotype, it can be deduced that the minority of the estrogen response element-dependent genes/pathways play critical roles in the regulation of bone physiology. These data demonstrate the importance of classical ERα signaling in regulating bone metabolism.

  14. Facial growth and development in unilateral cleft lip and palate: comparison between secondary alveolar bone grafting and primary periosteoplasty.

    PubMed

    Cagáňová, Veronika; Borský, Jiří; Smahel, Zbyněk; Velemínská, Jana

    2014-01-01

    To describe the effect of secondary alveolar bone grafting in patients with unilateral cleft lip and palate by comparison with a sample of patients who have undergone primary periosteoplasty. Cephalometric analysis of lateral x-ray films in a retrospective semilongitudinal study. Lateral x-ray films of 18 secondary alveolar bone grafting patients and 48 primary periosteoplasty patients at 10 years of age and again at 15 years of age. The treatment of secondary alveolar bone grafting patients included lip repair according to Tennison, palatoplasty including retropositioning, pharyngeal flap surgery, and secondary alveolar bone grafting. The lips of primary periosteoplasty patient were repaired using the methods of Tennison and Veau, followed by primary periosteoplasty, palatoplasty including retropositioning, and pharyngeal flap surgery. Lateral radiographs were assessed using classical morphometry. There were few significant differences at 10 years of age between the secondary alveolar bone grafting and primary periosteoplasty patients. At 15 years of age, there were several significant differences. Compared with primary periosteoplasty patients, subsequent development in patients who had undergone secondary alveolar bone grafting was characterized by a significantly better position of the upper and lower dentoalveolar components in relation to the facial plane, a higher increase in the global convexity of the soft profile, a significantly better maxillary inclination, and a more favorable development of vertical intermaxillary relationships. Craniofacial development in secondary alveolar bone grafting patients was better than that in primary periosteoplasty patients due to the more marked facial convexity, the increased prominence of the nose, and better vertical intermaxillary relationships.

  15. Primary stability of implants placed at different angulations in artificial bone.

    PubMed

    Kashi, Ajay; Gupta, Bhumija; Malmstrom, Hans; Romanos, Georgios E

    2015-02-01

    The aim of this study was to evaluate the primary stability (PS) of titanium implants with a progressive thread design and more thread stability in the apical threads placed in artificial bone materials. A total of 120 implants were placed in commercially available polyurethane composite bone blocks. The angulations that were chosen to place the implants in bone types II and IV were 0, 10, and 20 degrees, respectively. The implant dimensions were 11 mm in length and 3.5 mm in diameter. Two clinicians placed all implants, and an independent examiner evaluated the PS using the Osstell (ISQ) and Periotest devices. The χ test was used to evaluate the statistical differences between the PS at different angulations. This study showed that there was a statistically significant difference (P = 0.02) of the PS values, when measured using the Periotest values, among all 3 angulations in both bone qualities. Tilted implants with 10 degrees, angulation had a better stability than conventionally placed implants. The PS of dental implants is higher for implants placed in type II when compared with type IV artificial bone. A higher stability was found for implants placed with 10-degree angulations.

  16. The solitary (primary) cilium--a mechanosensory toggle switch in bone and cartilage cells.

    PubMed

    Whitfield, J F

    2008-06-01

    Osteocytes and articular chondrocytes sense and respond to the strains imposed on bones and joints by various activities such as breathing and walking. This mechanoresponsiveness is needed to maintain bone and cartilage microstructure and strength. In bone the large number of osteocytes form a vast osteointernet in which the gap junctionally interconnected members are lodged in an extensive lacunocanalicular network. The much smaller number of articular chondrocytes are not interconnected in a chondrointernet. Instead, they are separately lodged in capsules called chondrons. While there are many possible strain-sensing devices, it now appears that the non-motile solitary (primary) cilia protruding like aerials from osteocytes (as well as osteoblasts) and chondrocytes are switches that when toggled by cyclical pulses of lacunocanalicular fluid or cartilage compression send signals such as Ca(2+) surges into the cell to trigger a cascade of events that include appropriate gene activations to maintain and strengthen bone and cartilage. Moreover, the chondrocyte cilium with its Ihh(Indian hedgehog)-activated Smo receptor is a key player along with PTHrP in endochondral bone formation.

  17. [Bone metastases from unknown primary. FDG-PET detection of two synchronous tumors].

    PubMed

    Cuenca Cuenca, J I; Borrego Dorado, I; Rodríguez Rodríguez, J R; Navarro González, E; Vázquez Albertino, R

    2007-01-01

    A 60-year-old woman presented with noncardiac chest pain over months and negative laboratory findings. Conventional imaging methods and bone scintigraphy detected bone lesions suggesting metastatic disease from an unknown primary tumor. An 18FDG-PET scan performed to orient the search for the primary tumor found focal lesions suggesting lymphoma and identified a focal thyroid lesion and a cervical lymph node accessible for biopsy. The biopsy of this lymph node incidentally detected a papillary differentiated thyroid cancer (DTC), since the existence of a non-Hodgkin's lymphoma was confirmed after a new biopsy. After confirming the presence of a lymphoma, 18FDG-PET enabled the initial staging of the tumor, the evaluation of the response to treatment, and follow-up for detection of recurrence. On the other hand, 18FDG-PET incidentally detected a DTC.

  18. Effect of Coadministration of Vancomycin and BMP-2 on Cocultured Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells in Vitro

    DTIC Science & Technology

    2012-07-01

    Hudson MC. 2000. Intracellular Staph - ylococcus aureus induces apoptosis in mouse osteoblasts. FEMS Micro- biol. Lett. 186:151–156. 45. Wang Y, et al...Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells In Vitro 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Cocultured Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells In Vitro A. H. Nguyen,a S. Kim,a,b W. J. Maloney,b J. C. Wenke,c and Y. Yanga

  19. Primary Neoplasms of Bones in Mice: Retrospective Study and Review of Literature

    PubMed Central

    Kavirayani, A. M.; Sundberg, J. P.; Foreman, O.

    2011-01-01

    To compare and summarize the mechanisms, frequencies of occurrence, and classification schemes of spontaneous, experimental, and genetically engineered, mouse skeletal neoplasms, the literature was reviewed and archived case material at The Jackson Laboratory examined. The frequency of occurrence of spontaneous bone neoplasms was less than 1% for most strains, with the exceptions of osteomas in CF-1 (5.5% and 10% in two studies) and OF-1 outbred strains (35%), and osteosarcomas in NOD/ShiLtJ (11.5%) and NOD derived (7.1%) mice. The frequency was 100% for osteochondromas induced by conditional inactivation of exostoses (multiple) 1 (Ext1) in chondrocytes, osteosarcomas induced by tibial intramedullary inoculation of Moloney’s murine sarcoma virus, and osteosarcomas induced by conditional inactivation of Trp53-with or without inactivation of Rb1-in osteoblast precursors. Spontaneous osteogenic neoplasms were more frequent than spontaneous cartilaginous and vascular types. Malignant neoplasms were more frequent than benign ones. The age of occurrence for spontaneous neoplasms ranged from 37 to 720 (Mean 316.35) days for benign, and 35 to 990 (Mean 299.28) days for malignant neoplasms. In genetically engineered mice, the average age of occurrence ranged from 28 to 70 days for benign, and from 35 to 690 days for malignant neoplasms. Histologically, non-osteogenic neoplasms were similar across strains and mutant stocks; osteogenic neoplasms exhibited greater diversity. This comparison and summarization of mouse bone neoplasms provides valuable information for the selection of strains to create, compare, and validate models of bone neoplasms. PMID:21343597

  20. RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

    PubMed

    Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

    2014-10-01

    Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM fMLF and 1μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells.

  1. Longitudinal evaluation of mouse hind limb bone loss after spinal cord injury using novel, in vivo, methodology.

    PubMed

    McManus, Madonna M; Grill, Raymond J

    2011-12-07

    Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse, aberrant neuronal signaling and hormonal changes. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies. Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis. An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (naïve) mice (13% decrease, p < 0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal

  2. The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

    SciTech Connect

    Taguchi, Kazuhiro . E-mail: s3061@nms.ac.jp; Ogawa, Rei; Migita, Makoto; Hanawa, Hideki; Ito, Hiromoto; Orimo, Hideo

    2005-05-27

    We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses.

  3. Adherent Primary Cultures of Mouse Intercostal Muscle Fibers for Isolated Fiber Studies

    PubMed Central

    Robison, Patrick; Hernández-Ochoa, Erick O.; Schneider, Martin F.

    2011-01-01

    Primary culture models of single adult skeletal muscle fibers dissociated from locomotor muscles adhered to glass coverslips are routine and allow monitoring of functional processes in living cultured fibers. To date, such isolated fiber cultures have not been established for respiratory muscles, despite the fact that dysfunction of core respiratory muscles leading to respiratory arrest is the most common cause of death in many muscular diseases. Here we present the first description of an adherent culture system for single adult intercostal muscle fibers from the adult mouse. This system allows for monitoring functional properties of these living muscle fibers in culture with or without electrical field stimulation to drive muscle fiber contraction at physiological or pathological respiratory firing patterns. We also provide initial characterization of these fibers, demonstrating several common techniques in this new model system in the context of the established Flexor Digitorum Brevis muscle primary culture model. PMID:21869860

  4. Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

    PubMed Central

    Alterman, Julia F; Hall, Lauren M; Coles, Andrew H; Hassler, Matthew R; Didiot, Marie-Cecile; Chase, Kathryn; Abraham, Jasmin; Sottosanti, Emily; Johnson, Emily; Sapp, Ellen; Osborn, Maire F; Difiglia, Marian; Aronin, Neil; Khvorova, Anastasia

    2015-01-01

    Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context. PMID:26623938

  5. Extensive primary Ewings' sarcoma in the greater wing of the sphenoid bone.

    PubMed

    Apostolopoulos, Kostas; Ferekidis, Eleftherios

    2003-01-01

    We describe a rare case of an extensive primary cranial Ewing's sarcoma located in the greater wing of the sphenoid bone with extension to the orbit, the endocranium, the parapharyngeal and infratemporal space. The patient presented with diplopia, anosmia and prolapse of the left eye. He was given chemo- and radiotherapy and was free of symptoms on re-examination 1.5 years later. The prognosis of Ewing's sarcoma in the absence of surgery is uncertain, but prompt treatment appears to have a satisfactory therapeutic outcome. In the future, more cases should be studied in order to investigate the biological behaviour of a primary cranial Ewing's sarcoma.

  6. Turnover of bone marrow-derived cells in the irradiated mouse cornea

    PubMed Central

    Chinnery, Holly R; Humphries, Timothy; Clare, Adam; Dixon, Ariane E; Howes, Kristen; Moran, Caitlin B; Scott, Danielle; Zakrzewski, Marianna; Pearlman, Eric; McMenamin, Paul G

    2008-01-01

    In light of an increasing awareness of the presence of bone marrow (BM)-derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1gfp transgenic mice in which all monocyte-derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM-derived cells from transgenic eGFP mice were transplanted into whole-body irradiated wild-type recipients. Additionally, wild-type BM-derived cells were injected into irradiated Cx3cr1+/gfp recipients, creating reverse chimeras. At 2, 4 and 8 weeks post-reconstitution, the number of eGFP+ cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid-derived cells in the normal Cx3cr1+/gfp cornea was 366 cells/mm2. In BM chimeras 2 weeks post-reconstitution, 24% of the myeloid-derived cells had been replenished and were predominantly located in the anterior stroma. By 8 weeks post-reconstitution 75% of the myeloid-derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP+ cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid-derived cells in the mouse corneal stroma are replenished after 8 weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non-haematopoietic cells using BM chimeric mice in models of corneal inflammation. PMID:18540963

  7. Primary Neuroendocrine Carcinoma of the Breast Metastatic to the Bones, Which Chemotherapy?

    PubMed

    Soe, Aye M; Joseph, Gardith; Guevara, Elizabeth; Xiao, Philip

    2017-09-01

    The primary neuroendocrine carcinoma (NEC) of the breast is defined as immunohistochemical expression of neuroendocrine markers (chromogranin and synoptophysin) in more than 50% of the neoplastic cells according to World Health Organization (WHO) classification of tumors in 2003 (Tumours of the Breast and Female Genital Organs, 2003, Lyon: IARC Press). It accounts for less than 5% of all cancers arising from the breast (Tumours of the Breast and Female Genital Organs, 2003, Lyon, France: IARC Press). However, based on the study conducted by Wang et al., the primary NEC of breast comprises less than 0.1% of all mammary carcinomas (Frankf Z Pathol, 73, 1963, 24). Because of the rarity of the disease and absence of the prospective trials, there is no standard treatment for primary NEC of the breast. Herein, we report the case of a middle age woman with primary NEC with bone metastasis. © 2017 Wiley Periodicals, Inc.

  8. In vivo visualizing the dynamics of bone marrow stem cells in mouse retina and choroidal-retinal circulation

    NASA Astrophysics Data System (ADS)

    Wang, Heuy-Ching H.; Zwick, Harry; Edsall, Peter R.; Cheramie, Rachel D.; Lund, David J.; Stuck, Bruce

    2007-02-01

    It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. Therefore it is an attractive therapeutic intervention to apply autologous bone marrow-derived stem cells that may offer neuroprotection to laser-induced retinal injuries. The purpose of this study is to develop a method with which to visualize bone marrow stem cells dynamics in mouse retinal circulation. We have used a physiological method, confocal scanning laser ophthalmoscope (SLO), to track the highly enriched stem/progenitor cells circulating in the retina. Stem cells were enriched by immunomagnetic depletion of cells committed to the T- and B lymphocytic, myeloid and erythorid lineages. CellTracker TM Green-labeled stem cells were injected into the tail veins of mice with laser-induced focal retinal injuries. Bone marrow stem cells labeled with CellTracker TM Green were visible in the retinal circulation for as long as 1 hour and 30 minutes. These studies suggest that stem cell-enriched bone marrow cells may have the ability to mobilize into laser-induced retinal injuries and possibly further proliferate, differentiate and functionally integrate into the retina.

  9. Enumeration of the colony-forming units–fibroblast from mouse and human bone marrow in normal and pathological conditions

    PubMed Central

    Kuznetsov, Sergei A.; Mankani, Mahesh H.; Bianco, Paolo; Robey, Pamela G.

    2009-01-01

    Bone marrow stromal cell populations, containing a subset of multipotential skeletal stem cells, are increasingly contemplated for use in tissue engineering and stem cell therapy, whereas their involvement in the pathogenetic mechanisms of skeletal disorders is far less recognized. We compared the concentrations of stromal clonogenic cells, colony forming units–fibroblast (CFU-Fs), in norm and pathology. Initially, culture conditions were optimized by demonstrating that fetal bovine serum heat inactivation could significantly repress colony formation. Using non-heat-inactivated fetal bovine serum, the concentration of CFU-Fs (colony-forming efficiency, CFE) ranged from 3.5 ± 1.0 to 11.5 ± 4.0 per 1 × 105 nucleated cells in five inbred mouse strains. In four transgenic lines with profound bone involvement, CFE was either significantly reduced or increased compared to wild-type littermates. In normal human donors, CFE decreased slightly with age and averaged 52.2 ± 4.1 for children and 32.3 ± 3.0 for adults. CFE was significantly altered in patients with several skeletal, metabolic, and hematological disorders: reduced in congenital generalized lipodystrophy, achondroplasia (SADDAN), pseudoachondroplasia, and Paget disease of bone and elevated in alcaptonuria and sickle cell anemia. Our findings indicate that under appropriate culture conditions, CFE values may provide useful insights into bone/bone marrow pathophysiology. PMID:19383412

  10. Constructing a multi-scan synchrotron X-ray microscope to study the function of osteocyte canaliculi in mouse bone

    NASA Astrophysics Data System (ADS)

    Nango, Nobuhito; Kubota, Shogo; Yashiro, Wataru; Momose, Atsushi; Takada, Yasunari; Matsuo, Koichi

    2012-07-01

    Formulating a multi-scan method applied to an X-ray microscope CT with synchrotron radiation, we attempted to analyze the 3D functional structure of osteocyte canaliculi inside the cortical bone of a mouse tibia. We employed a two-method combination to scan the same position of the specimen. To extract the internal bone canalicular structure, we first combined a Talbot interferometer with an X-ray microscope, and applied a differential phase imaging method to measure the absolute value of bone mineral around the canaliculi. Next, we used the X-ray microscope without the Talbot interferometer under a defocus condition, moving the specimen toward the zone plate by 6 mm. This defocus contrast method visualizes the canaliculi by emphasizing the edges of the bone. We performed CT scans by the two configurations and precisely aligned resultant 3D images so that the same position in the specimen is compared. We could extract the osteocyte canaliculi and evaluate the mineral density of their surroundings. The degree of mineralization varied for each osteocyte lacuna and canaliculus. The multi-scan microscopic X-ray CT is a powerful tool for analyzing bone mineralization.

  11. Constructing a multi-scan synchrotron X-ray microscope to study the function of osteocyte canaliculi in mouse bone

    SciTech Connect

    Nango, Nobuhito; Kubota, Shogo; Yashiro, Wataru; Momose, Atsushi; Takada, Yasunari; Matsuo, Koichi

    2012-07-31

    Formulating a multi-scan method applied to an X-ray microscope CT with synchrotron radiation, we attempted to analyze the 3D functional structure of osteocyte canaliculi inside the cortical bone of a mouse tibia. We employed a two-method combination to scan the same position of the specimen. To extract the internal bone canalicular structure, we first combined a Talbot interferometer with an X-ray microscope, and applied a differential phase imaging method to measure the absolute value of bone mineral around the canaliculi. Next, we used the X-ray microscope without the Talbot interferometer under a defocus condition, moving the specimen toward the zone plate by 6 mm. This defocus contrast method visualizes the canaliculi by emphasizing the edges of the bone. We performed CT scans by the two configurations and precisely aligned resultant 3D images so that the same position in the specimen is compared. We could extract the osteocyte canaliculi and evaluate the mineral density of their surroundings. The degree of mineralization varied for each osteocyte lacuna and canaliculus. The multi-scan microscopic X-ray CT is a powerful tool for analyzing bone mineralization.

  12. PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes.

    PubMed

    Kandil, E; Kohda, K; Ishibashi, T; Tanaka, K; Kasahara, M

    1997-01-01

    The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-gamma (IFN-gamma)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 alpha- and beta-subunits. The deduced amino acid sequences of the alpha- and beta-subunits were 49% identical. We also determined the primary structure of the mouse PA28 gamma-subunit (Ki antigen), a protein of unknown function structurally related to the alpha- and beta-subunits. The amino acid sequence identity of the gamma-subunit to the alpha- and beta-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the alpha- and beta-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-gamma-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the gamma-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a gamma-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-gamma-inducible alpha- and beta-subunits emerged by gene duplication from a gamma-subunit-like precursor.

  13. Endogenous retrovirus induces leukemia in a xenograft mouse model for primary myelofibrosis

    PubMed Central

    Triviai, Ioanna; Ziegler, Marion; Bergholz, Ulla; Oler, Andrew J.; Stübig, Thomas; Prassolov, Vladimir; Fehse, Boris; Kozak, Christine A.; Kröger, Nicolaus; Stocking, Carol

    2014-01-01

    The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdcscid and Il2rgnull alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation. PMID:24912157

  14. Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes.

    PubMed

    Akie, Thomas E; Cooper, Marcus P

    2015-08-27

    Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via β-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying β-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of β-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

  15. Chemokine-Targeted Mouse Models of Human Primary and Metastatic Colorectal Cancer

    PubMed Central

    Chen, Huanhuan Joyce; Sun, Jian; Huang, Zhiliang; Hou, Harry; Arcilla, Myra; Rakhilin, Nikolai; Joe, Daniel J.; Choi, Jiahn; Gadamsetty, Poornima; Milsom, Jeff; Nandakumar, Govind; Longman, Randy; Zhou, Xi Kathy; Edwards, Robert; Chen, Jonlin; Chen, Kai Yuan; Bu, Pengcheng; Wang, Lihua; Xu, Yitian; Munroe, Robert; Abratte, Christian; Miller, Andrew D.; Gümüş, Zeynep H.; Shuler, Michael; Nishimura, Nozomi; Edelmann, Winfried; Shen, Xiling; Lipkin, Steven M.

    2015-01-01

    Current orthotopic xenograft models of human colorectal cancer (CRC) require surgery and do not robustly form metastases in the liver, the most common site clinically. CCR9 traffics lymphocytes to intestine and colorectum. We engineered use of the chemokine receptor CCR9 in CRC cell lines and patient-derived cells to create primary gastrointestinal (GI) tumors in immunodeficient mice by tail-vein injection rather than surgery. The tumors metastasize inducibly and robustly to the liver. Metastases have higher DKK4 and NOTCH signaling levels and are more chemoresistant than paired sub-cutaneous xenografts. Using this approach, we generated 17 chemokine-targeted mouse models (CTMMs) that recapitulate the majority of common human somatic CRC mutations. We also show that primary tumors can be modeled in immunocompetent mice by microinjecting CCR9-expressing cancer cell lines into early-stage mouse blastocysts, which induces central immune tolerance. We expect that CTMMs will facilitate investigation of the biology of CRC metastasis and drug screening. PMID:26006007

  16. Establishment of primary cultures for mouse ameloblasts as a model of their lifetime

    SciTech Connect

    Suzawa, Tetsuo . E-mail: suzawa@dent.showa-u.ac.jp; Itoh, Nao; Takahashi, Naoyuki; Katagiri, Takenobu; Morimura, Naoko; Kobayashi, Yasuna; Yamamoto, Toshinori; Kamijo, Ryutaro

    2006-07-07

    To understand how the properties of ameloblasts are spatiotemporally regulated during amelogenesis, two primary cultures of ameloblasts in different stages of differentiation were established from mouse enamel epithelium. Mouse primary ameloblasts (MPAs) prepared from immature enamel epithelium (MPA-I) could proliferate, whereas those from mature enamel epithelium (MPA-M) could not. MPA-M but not MPA-I caused apoptosis during culture. The mRNA expression of amelogenin, a marker of immature ameloblasts, was down-regulated, and that of enamel matrix serine proteiase-1, a marker of mature ameloblasts, was induced in MPA-I during culture. Using green fluorescence protein as a reporter, a visualized reporter system was established to analyze the promoter activity of the amelogenin gene. The region between -1102 bp and -261 bp was required for the reporter expression in MPA-I. These results suggest that MPAs are valuable in vitro models for investigation of ameloblast biology, and that the visualized system is useful for promoter analysis in MPAs.

  17. Characterization of the primary spinal afferent innervation of the mouse colon using retrograde labelling.

    PubMed

    Robinson, D R; McNaughton, P A; Evans, M L; Hicks, G A

    2004-02-01

    Visceral pain is the most common form of pain produced by disease and is thus of interest in the study of gastrointestinal (GI) complaints such as irritable bowel syndrome, in which sensory signals perceived as GI pain travel in extrinsic afferent neurones with cell bodies in the dorsal root ganglia (DRG). The DRG from which the primary spinal afferent innervation of the mouse descending colon arises are not well defined. This study has combined retrograde labelling and immunohistochemistry to identify and characterize these neurones. Small to medium-sized retrogradely labelled cell bodies were found in the DRG at levels T8-L1 and L6-S1. Calcitonin gene-related peptide (CGRP)- and P2X3-like immunoreactivity (LI) was seen in 81 and 32%, respectively, of retrogradely labelled cells, and 20% bound the Griffonia simplicifolia-derived isolectin IB4. CGRP-LI and IB4 were co-localized in 22% of retrogradely labelled cells, whilst P2X3-LI and IB4 were co-localized in 7% (vs 34% seen in the whole DRG population). Eighty-two per cent of retrogradely labelled cells exhibited vanilloid receptor 1-like immunoreactivity (VR1-LI). These data suggest that mouse colonic spinal primary afferent neurones are mostly peptidergic CGRP-containing, VR1-LI, C fibre afferents. In contrast to the general DRG population, a subset of neurones exist that are P2X3 receptor-LI but do not bind IB4.

  18. Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes.

    PubMed

    Hwang, Geun Hye; Jeon, Yu Jin; Han, Ho Jae; Park, Soo Hyun; Baek, Kyoung Min; Chang, Woochul; Kim, Joong Sun; Kim, Lark Kyun; Lee, You-Mie; Lee, Sangkyu; Bae, Jong-Sup; Jee, Jun-Goo; Lee, Min Young

    2015-01-01

    Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation.

  19. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication.

    PubMed

    Wang, Gefei; Li, Rui; Jiang, Zhiwu; Gu, Liming; Chen, Yanxia; Dai, Jianping; Li, Kangsheng

    2016-01-01

    Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy.

  20. Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

    PubMed Central

    Hwang, Geun Hye; Jeon, Yu Jin; Han, Ho Jae; Park, Soo Hyun; Baek, Kyoung Min; Chang, Woochul; Kim, Joong Sun; Kim, Lark Kyun; Lee, You-Mie; Lee, Sangkyu; Bae, Jong-Sup; Jee, Jun-Goo

    2015-01-01

    Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation. PMID:25798044

  1. Synaptic Properties of Corticocortical Connections between the Primary and Secondary Visual Cortical Areas in the Mouse

    PubMed Central

    De Pasquale, Roberto; Sherman, S. Murray

    2011-01-01

    Despite the importance of corticocortical connections, few published studies have investigated the functional, synaptic properties of such connections in any species, because most studies have been purely anatomical or aimed at functional features other than synaptic properties. We recently published a study of synaptic properties of connections between the primary and secondary cortical auditory areas in brain slices from the mouse, and in the present study we aimed to extend this by performing analogous studies of the primary and secondary visual areas (V1 and V2). We found effectively the same results. That is, connections between V1 and V2 in both directions were quite similar; in each case the glutamatergic inputs could be classified as one of two types, Classes 1B (formerly “driver”) and 2 (formerly “modulator”). There is a clear laminar correlation for these different inputs, both in terms of the laminae of origin and those in which the recorded cells were located. Our data suggest common pattern to the functional organization of corticocortical connectivity in the mouse cortex. PMID:22090476

  2. Alpha-1 antitrypsin gene therapy prevented bone loss in ovariectomy induced osteoporosis mouse model

    USDA-ARS?s Scientific Manuscript database

    Osteoporosis is a major healthcare burden affecting mostly postmenopausal women characterized by compromised bone strength and increased risk of fragility fracture. Although pathogenesis of this disease is complex, elevated proinflammatory cytokine production is clearly involved in bone loss at meno...

  3. CXC receptor knockout mice: characterization of skeletal features and membranous bone healing in the adult mouse.

    PubMed

    Bischoff, David S; Sakamoto, Taylor; Ishida, Kenji; Makhijani, Nalini S; Gruber, Helen E; Yamaguchi, Dean T

    2011-02-01

    The potential role of CXC chemokines bearing the glu-leu-arg (ELR) motif in bone repair was studied using a cranial defect (CD) model in mice lacking the CXC receptor (mCXCR(-/-) knockout mice), which is homologous to knockout of the human CXC receptor 2 (CXCR2) gene. During the inflammatory stage of bone repair, ELR CXC chemokines are released by inflammatory cells and serve as chemotactic and angiogenic factors. mCXCR(-/-) mice were smaller in weight and length from base of tail to nose tip, compared to WT littermates. DEXA analysis indicated that bone mineral density (BMD), bone mineral content (BMC), total area (TA), bone area (BA), and total tissue mass (TTM) were decreased in the mCXCR(-/-) mice at 6, 12, and 18 weeks of age. Trabecular bone characteristics in mCXCR(-/-) (% bone, connectivity, number, and thickness) were reduced, and trabecular spacing was increased as evidenced by μCT. There was no difference in bone formation or resorption indices measured by bone histomorphometry. Trabecular BMD was not altered. Cortical bone volume, BMD, and thickness were reduced; whereas, bone marrow volume was increased in mCXCR(-/-). Decreased polar moment of inertia (J) in the tibias/femurs suggested that the mCXCR(-/-) long bones are weaker. This was confirmed by three-point bending testing of the femurs. CDs created in 6-week-old male mCXCR(-/-) and WT littermates were not completely healed at 12 weeks; WT animals, however, had significantly more bone in-growth than mCXCR(-/-). New bone sites were identified using polarized light and assessed for numbers of osteocyte (OCy) lacunae and blood vessels (BlV) around the original CD. In new bone, the number of BlV in WT was >2× that seen in mCXCR(-/-). Bone histomorphometry parameters in the cranial defect did not show any difference in bone formation or resorption markers. In summary, studies showed that mCXCR(-/-) mice have (1) reduced weight and size; (2) decreased BMD and BMC; (3) decreased amounts of trabecular

  4. Bone marrow transplantation reverses new-onset immunoinflammatory diabetes in a mouse model.

    PubMed

    Lv, Cheng-Lan; Wang, Jing; Xie, Ting; Ouyang, Jian

    2014-01-01

    Bone marrow transplantation might be an effective method to cure type 1 diabetes mellitus. This study aimed to investigate whether bone marrow transplantation could reverse hyperglycemia in diabetic mice and whether high-dose total body irradiation followed by high-dose bone marrow mononuclear cell infusion could improve the efficiency of bone marrow transplantation in treating diabetic mice. Diabetic mice after multiple low doses of streptozotocin injection were irradiated followed by infusion with approximately 1×10(7) bone marrow mononuclear cells intravenously. Before and after bone marrow transplantation, fasting blood glucose, intraperitoneal glucose tolerance test, serum insulin, pancreatic histology, and the examination of insulin and glucagon in islets were processed. All recipients returned to near euglycemic within 1 week after undergoing bone marrow transplantation. No mice became hyperglycemia again during investigation period. The change of serum insulin, glucose tolerance test, pancreatic histology and the expression of insulin and glucagon in recipient islets after bone marrow transplantation all revealed islets regeneration and significant amelioration when compared respectively with those of diabetic mice without bone marrow transplantation. Bone marrow transplantation contributed to reduce blood glucose, prevent further blood glucose hike in diabetic recipients, and promote islets regeneration. High-dose total body irradiation in combination with high-dose bone marrow monoclear cell infusion could improve the efficiency of bone marrow transplantation in treating streptozotocin-induced diabetes.

  5. Systemically Transplanted Bone Marrow-derived Cells Contribute to Dental Pulp Regeneration in a Chimeric Mouse Model.

    PubMed

    Xu, Wenan; Jiang, Shan; Chen, Qiuyue; Ye, Yanyan; Chen, Jiajing; Heng, Boon Chin; Jiang, Qianli; Wu, Buling; Ding, Zihai; Zhang, Chengfei

    2016-02-01

    Migratory cells via blood circulation or cells adjacent to the root apex may potentially participate in dental pulp tissue regeneration or renewal. This study investigated whether systemically transplanted bone marrow cells can contribute to pulp regeneration in a chimeric mouse model. A chimeric mouse model was created through the injection of bone marrow cells from green fluorescent protein (GFP) transgenic C57BL/6 mice into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 8.5 Gy from a high-frequency linear accelerator. These mice were subjected to pulpectomy and pulp revascularization. At 1, 4, and 8 weeks after surgery, in vivo animal imaging and histologic analyses were conducted. In vivo animal imaging showed that the green biofluorescence signal from the transplanted GFP+ cells increased significantly and was maintained at a high level during the first 4 weeks after surgery. Immunofluorescence analyses of tooth specimens collected at 8 weeks postsurgery showed the presence of nestin+/GFP+, α smooth muscle actin (α-SMA)/GFP+, and NeuN/GFP+ cells within the regenerated pulplike tissue. These data confirm that transplanted bone marrow-derived cells can contribute to dental pulp regeneration. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  6. Taurine, a major amino acid of oyster, enhances linear bone growth in a mouse model of protein malnutrition.

    PubMed

    Moon, Phil-Dong; Kim, Min-Ho; Lim, Hun-Sun; Oh, Hyun-A; Nam, Sun-Young; Han, Na-Ra; Kim, Myong-Jo; Jeong, Hyun-Ja; Kim, Hyung-Min

    2015-05-06

    Oysters (Oys) contain various beneficial components, such as, antioxidants and amino acids. However, the effects of Oys or taurine (Tau), a major amino acid in Oys on bone growth have not been determined. In the present study, we evaluated the effects of Oys or Tau on linear bone growth in a mouse model of protein malnutrition. To make the protein malnutrition in a mouse, we used a low protein diet. Growth plate thickness was increased by Oys or Tau. Bone volume/tissue volume, trabecular thickness, trabecular number, connection density, and total porosity were also improved by Oys or Tau. Oys or Tau increased insulin-like growth factor-1 (IGF-1) levels in serum, liver, and tibia-growth plate. Phosphorylations of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) were increased by Oys and by Tau. These findings show that Oys or Tau may increase growth plate thickness by elevating IGF-1 levels and by promoting the phosphorylations of JAK2-STAT5, and suggest that Oys or Tau are growth-promoting substances of potential use in the food and pharmaceutical industries. © 2015 International Union of Biochemistry and Molecular Biology.

  7. Contemporary adjuvant polymethyl methacrylate cementation optimally limits recurrence in primary giant cell tumor of bone patients compared to bone grafting: a systematic review and meta-analysis.

    PubMed

    Zuo, Dongqing; Zheng, Longpo; Sun, Wei; Fu, Dong; Hua, Yingqi; Cai, Zhengdong

    2013-07-16

    Reports of recurrence following restructuring of primary giant cell tumor (GCT) defects using polymethyl methacrylate (PMMA) bone cementation or allogeneic bone graft with and without adjuvants for intralesional curettage vary widely. Systematic review and meta-analysis were conducted to investigate efficacy of PMMA bone cementation and allogeneic bone grafting following intralesional curettage for GCT. Medline, EMBASE, Google Scholar, and Cochrane databases were searched for studies reporting GCT of bone treatment with PMMA cementation and/or bone grafting with or without adjuvant therapy following intralesional curettage of primary GCTs. Pooled risk ratios and 95% confidence intervals (CIs) for local recurrence risks were calculated by fixed-effects methods. Of 1,690 relevant titles, 6 eligible studies (1,293 patients) spanning March 2008 to December 2011 were identified in published data. Treatment outcomes of PMMA-only (n = 374), bone graft-only (n = 436), PMMA with or without adjuvant (PMMA + adjuvant; n = 594), and bone graft filling with or without adjuvant (bone graft + adjuvant; n = 699) were compared. Bone graft-only patients exhibited higher recurrence rates than PMMA-treated patients (RR 2.09, 95% CI (1.64, 2.66), Overall effect: Z = 6.00; P <0.001), and bone graft + adjuvant patients exhibited higher recurrence rates than PMMA + adjuvant patients (RR 1.66, 95% CI (1.21, 2.28), Overall effect: Z = 3.15, P = 0.002). Local recurrence was minimal in PMMA cementation patients, suggesting that PMMA is preferable for routine clinical restructuring in eligible GCT patients. Relationships between tumor characteristics, other modern adjuvants, and recurrence require further exploration.

  8. Contemporary adjuvant polymethyl methacrylate cementation optimally limits recurrence in primary giant cell tumor of bone patients compared to bone grafting: a systematic review and meta-analysis

    PubMed Central

    2013-01-01

    Background Reports of recurrence following restructuring of primary giant cell tumor (GCT) defects using polymethyl methacrylate (PMMA) bone cementation or allogeneic bone graft with and without adjuvants for intralesional curettage vary widely. Systematic review and meta-analysis were conducted to investigate efficacy of PMMA bone cementation and allogeneic bone grafting following intralesional curettage for GCT. Methods Medline, EMBASE, Google Scholar, and Cochrane databases were searched for studies reporting GCT of bone treatment with PMMA cementation and/or bone grafting with or without adjuvant therapy following intralesional curettage of primary GCTs. Pooled risk ratios and 95% confidence intervals (CIs) for local recurrence risks were calculated by fixed-effects methods. Results Of 1,690 relevant titles, 6 eligible studies (1,293 patients) spanning March 2008 to December 2011 were identified in published data. Treatment outcomes of PMMA-only (n = 374), bone graft-only (n = 436), PMMA with or without adjuvant (PMMA + adjuvant; n = 594), and bone graft filling with or without adjuvant (bone graft + adjuvant; n = 699) were compared. Bone graft-only patients exhibited higher recurrence rates than PMMA-treated patients (RR 2.09, 95% CI (1.64, 2.66), Overall effect: Z = 6.00; P <0.001), and bone graft + adjuvant patients exhibited higher recurrence rates than PMMA + adjuvant patients (RR 1.66, 95% CI (1.21, 2.28), Overall effect: Z = 3.15, P = 0.002). Conclusions Local recurrence was minimal in PMMA cementation patients, suggesting that PMMA is preferable for routine clinical restructuring in eligible GCT patients. Relationships between tumor characteristics, other modern adjuvants, and recurrence require further exploration. PMID:23866921

  9. Effect of Low Vitamin D on Volumetric Bone Mineral Density, Bone Microarchitecture, and Stiffness in Primary Hyperparathyroidism

    PubMed Central

    Nishiyama, Kyle K.; Zhou, Bin; Cong, Elaine; Wang, Ji; Lee, James A.; Kepley, Anna; Zhang, Chengchen; Guo, X. Edward; Silverberg, Shonni J.

    2016-01-01

    Context: Patients with 25-hydroxyvitamin D deficiency (25OHD <20 ng/ml) and primary hyperparathyroidism (PHPT) have more severe disease reflected by higher serum PTH levels compared to those with vitamin D levels in the insufficient (20–29 ng/ml) or replete range (≥30 ng/ml). Objective: To study the effect of low vitamin D in PHPT on volumetric bone mineral density (vBMD), bone microarchitecture, and bone strength. Design, Setting, and Participants: This is a cross-sectional analysis of 99 PHPT patients with and without 25OHD insufficiency and deficiency from a university hospital. Outcome Measures: Bone microarchitecture and strength were assessed with high-resolution peripheral quantitative computed tomography (HRpQCT), microfinite element analysis, and individual trabecula segmentation. Results: In this cohort, 25OHD levels were deficient in 18.1%, insufficient in 35.4% and replete in 46.5%. Those with lower 25OHD levels had higher PTH (P < .0001), were younger (P = .001) and tended to weigh more (P = .053). There were no age-, weight- and sex-adjusted between-group differences (<20 vs 20–29 vs ≥30 ng/ml) in any HRpQCT, microfinite element analysis, or individual trabecula segmentation indices. Because few participants had 25OHD below 20 ng/ml, we also compared those with 25OHD below 30 vs at least 30 ng/ml and found only a trend toward lower adjusted cortical vBMD (3.1%, P = .08) and higher cortical porosity (least squares mean ± SEM 7.5 ± 0.3 vs 6.6 ± 0.3%, P = .07) at the tibia but not the radius. Stiffness did not differ at either site. In multiple regression analysis, 25OHD accounted for only three of the 49.2% known variance in cortical vBMD; 25OHD was not significant in the model for cortical porosity at the tibia. Conclusion: Low 25OHD levels are associated with higher PTH levels in PHPT, but contrary to our hypothesis, these differences did not significantly affect vBMD or microarchitecture, nor did they result in lower stiffness. Low vitamin

  10. [Clinical perspectives of the study of RANK/RANKL/OPG system components in primary and metastatic bone tumor].

    PubMed

    Kushlinskiĭ, N E; Timofeev, Iu S; Gershteĭn, E S; Solov'ev, Iu N

    2014-01-01

    Disbalance of bone homeostasis, associated with malfunctioning of RANK/RANKL/OPG system underlies the oncological processes such as the destruction of bone, metastasis development, tumor progression. Pathological activity of system was described in such conditions, as breast cancer, prostate cancer, multiple myeloma, squamous cell carcinoma, Hodgkin's disease, and also metastasis in bones from lung cancer and other malignant diseases. In the literature, there is evidence of involvement of RANK/RANKL/OPG system in the pathogenesis of bone tumors (osteosarcoma, giant cell tumor of bone, chondroblastoma). Experimental data show that RANKL inhibitors can play a role in reducing tumor-induced lesions of bone in multiple myeloma, breast cancer, prostate cancer and lung cancer. Also this review presents data from clinical studies of the drug efficacy targeted on RANK/RANKL/OPG system and results of authors' study of the levels of this system's components and proinflammatory cytokines in blood serum of primary bone sarcoma patients.

  11. Primary bone marrow lymphoma: an uncommon extranodal presentation of aggressive non-hodgkin lymphomas.

    PubMed

    Martinez, Antonio; Ponzoni, Maurilio; Agostinelli, Claudio; Hebeda, Konnie M; Matutes, Estella; Peccatori, Jacopo; Campidelli, Cristina; Espinet, Blanca; Perea, Granada; Acevedo, Agustin; Mehrjardi, Ali Zare; Martinez-Bernal, Monica; Gelemur, Marta; Zucca, Emanuele; Pileri, Stefano; Campo, Elias; López-Guillermo, Armando; Rozman, Maria

    2012-02-01

    Bone marrow involvement by lymphoma is considered a systemic dissemination of the disease arising elsewhere, although some tumors may arise primarily in the bone marrow microenvironment. Primary bone marrow lymphoma (PBML) is a rare entity whose real boundaries and clinicobiological significance are not well defined. Criteria to diagnose PBML encompass isolated bone marrow infiltration, with no evidence of nodal or extranodal involvement, including the bone, and the exclusion of leukemia/lymphomas that are considered to primarily involve the bone marrow. Twenty-one out of 40 lymphomas retrospectively reviewed by the International Extranodal Lymphoma Study Group from 12 institutions in 7 different countries over a 25-year period fulfilled the inclusion criteria. These cases comprised 4 follicular lymphomas (FLs), 15 diffuse large B-cell lymphomas (DLBCLs), and 2 peripheral T-cell lymphomas, not otherwise specified. The FL cases showed paratrabecular infiltration, BCL2 protein and CD10 expression, and BCL2 gene rearrangement. DLBCL showed nodular infiltration in 6 cases and was diffuse in 9 cases; it also showed positivity for BCL2 protein (9/10) and IRF4 (6/8). Median age was 65 years with male predominance. All but 3 FL patients were symptomatic. Most cases presented with cytopenias and high lactate dehydrogenase. Four patients (3 FL cases and 1 DLBCL case) had leukemic involvement. Most DLBCL patients received CHOP-like or R-CHOP-like regimens. The outcome was unfavorable, with a median overall survival of 1.8 years. In conclusion, PBML is a very uncommon lymphoma with particular clinical features and heterogenous histology. Its recognition is important to establish accurate diagnosis and adequate therapy.

  12. Analysis of differences in bone removal during femoral box osteotomy for primary total knee arthroplasty

    PubMed Central

    GRACEFFA, ANGELO; INDELLI, PIER FRANCESCO; BASNETT, KAITLYN; MARCUCCI, MASSIMILIANO

    2014-01-01

    Purpose this study was conducted to compare the quantity of intercondylar bone removed during femoral box osteotomy for implantation of three contemporary posterior stabilized (PS) total knee arthroplasty designs: Sigma PS (DePuy), Vanguard (Biomet) and Persona (Zimmer). Methods we compared the maximum volumetric bone resection required for the housing of the PS mechanism of these three designs. Bone removal by each PS box cutting jig was three-dimensionally measured. The differences between the three designs were analyzed by the Kruskal-Wallis test. The Mann-Whitney U-test was used for pairwise comparisons. The level of significance was set at p<0.05. Results for small-size implants, the average box osteotomy volume of Persona was significantly smaller than the Vanguard and Sigma PS volumes (p=0.003). The mean difference between Vanguard and Sigma PS (p=0.01) was also significant. For medium size implants, the mean difference between Persona and Sigma PS (p=0.008) and the mean difference between Vanguard and Sigma PS (p=0.01) were statistically significant. For large size implants, the mean difference between Vanguard and Sigma PS (p=0.01) and the mean difference between Sigma PS and Persona (p=0.008) were statistically significant. Conclusions irrespective of implant size, the Persona cutting jig always resected significantly less bone than did Vanguard and Sigma PS. Clinical Relevance although this study does not establish any clinical relevance of removing more or less bone at primary TKA, its results suggest that if a PS design is indicated, it is preferable to select a model which resects less distal femoral bone. PMID:25606547

  13. Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2.

    PubMed

    Wang, Feng; Wu, Li-An; Li, Wentong; Yang, Yuan; Guo, Feng; Gao, Qingping; Chuang, Hui-Hsiu; Shoff, Lisa; Wang, Wei; Chen, Shuo

    2013-09-01

    Odontogenesis is the result of the reciprocal interactions between epithelial-mesenchymal cells leading to terminally differentiated odontoblasts. This process from dental papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the dental mesenchymal cell differentiation into odontoblasts and the molecular events of dentin formation. In this study, immortalized dental papilla mesenchymal cell lines were generated from the first mouse mandibular molars at postnatal day 3 using pSV40. These transformed cells were characterized by RT-PCR, immunohistochemistry, Western blot, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iMDP-3, displayed a high proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralized nodules. Furthermore, iMDP-3 cells had high transfection efficiency as well as were inducible and responded to BMP2 stimulation. We conclude that the establishment of the stable murine dental papilla mesenchymal cell line might be used for studying the mechanisms of dental cell differentiation and dentin formation.

  14. Influence of surgical technique, implant shape and diameter on the primary stability in cancellous bone.

    PubMed

    Bilhan, H; Geckili, O; Mumcu, E; Bozdag, E; Sünbüloğlu, E; Kutay, O

    2010-12-01

    Achievement of primary stability during surgical placement of dental implants is one of the most important factors for successful osseointegration depending on various anatomical, surgical and implant-related factors. Resonance frequency analysis (RFA) has been shown as a non-invasive and objective technique for measuring the stability of implants. The aim of this study was to evaluate the effect of some surgical and implant-related factors in enhancing primary stability and to estimate a correlation between RFA and insertion torque (IT) in proximal regions of cow ribs representing cancellous bone. Fifteen implant beds were prepared in the most proximal region of six fresh cow ribs. Ninety implants with three different shapes and two different diameters were placed with two different surgical techniques, and the primary stability was compared using RFA and IT. Significantly higher RFA and IT values were achieved when under-dimensioned drilling was used as the surgical method (P<0·01); significantly higher IT values were obtained with the use of wider implants (P<0·01) and partially conical Astra Tech implants showed the highest IT values (P<0·01). When all the implants were considered, significant correlations between the IT and RFA values were noted (%40·6, P<0·05). Partially conical implants with a wide diameter to be placed with the modified surgical technique proposed appear to be useful in enhancing the primary stability in cancellous bone.

  15. Bone fracture toughness and strength correlate with collagen cross-link maturity in a dose-controlled lathyrism mouse model

    PubMed Central

    McNerny, Erin M. B.; Gong, Bo; Morris, Michael D.; Kohn, David H.

    2014-01-01

    Collagen cross-linking is altered in many diseases of bone, and enzymatic collagen cross-links are important to bone quality as evidenced by losses of strength following lysyl oxidase inhibition (lathyrism). We hypothesized that cross-links also contribute directly to bone fracture toughness. A mouse model of lathyrism using subcutaneous injection of up to 500mg/kg β-aminopropionitrile (BAPN) was developed and characterized (60 animals across 4 dosage groups). Three weeks of 150 or 350 mg/kg BAPN treatment in young growing mice significantly reduced cortical bone fracture toughness, strength, and pyridinoline cross-link content. Ratios reflecting relative cross-link maturity were positive regressors of fracture toughness (HP/[DHLNL+HLNL] r2=0.208, p<0.05; [HP+LP]/[DHNL+HLNL] r2=0.196, p<0.1), whereas quantities of mature pyridinoline cross-links were significant positive regressors of tissue strength (lysyl pyridinoline r2=0.159, p=0.014; hydroxylysyl pyridinoline r2=0.112, p<0.05). Immature and pyrrole cross-links, which were not significantly reduced by BAPN, did not correlate with mechanical properties. The effect of BAPN treatment on mechanical properties was dose specific, with the greatest impact found at the intermediate (350mg/kg) dose. Calcein labeling was used to define locations of new bone formation, allowing for the identification of regions of normally cross-linked (preexisting) and BAPN treated (newly formed, cross-link-deficient) bone. Raman spectroscopy revealed spatial differences due to relative tissue age and effects of cross-link inhibition. Newly deposited tissues had lower mineral/matrix, carbonate/phosphate and Amide I cross-link (matrix maturity) ratios compared to preexisting tissues. BAPN treatment did not affect mineral measures, but significantly increased the cross-link (matrix maturity) ratio compared to newly formed control tissue. Our study reveals that spatially localized effects of short term BAPN cross-link inhibition can alter

  16. Modeling pathogenesis of primary liver cancer in lineage-specific mouse cell types.

    PubMed

    Holczbauer, Agnes; Factor, Valentina M; Andersen, Jesper B; Marquardt, Jens U; Kleiner, David E; Raggi, Chiara; Kitade, Mitsuteru; Seo, Daekwan; Akita, Hirofumi; Durkin, Marian E; Thorgeirsson, Snorri S

    2013-07-01

    Human primary liver cancer is classified into biologically distinct subgroups based on cellular origin. Liver cancer stem cells (CSCs) have been recently described. We investigated the ability of distinct lineages of hepatic cells to become liver CSCs and the phenotypic and genetic heterogeneity of primary liver cancer. We transduced mouse primary hepatic progenitor cells, lineage-committed hepatoblasts, and differentiated adult hepatocytes with transgenes encoding oncogenic H-Ras and SV40LT. The CSC properties of transduced cells and their ability to form tumors were tested by standard in vitro and in vivo assays and transcriptome profiling. Irrespective of origin, all transduced cells acquired markers of CSC/progenitor cells, side populations, and self-renewal capacity in vitro. They also formed a broad spectrum of liver tumors, ranging from cholangiocarcinoma to hepatocellular carcinoma, which resembled human liver tumors, based on genomic and histologic analyses. The tumor cells coexpressed hepatocyte (hepatocyte nuclear factor 4α), progenitor/biliary (keratin 19, epithelial cell adhesion molecule, A6), and mesenchymal (vimentin) markers and showed dysregulation of genes that control the epithelial-mesenchymal transition. Gene expression analyses could distinguish tumors of different cellular origin, indicating the contribution of lineage stage-dependent genetic changes to malignant transformation. Activation of c-Myc and its target genes was required to reprogram adult hepatocytes into CSCs and for tumors to develop. Stable knockdown of c-Myc in transformed adult hepatocytes reduced their CSC properties in vitro and suppressed growth of tumors in immunodeficient mice. Any cell type in the mouse hepatic lineage can undergo oncogenic reprogramming into a CSC by activating different cell type-specific pathways. Identification of common and cell of origin-specific phenotypic and genetic changes could provide new therapeutic targets for liver cancer. Copyright

  17. CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization

    PubMed Central

    Tormin, Ariane; Li, Ou; Brune, Jan Claas; Walsh, Stuart; Schütz, Birgit; Ehinger, Mats; Ditzel, Nicholas; Kassem, Moustapha

    2011-01-01

    Nonhematopoietic bone marrow mesenchymal stem cells (BM-MSCs) are of central importance for bone marrow stroma and the hematopoietic environment. However, the exact phenotype and anatomical distribution of specified MSC populations in the marrow are unknown. We characterized the phenotype of primary human BM-MSCs and found that all assayable colony-forming units-fibroblast (CFU-Fs) were highly and exclusively enriched not only in the lin−/CD271+/CD45−/CD146+ stem-cell fraction, but also in lin−/CD271+/CD45−/CD146−/low cells. Both populations, regardless of CD146 expression, shared a similar phenotype and genotype, gave rise to typical cultured stromal cells, and formed bone and hematopoietic stroma in vivo. Interestingly, CD146 was up-regulated in normoxia and down-regulated in hypoxia. This was correlated with in situ localization differences, with CD146 coexpressing reticular cells located in perivascular regions, whereas bone-lining MSCs expressed CD271 alone. In both regions, CD34+ hematopoietic stem/progenitor cells were located in close proximity to MSCs. These novel findings show that the expression of CD146 differentiates between perivascular versus endosteal localization of non-hematopoietic BM-MSC populations, which may be useful for the study of the hematopoietic environment. PMID:21415267

  18. Primary cilia are present on human blood and bone marrow cells and mediate Hedgehog signaling.

    PubMed

    Singh, Mohan; Chaudhry, Parvesh; Merchant, Akil A

    2016-12-01

    Primary cilia are nonmotile, microtubule-based organelles that are present on the cellular membrane of all eukaryotic cells. Functional cilia are required for the response to developmental signaling pathways such as Hedgehog (Hh) and Wnt/β-catenin. Although the Hh pathway has been shown to be active in leukemia and other blood cancers, there have been no reports describing the presence of primary cilia in human blood or leukemia cells. In the present study, we show that nearly all human blood and bone marrow cells have primary cilia (97-99%). In contrast, primary cilia on AML cell lines (KG1, KG1a, and K562) were less frequent (10-36% of cells) and were often shorter and dysmorphic, with less well-defined basal bodies. Finally, we show that treatment of blood cells with the Hh pathway ligand Sonic Hedgehog (SHh) causes translocation of Smoothened (SMO) to the primary cilia and activation of Hh target genes, demonstrating that primary cilia in blood cells are functional and participate in Hh signaling. Loss of primary cilia on leukemia cells may have important implications for aberrant pathway activation and response to SMO inhibitors currently in clinical development. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  19. Stage IE Primary Bone Lymphoma:Limb Salvage for Local Recurrence.

    PubMed

    Jamshidi, Khodamorad; Jabalameli, Mahmoud; Hoseini, Mohammad Ghorban; Bagherifard, Abolfazl

    2015-01-01

    Primary bone lymphoma or non-Hodgkin lymphoma of bone is a rare disease. There are only a few case series of stage IE of this condition in medical literature. The aim of this study is to determine the rate of survival for stage IE after combined modality treatment, the rate of local recurrence, and the results of limb salvage in cases of local recurrence. We collected data from 61 patients with histologically confirmed PBL treated at the Musculoskeletal Oncology Department of our hospital from 2000 to 2010. Retrospective evaluation included demographics, symptoms, tumor locations, outcomes of surgical treatment for local recurrence and survival rates. All patients received Combined Modality Therapy. Overall,five year survival was 89% and five year disease free survival rate was 78%. Local recurrence occurred in 6 patients during follow up period, which was treated surgically by wide excision and reconstruction. The mean follow-up for the local recurrence group was 36(24-54) months and mortality rate in this group was 17%. Combined Modality Therapy for stage IE primary bone lymphomaresults in good survival rate. In case of local recurrence, wide excision and reconstruction improves the outcomes.

  20. Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes

    EPA Science Inventory

    Abstract: Propiconazole is a triazole-containing fungicide that is used agriculturally on grasses, fruits, grains, seeds, hardwoods, and conifers. Propiconazole is a mouse liver hepatotoxicant and a hepatocarcinogen and has adverse reproductive and developmental toxicities in exp...

  1. Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes

    EPA Science Inventory

    Abstract: Propiconazole is a triazole-containing fungicide that is used agriculturally on grasses, fruits, grains, seeds, hardwoods, and conifers. Propiconazole is a mouse liver hepatotoxicant and a hepatocarcinogen and has adverse reproductive and developmental toxicities in exp...

  2. Identification and Characterization of Lineage(-)CD45(-)Sca-1(+) VSEL Phenotypic Cells Residing in Adult Mouse Bone Tissue.

    PubMed

    Nakatsuka, Ryusuke; Iwaki, Ryuji; Matsuoka, Yoshikazu; Sumide, Keisuke; Kawamura, Hiroshi; Fujioka, Tatsuya; Sasaki, Yutaka; Uemura, Yasushi; Asano, Hiroaki; Kwon, A-Hon; Sonoda, Yoshiaki

    2016-01-01

    Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

  3. Deletion of bone-marrow-derived receptor for AGEs (RAGE) improves renal function in an experimental mouse model of diabetes.

    PubMed

    Tesch, Greg; Sourris, Karly C; Summers, Shaun A; McCarthy, Domenica; Ward, Micheal S; Borg, Danielle J; Gallo, Linda A; Fotheringham, Amelia K; Pettit, Allison R; Yap, Felicia Y T; Harcourt, Brooke E; Tan, Adeline L Y; Kausman, Joshua Y; Nikolic-Paterson, David; Kitching, Arthur R; Forbes, Josephine M

    2014-09-01

    The AGEs and the receptor for AGEs (RAGE) are known contributors to diabetic complications. RAGE also has a physiological role in innate and adaptive immunity and is expressed on immune cells. The aim of this study was to determine whether deletion of RAGE from bone-marrow-derived cells influences the pathogenesis of experimental diabetic nephropathy. Groups (n = 8/group) of lethally irradiated 8 week old wild-type (WT) mice were reconstituted with bone marrow from WT (WT → WT) or RAGE-deficient (RG) mice (RG → WT). Diabetes was induced using multiple low doses of streptozotocin after 8 weeks of bone marrow reconstitution and mice were followed for a further 24 weeks. Compared with diabetic WT mice reconstituted with WT bone marrow, diabetic WT mice reconstituted with RG bone marrow had lower urinary albumin excretion and podocyte loss, more normal creatinine clearance and less tubulo-interstitial injury and fibrosis. However, glomerular collagen IV deposition, glomerulosclerosis and cortical levels of TGF-β were not different among diabetic mouse groups. The renal tubulo-interstitium of diabetic RG → WT mice also contained fewer infiltrating CD68(+) macrophages that were activated. Diabetic RG → WT mice had lower renal cortical concentrations of CC chemokine ligand 2 (CCL2), macrophage inhibitory factor (MIF) and IL-6 than diabetic WT → WT mice. Renal cortical RAGE ligands S100 calgranulin (S100A)8/9 and AGEs, but not high mobility box protein B-1 (HMGB-1) were also decreased in diabetic RG → WT compared with diabetic WT → WT mice. In vitro, bone-marrow-derived macrophages from WT but not RG mice stimulated collagen IV production in cultured proximal tubule cells. These studies suggest that RAGE expression on haemopoietically derived immune cells contributes to the functional changes seen in diabetic nephropathy by promoting macrophage infiltration and renal tubulo-interstitial damage.

  4. Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes.

    PubMed

    White, Jennifer; Barro, Marietta V; Makarenkova, Helen P; Sanger, Joseph W; Sanger, Jean M

    2014-09-01

    It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, nonmuscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z-bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. © 2014 Wiley Periodicals, Inc.

  5. Dura Mater Stimulates Human Adipose-Derived Stromal Cells to Undergo Bone Formation in Mouse Calvarial Defects

    PubMed Central

    Levi, Benjamin; Nelson, Emily R.; Li, Shuli; James, Aaron W.; Hyun, Jeong S.; Montoro, Daniel T.; Lee, Min; Glotzbach, Jason P.; Commons, George W.; Longaker, Michael T.

    2015-01-01

    Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2–Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair. PMID:21656608

  6. Mouse embryonic fibroblasts (MEF) exhibit a similar but not identical phenotype to bone marrow stromal stem cells (BMSC).

    PubMed

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M; Abdallah, Basem M; Kassem, Moustapha

    2012-06-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been reported. Utilizing standard in vitro and in vivo assays we performed a side-by-side comparison of MEF and BMSC to determine their ability to differentiate into mesoderm-type cells. BMSC were isolated from 8-10 weeks old mouse bone marrow by plastic adherence. MEF were established by trypsin/EDTA digestion from E13.5 embryos after removing heads and viscera, followed by plastic adherence. Compared to BMSC, MEF exhibited telomerase activity and improved cell proliferation as assessed by q-PCR based TRAP assay and cell number quantification, respectively. FACS analysis revealed that MEF exhibited surface markers characteristic of the BMSC: Sca-1(+), CD73(+), CD105(+), CD29(+), CD44(+), CD106(+), CD11b(-), and CD45(-). In contrast to BMSC, ex vivo osteoblast (OB) differentiation of MEF exhibited a less mature osteoblastic phenotype (less alkaline phosphatase, collagen type I and osteocalcin) as assessed by real-time PCR analysis. Compared to BMSC, MEF exhibited a more enhanced differentiation into adipocyte and chondrocyte lineages. Interestingly, both MEF and BMSC formed the same amount of heterotopic bone and bone marrow elements upon in vivo subcutaneous implantation with hydroxyapatite/tricalcium phosphate, in immune deficient mice. In conclusion, MEF contain a population of stem cells that behave in ex vivo and in vivo assays, similar but not identical, to BMSC. Due to their enhanced cell growth, they may represent a good alternative for BMSC in studying molecular mechanisms of stem cell commitment and differentiation to osteoblasts, adipocytes and chondrocytes.

  7. Effect of peripheral lymphoid cells on the incidence of lethal graft versus host disease following allogeneic mouse bone marrow transplantation

    SciTech Connect

    Almaraz, R.; Ballinger, W.; Sachs, D.H.; Rosenberg, S.A.

    1983-02-01

    Experiments were performed to study the role of circulating lymphoid cells in the incidence of lethal graft versus host disease (GVHD) in radiation-induced fully allogeneic mouse chimeras. The incidence of GVHD was reduced significantly in BALB/c leads to C57BL/6 radiation chimeras if bone marrow donors were exsanguinated immediately prior to marrow harvest. Chimeras resulting from the injection of bone marrow from bled donors exhibited only donor cells in spleen, bone marrow and peripheral blood and normal levels of Thy 1+ and Ia+ cells were found in each of these lymphoid compartments. The addition of as few as 3 X 10(4) peripheral mononuclear cells to the marrow from exsanguinated donors uniformly led to lethal GVHD. /sup 51/Cr-labeled cell traffic studies revealed that prior exsanguination of marrow donors led to about a 70% reduction in the number of circulating mononuclear cells contaminating the bone marrow at the time of marrow harvest. This decrease in contaminating peripheral cells was calculated to be in the appropriate range to account for the decreased GVHD seen when marrow from exsanguinated donors was used. It thus appears that peripheral cells contaminating marrow can be an important factor in causing lethal GVHD in allogeneic radiation chimeras.

  8. Administration of soluble activin receptor 2B increases bone and muscle mass in a mouse model of osteogenesis imperfecta

    PubMed Central

    DiGirolamo, Douglas J.; Singhal, Vandana; Chang, Xiaoli; Lee, Se-Jin; Germain-Lee, Emily L.

    2015-01-01

    Osteogenesis imperfecta (OI) comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the skeletal complications of OI, many patients also report intolerance to physical activity, fatigue and muscle weakness. Indeed, recent studies have demonstrated that skeletal muscle is also negatively affected by OI, both directly and indirectly. Given the well-established interdependence of bone and skeletal muscle in both physiology and pathophysiology and the observations of skeletal muscle pathology in patients with OI, we investigated the therapeutic potential of simultaneous anabolic targeting of both bone and skeletal muscle using a soluble activin receptor 2B (ACVR2B) in a mouse model of type III OI (oim). Treatment of 12-week-old oim mice with ACVR2B for 4 weeks resulted in significant increases in both bone and muscle that were similar to those observed in healthy, wild-type littermates. This proof of concept study provides encouraging evidence for a holistic approach to treating the deleterious consequences of OI in the musculoskeletal system. PMID:26161291

  9. Thermal response and hyperthermic radiosensitization of scid mouse bone marrow CFU-C.

    PubMed

    O'Hara, M D; Pollard, M D; Wheatley, G; Regine, W F; Mohiuddin, M; Leeper, D B

    1995-02-15

    Scid mice are severely immunodeficient as a result of a defective recombinase system. Mice with the scid mutation have been shown to have an increased sensitivity to ionizing radiation, presumably as a result of an inability to repair DNA damage. Little is known of the impact of this mutation on the thermal response and on hyperthermic radiosensitization. This investigation established the thermal response (42-44 degrees C), patterns of thermotolerance development, and the impact of hyperthermia (60 min at 40 degrees C or 42 degrees C) on the radiation response of bone marrow colony forming unit-culture cells (CFU-C) in scid mice. Anesthetized scid mice (pentobarbital, 90 mg/kg) were killed by cervical dislocation and the nucleated marrow obtained from both tibia and femora by passing 2 ml of cold McCoy's 5A medium supplemented with 15% fetal bovine serum through each bone. Single cell suspensions of nucleated marrow were heated in 12 x 75 mm sterile tissue culture tubes at a concentration of approximately 5 x 10(6) cells/ml. Radiation, when used, was delivered immediately prior to hyperthermia by a 137Cs irradiator (dose rate of 1.20 Gy/min). Colony forming unit-culture were cultured in semisolid agar in the presence of colony stimulating factor (conditioned medium from L929 cells) for 7 days. The slope of the radiation dose-response curve for CFU-C in scid mice was biphasic, the Dos (+/- SE) were 0.29 +/- 0.03 Gy and 1.09 +/- 0.20 Gy, respectively. The Dos of the radiation dose-response curve for wild type marrow from CB-17 and Balb/c mice were 1.28 +/- 0.05 Gy and 1.47 +/- 0.15 Gy, respectively. The Dos of the hyperthermia dose-response curves for scid mice were 75 +/- 5, 10 +/- 1.4, and 4 +/- 0.2 min, respectively, for temperatures of 42 degrees, 43 degrees, and 44 degrees C. Thermotolerance development at 37 degrees C increased to a maximum at approximately 240 min after acute hyperthermia (15 min at 44 degrees C) and thereafter, decreased to control levels

  10. Receptor-Activator of Nuclear KappaB Ligand Expression as a New Therapeutic Target in Primary Bone Tumors

    PubMed Central

    Yamagishi, Tetsuro; Kawashima, Hiroyuki; Ogose, Akira; Ariizumi, Takashi; Sasaki, Taro; Hatano, Hiroshi; Hotta, Tetsuo; Endo, Naoto

    2016-01-01

    The receptor-activator of nuclear kappaB ligand (RANKL) signaling pathway plays an important role in the regulation of bone growth and mediates the formation and activation of osteoclasts. Osteoclasts are involved in significant bone resorption and destruction. Denosumab is a fully human monoclonal antibody against RANKL that specifically inhibits osteoclast differentiation and bone resorption. It has been approved for use for multiple myeloma and bone metastases, as well as for giant cell tumor of bone. However, there is no previous report quantitatively, comparing RANKL expression in histologically varied bone tumors. Therefore, we analyzed the mRNA level of various bone tumors and investigated the possibility of these tumors as a new therapeutic target for denosumab. We examined RANKL mRNA expression in 135 clinical specimens of primary and metastatic bone tumors using real-time PCR. The relative quantification of mRNA expression levels was performed via normalization with RPMI8226, a human multiple myeloma cell line that is recognized to express RANKL. Of 135 cases, 64 were also evaluated for RANKL expression by using immunohistochemistry. Among all of the tumors investigated, RANKL expression and the RANKL/osteoprotegerin ratio were highest in giant cell tumor of bone. High RANKL mRNA expression was observed in cases of aneurysmal bone cyst, fibrous dysplasia, osteosarcoma, chondrosarcoma, and enchondroma, as compared to cases of multiple myeloma and bone lesions from metastatic carcinoma. RANKL-positive stromal cells were detected in six cases: five cases of GCTB and one case of fibrous dysplasia. The current study findings indicate that some primary bone tumors present new therapeutic targets for denosumab, particularly those tumors expressing RANKL and those involving bone resorption by osteoclasts. PMID:27163152

  11. Establishment and Genomic Characterization of Mouse Xenografts of Human Primary Prostate Tumors

    PubMed Central

    Priolo, Carmen; Agostini, Michelle; Vena, Natalie; Ligon, Azra H.; Fiorentino, Michelangelo; Shin, Eyoung; Farsetti, Antonella; Pontecorvi, Alfredo; Sicinska, Ewa; Loda, Massimo

    2010-01-01

    Serum prostate-specific antigen screening has led to earlier detection and surgical treatment of prostate cancer, favoring an increasing incidence-to-mortality ratio. However, about one third of tumors that are diagnosed when still confined to the prostate can relapse within 10 years from the first treatment. The challenge is therefore to identify prognostic markers of aggressive versus indolent tumors. Although several preclinical models of advanced prostate tumors are available, a model that recapitulates the genetic and growth behavior of primary tumors is still lacking. Here, we report a complete histopathological and genomic characterization of xenografts derived from primary localized low- and high-grade human prostate tumors that were implanted under the renal capsule of immunodeficient mice. We obtained a tumor take of 56% and show that these xenografts maintained the histological as well as most genomic features of the parental tumors. Serum prostate-specific antigen levels were measurable only in tumor xenograft-bearing mice, but not in those implanted with either normal prostate tissue or in tumors that likely regressed. Finally, we show that a high proliferation rate, but not the pathological stage or the Gleason grade of the original tumor, was a fundamental prerequisite for tumor take in mice. This mouse xenograft model represents a useful preclinical model of primary prostate tumors for their biological characterization, biomarker discovery, and drug testing. PMID:20167861

  12. Histamine H3 receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion.

    PubMed

    Iida, Tomomitsu; Yoshikawa, Takeo; Matsuzawa, Takuro; Naganuma, Fumito; Nakamura, Tadaho; Miura, Yamato; Mohsen, Attayeb S; Harada, Ryuichi; Iwata, Ren; Yanai, Kazuhiko

    2015-07-01

    Histamine is a physiological amine which initiates a multitude of physiological responses by binding to four known G-protein coupled histamine receptor subtypes as follows: histamine H1 receptor (H1 R), H2 R, H3 R, and H4 R. Brain histamine elicits neuronal excitation and regulates a variety of physiological processes such as learning and memory, sleep-awake cycle and appetite regulation. Microglia, the resident macrophages in the brain, express histamine receptors; however, the effects of histamine on critical microglial functions such as chemotaxis, phagocytosis, and cytokine secretion have not been examined in primary cells. We demonstrated that mouse primary microglia express H2 R, H3 R, histidine decarboxylase, a histamine synthase, and histamine N-methyltransferase, a histamine metabolizing enzyme. Both forskolin-induced cAMP accumulation and ATP-induced intracellular Ca(2+) transients were reduced by the H3 R agonist imetit but not the H2 R agonist amthamine. H3 R activation on two ubiquitous second messenger signalling pathways suggests that H3 R can regulate various microglial functions. In fact, histamine and imetit dose-dependently inhibited microglial chemotaxis, phagocytosis, and lipopolysaccharide (LPS)-induced cytokine production. Furthermore, we confirmed that microglia produced histamine in the presence of LPS, suggesting that H3 R activation regulate microglial function by autocrine and/or paracrine signalling. In conclusion, we demonstrate the involvement of histamine in primary microglial functions, providing the novel insight into physiological roles of brain histamine.

  13. Physiology of Layer 5 Pyramidal Neurons in Mouse Primary Visual Cortex: Coincidence Detection through Bursting

    PubMed Central

    Shai, Adam S.; Anastassiou, Costas A.; Larkum, Matthew E.; Koch, Christof

    2015-01-01

    L5 pyramidal neurons are the only neocortical cell type with dendrites reaching all six layers of cortex, casting them as one of the main integrators in the cortical column. What is the nature and mode of computation performed in mouse primary visual cortex (V1) given the physiology of L5 pyramidal neurons? First, we experimentally establish active properties of the dendrites of L5 pyramidal neurons of mouse V1 using patch-clamp recordings. Using a detailed multi-compartmental model, we show this physiological setup to be well suited for coincidence detection between basal and apical tuft inputs by controlling the frequency of spike output. We further show how direct inhibition of calcium channels in the dendrites modulates such coincidence detection. To establish the singe-cell computation that this biophysics supports, we show that the combination of frequency-modulation of somatic output by tuft input and (simulated) calcium-channel blockage functionally acts as a composite sigmoidal function. Finally, we explore how this computation provides a mechanism whereby dendritic spiking contributes to orientation tuning in pyramidal neurons. PMID:25768881

  14. Regulation of protein kinase D during differentiation and proliferation of primary mouse keratinocytes.

    PubMed

    Ernest Dodd, M; Ristich, Vladimir L; Ray, Sagarika; Lober, Robert M; Bollag, Wendy B

    2005-08-01

    Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.

  15. Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cells in scaffold

    PubMed Central

    Im, Jin-Young; Min, Woo-Kie; You, Changkook; Kim, Hyun-Ok; Jin, Hee-Kyung

    2013-01-01

    Combination of tissue engineering and cell therapy represents a promising approach for bone regeneration. Human mesenchymal stem cells (hMSCs) have properties that include low immunogenicity, high proliferation rate, and multi-differentiation potential; therefore, they are an attractive seeding source for tissue engineering therapy. Here we found that hMSCs with a scaffold did not affect cell viability and osteogenic differentiation. We also investigated regenerative effect of hMSCs with the scaffold in a calvarial bone defect model. Formation of new bone was evaluated by micro-CT, histology and expression of osteogenic markers. The results clearly showed interesting evidence indicating that hMSCs with scaffold increased the formation of new bone and expression of osteogenic markers, compared to the empty and scaffold only groups. Overall, our results suggest that hMSCs with scaffold are suitable for stimulation of intense bone regeneration in critical-sized bone defects. PMID:24396384

  16. In vitro comparison of primary stability of two implant designs in D3 bone.

    PubMed

    González-Serrano, J; Ortega-Aranegui, R; López-Quiles, J

    2017-07-01

    Primary stability (PS) is a key factor for implant survival rate and depends on implant design or bone quality. The aim of this study was to compare different thread designs implants, evaluating PS with periotest values (PV) and implant stability quotient (ISQ) values through resonance frequency analysis (RFA). A total of 60 implants (Radhex®, Inmet-Garnick S.A., Guadalajara, Spain) were placed in freshly bovine ribs in vitro. Two designs were used: 30 tapered body with single thread design (PHI) and 30 tapered body with double thread design implants (PHIA). Both designs were 4mm wide and 12mm long. Implants were placed according to manufacturer's guidelines. Osstell™ and Periotest® devices were used to evaluate PS by a blinded independent observer. Computed tomographies (CTs) of the ribs were made (BrightSpeed Series CT systems, GE Healthcare, Milwaukee, WI, USA) and bone quality surrounding each implant was evaluated in Hounsfield Units (HU) using Ez3D Plus software (Vatech Co., Korea). Bone quality was classified according to Misch and Kircos in D1, D2, D3 or D4. All implants were mechanically stable. Only implants placed in D3 bone (350-850 HU) were selected for the study: 28 PHI and 26 PHIA. The one way ANOVA showed significant difference (p < 0.005) among two implants designs in ISQ values (61,55 ± 6,67 in PHI and 68,94 ± 5,82 in PHIA). No significant difference (p = 0,171) was shown in PV between two designs (-4,47 ± 1,39 in PHI and -4,77 ± 0,87 in PHIA). Higher PS was found using Osstell™ device in implants with double thread design (PHIA) in comparison to implants with single thread design (PHI) in D3 bone.

  17. Relationship between histologic grade and cytofluorometric cellular DNA and RNA content in primary bone tumors.

    PubMed

    Takeshita, H; Kusuzaki, K; Kuzuhara, A; Tsuji, Y; Ashihara, T; Gebhardt, M C; Mankin, H J; Springfield, D S; Hirasawa, Y

    2001-01-01

    The diagnosis and grading of bone tumors remains a challenging problem. We studied the relationship between histologic grade and cytofluorometric cellular DNA and RNA content in 108 primary bone tumors. The data included DNA ploidy, mean DNA content (MDC), S-phase fraction (SPF), mean RNA content (MRC) and RNA/DNA ratio (RDR; MRC/MDC) which represents the RNA content normalized for the DNA content. Benign tumors had a diploid stem line with low MDC (mean; 1.04), low SPF (0.9), high MRC (2.41) and high RDR (2.31). Giant cell tumors of bone, which are locally aggressive benign tumors, showed diploidy with relatively higher MDC (1.07, p < 0.01) and SPF (2.6, p < 0.01) and lower MRC (1.81, p < 0.01) and RDR (1.69, p < 0.01). Similar results were obtained in low-grade sarcomas. In high-grade sarcomas, the data depended on the histologic findings. Pleomorphic sarcomas such as osteosarcomas revealed aneuploidy with remarkably higher MDC (1.70 in osteosarcomas, p < 0.01) and SPF (6.5, p < 0.01), but lower RDR (1.70, p < 0.01). In contrast, small cell sarcomas, such as Ewing's sarcomas, showed diploidy with low MDC (1.11 in Ewing's sarcomas, N.S.) and SPF (2.5, p < 0.01) and extremely low RDR (1.34, p < 0.01). The RDR value was higher in well-differentiated tumors than in primitive tumors, rendering it useful in grading bone tumors with a diploid stem line. By combining the RDR value with the MDC value, 96% of diploid sarcomas could be distinguished from benign tumors. These results indicate that cellular DNA and RNA content analysis may be of value in assessing the malignant potential of diploid as well as aneuploid bone sarcomas.

  18. Deregulation of arginase induces bone complications in high-fat/high-sucrose diet diabetic mouse model.

    PubMed

    Bhatta, Anil; Sangani, Rajnikumar; Kolhe, Ravindra; Toque, Haroldo A; Cain, Michael; Wong, Abby; Howie, Nicole; Shinde, Rahul; Elsalanty, Mohammed; Yao, Lin; Chutkan, Norman; Hunter, Monty; Caldwell, Ruth B; Isales, Carlos; Caldwell, R William; Fulzele, Sadanand

    2016-02-15

    A balanced diet is crucial for healthy development and prevention of musculoskeletal related diseases. Diets high in fat content are known to cause obesity, diabetes and a number of other disease states. Our group and others have previously reported that activity of the urea cycle enzyme arginase is involved in diabetes-induced dysregulation of vascular function due to decreases in nitric oxide formation. We hypothesized that diabetes may also elevate arginase activity in bone and bone marrow, which could lead to bone-related complications. To test this we determined the effects of diabetes on expression and activity of arginase, in bone and bone marrow stromal cells (BMSCs). We demonstrated that arginase 1 is abundantly present in the bone and BMSCs. We also demonstrated that arginase activity and expression in bone and bone marrow is up-regulated in models of diabetes induced by HFHS diet and streptozotocin (STZ). HFHS diet down-regulated expression of healthy bone metabolism markers (BMP2, COL-1, ALP, and RUNX2) and reduced bone mineral density, bone volume and trabecular thickness. However, treatment with an arginase inhibitor (ABH) prevented these bone-related complications of diabetes. In-vitro study of BMSCs showed that high glucose treatment increased arginase activity and decreased nitric oxide production. These effects were reversed by treatment with an arginase inhibitor (ABH). Our study provides evidence that deregulation of l-arginine metabolism plays a vital role in HFHS diet-induced diabetic complications and that these complications can be prevented by treatment with arginase inhibitors. The modulation of l-arginine metabolism in disease could offer a novel therapeutic approach for osteoporosis and other musculoskeletal related diseases.

  19. Mixed functional microarchitectures for orientation selectivity in the mouse primary visual cortex

    PubMed Central

    Kondo, Satoru; Yoshida, Takashi; Ohki, Kenichi

    2016-01-01

    A minicolumn is the smallest anatomical module in the cortical architecture, but it is still in debate whether it serves as functional units for cortical processing. In the rodent primary visual cortex (V1), neurons with different preferred orientations are mixed horizontally in a salt and pepper manner, but vertical functional organization was not examined. In this study, we found that neurons with similar orientation preference are weakly but significantly clustered vertically in a short length and horizontally in the scale of a minicolumn. Interestingly, the vertical clustering is found only in a part of minicolumns, and others are composed of neurons with a variety of orientation preferences. Thus, the mouse V1 is a mixture of vertical clusters of neurons with various degrees of orientation similarity, which may be the compromise between the brain size and keeping the vertical clusters of similarly tuned neurons at least in a subset of clusters. PMID:27767032

  20. Primary Ovarian Insufficiency Induced by Fanconi Anemia E Mutation in a Mouse Model.

    PubMed

    Fu, Chun; Begum, Khurshida; Overbeek, Paul A

    2016-01-01

    In most cases of primary ovarian insufficiency (POI), the cause of the depletion of ovarian follicles is unknown. Fanconi anemia (FA) proteins are known to play important roles in follicular development. Using random insertional mutagenesis with a lentiviral transgene, we identified a family with reduced fertility in the homozygous transgenic mice. We identified the integration site and found that the lentivirus had integrated into intron 8 of the Fanconi E gene (Fance). By RT-PCR and in situ hybridization, we found that Fance transcript levels were significantly reduced. The Fance homozygous mutant mice were assayed for changes in ovarian development, follicle numbers and estrous cycle. Ovarian dysplasias and a severe lack of follicles were seen in the mutant mice. In addition, the estrous cycle was disrupted in adult females. Our results suggest that POI has been induced by the Fance mutation in this new mouse model.

  1. Primary Ovarian Insufficiency Induced by Fanconi Anemia E Mutation in a Mouse Model

    PubMed Central

    Fu, Chun; Begum, Khurshida; Overbeek, Paul A.

    2016-01-01

    In most cases of primary ovarian insufficiency (POI), the cause of the depletion of ovarian follicles is unknown. Fanconi anemia (FA) proteins are known to play important roles in follicular development. Using random insertional mutagenesis with a lentiviral transgene, we identified a family with reduced fertility in the homozygous transgenic mice. We identified the integration site and found that the lentivirus had integrated into intron 8 of the Fanconi E gene (Fance). By RT-PCR and in situ hybridization, we found that Fance transcript levels were significantly reduced. The Fance homozygous mutant mice were assayed for changes in ovarian development, follicle numbers and estrous cycle. Ovarian dysplasias and a severe lack of follicles were seen in the mutant mice. In addition, the estrous cycle was disrupted in adult females. Our results suggest that POI has been induced by the Fance mutation in this new mouse model. PMID:26939056

  2. Therapeutic impact of low amplitude high frequency whole body vibrations on the osteogenesis imperfecta mouse bone.

    PubMed

    Vanleene, Maximilien; Shefelbine, Sandra J

    2013-04-01

    Osteogenesis imperfecta (OI) is characterized by extremely brittle bone. Currently, bisphosphonate drugs allow a decrease of fracture by inhibiting bone resorption and increasing bone mass but with possible long term side effects. Whole body mechanical vibrations (WBV) treatment may offer a promising route to stimulate bone formation in OI patients as it has exhibited health benefits on both muscle and bone mass in human and animal models. The present study has investigated the effects of WBV (45Hz, 0.3g, 15minutes/days, 5days/week) in young OI (oim) and wild type female mice from 3 to 8weeks of age. Vibration therapy resulted in a significant increase in the cortical bone area and cortical thickness in the femur and tibia diaphysis of both vibrated oim and wild type mice compared to sham controls. Trabecular bone was not affected by vibration in the wild type mice; vibrated oim mice, however, exhibited significantly higher trabecular bone volume fraction in the proximal tibia. Femoral stiffness and yield load in three point bending were greater in the vibrated wild type mice than in sham controls, most likely attributed to the increase in femur cortical cross sectional area observed in the μCT morphology analyses. The vibrated oim mice showed a trend toward improved mechanical properties, but bending data had large standard deviations and there was no significant difference between vibrated and non-vibrated oim mice. No significant difference of the bone apposition was observed in the tibial metaphyseal trabecular bone for both the oim and wild type vibrated mice by histomorphometry analyses of calcein labels. At the mid diaphysis, the cortical bone apposition was not significantly influenced by the WBV treatment in both the endosteum and periosteum of the oim vibrated mice while a significant change is observed in the endosteum of the vibrated wild type mice. As only a weak impact in bone apposition between the vibrated and sham groups is observed in the

  3. Development of an efficient screening system to identify novel bone metabolism-related genes using the exchangeable gene trap mutagenesis mouse models

    PubMed Central

    Kurogi, Syuji; Sekimoto, Tomohisa; Funamoto, Taro; Ota, Tomomi; Nakamura, Shihoko; Nagai, Takuya; Nakahara, Mai; Yoshinobu, Kumiko; Araki, Kimi; Araki, Masatake; Chosa, Etsuo

    2017-01-01

    Despite numerous genetic studies on bone metabolism, understanding of the specific mechanisms is lacking. We developed an efficient screening system to identify novel genes involved in bone metabolism using mutant mouse strains registered with the Exchangeable Gene Trap Clones (EGTC) database. From 1278 trap clones in the EGTC database, 52 candidate lines were selected in the first screening, determined based on “EST profile”, “X-gal”, “Related article”, and “Novel gene”. For the second screening, bone morphometric analysis, biomechanical strength analysis, bone X-gal staining, etc. were performed on candidate lines. Forty-two male trap lines (80.8%) showed abnormalities with either bone morphometric analysis or biomechanical strength analysis. In the screening process, X-gal staining was significantly efficient (P = 0.0057). As examples, Lbr and Nedd4 trap lines selected using the screening system showed significant bone decrease and fragility, suggesting a relationship with osteoblast differentiation. This screening system using EGTC mouse lines is extremely efficient for identifying novel genes involved in bone metabolism. The gene trap lines identified as abnormal using this screening approach are highly likely to trap important genes for bone metabolism. These selected trap mice will be valuable for use as novel bio-resources in bone research. PMID:28106071

  4. Enhanced Wnt signaling improves bone mass and strength, but not brittleness, in the Col1a1(+/mov13) mouse model of type I Osteogenesis Imperfecta.

    PubMed

    Jacobsen, Christina M; Schwartz, Marissa A; Roberts, Heather J; Lim, Kyung-Eun; Spevak, Lyudmila; Boskey, Adele L; Zurakowski, David; Robling, Alexander G; Warman, Matthew L

    2016-09-01

    Osteogenesis Imperfecta (OI) comprises a group of genetic skeletal fragility disorders. The mildest form of OI, Osteogenesis Imperfecta type I, is frequently caused by haploinsufficiency mutations in COL1A1, the gene encoding the α1(I) chain of type 1 collagen. Children with OI type I have a 95-fold higher fracture rate compared to unaffected children. Therapies for OI type I in the pediatric population are limited to anti-catabolic agents. In adults with osteoporosis, anabolic therapies that enhance Wnt signaling in bone improve bone mass, and ongoing clinical trials are determining if these therapies also reduce fracture risk. We performed a proof-of-principle experiment in mice to determine whether enhancing Wnt signaling in bone could benefit children with OI type I. We crossed a mouse model of OI type I (Col1a1(+/Mov13)) with a high bone mass (HBM) mouse (Lrp5(+/p.A214V)) that has increased bone strength from enhanced Wnt signaling. Offspring that inherited the OI and HBM alleles had higher bone mass and strength than mice that inherited the OI allele alone. However, OI+HBM and OI mice still had bones with lower ductility compared to wild-type mice. We conclude that enhancing Wnt signaling does not make OI bone normal, but does improve bone properties that could reduce fracture risk. Therefore, agents that enhance Wnt signaling are likely to benefit children and adults with OI type 1.

  5. Positive Selection in Bone Morphogenetic Protein 15 Targets a Natural Mutation Associated with Primary Ovarian Insufficiency in Human

    PubMed Central

    Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  6. Assessment of bone dysplasia by micro-CT and glycosaminoglycan levels in mouse models for mucopolysaccharidosis type I, IIIA, IVA, and VII

    PubMed Central

    Rowan, Daniel J.; Tomatsu, Shunji; Grubb, Jeffrey H.; Montaño, Adriana M.; Sly, William S.

    2012-01-01

    Summary Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by mutations in lysosomal enzymes involved in degradation of glycosaminoglycans (GAGs). Patients with MPS grow poorly and become physically disabled due to systemic bone disease. While many of the major skeletal effects in mouse models for MPS have been described, no detailed analysis that compares GAGs levels and characteristics of bone by micro-CT has been done. The aims of this study were to assess severity of bone dysplasia among four MPS mouse models (MPS I, IIIA, IVA and VII), to determine the relationship between severity of bone dysplasia and serum keratan sulfate (KS) and heparan sulfate (HS) levels in those models, and to explore the mechanism of KS elevation in MPS I, IIIA, and VII mouse models. Clinically, MPS VII mice had the most severe bone pathology; however, MPS I and IVA mice also showed skeletal pathology. MPS I and VII mice showed severe bone dysplasia, higher bone mineral density, narrowed spinal canal, and shorter sclerotic bones by micro-CT and radiographs. Serum KS and HS levels were elevated in MPS I, IIIA, and VII mice. Severity of skeletal disease displayed by micro-CT, radiographs and histopathology correlated with the level of KS elevation. We showed that elevated HS levels in MPS mouse models could inhibit N-acetylgalactosamine-6-sulfate sulfatase enzyme. These studies suggest that KS could be released from chondrocytes affected by accumulation of other GAGs and that KS could be useful as a biomarker for severity of bone dysplasia in MPS disorders. PMID:22971960

  7. Targeting the LRP5 pathway improves bone properties in a mouse model of osteogenesis imperfecta.

    PubMed

    Jacobsen, Christina M; Barber, Lauren A; Ayturk, Ugur M; Roberts, Heather J; Deal, Lauren E; Schwartz, Marissa A; Weis, MaryAnn; Eyre, David; Zurakowski, David; Robling, Alexander G; Warman, Matthew L

    2014-10-01

    The cell surface receptor low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5(p.A214V) ) that is orthologous to a human HBM-causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5(+/p.A214V) mice to Col1a2(+/p.G610C) mice, which model human type IV OI. We found that Col1a2(+/p.G610C) ;Lrp5(+/p.A214V) offspring had significantly increased bone mass and strength compared to Col1a2(+/p.G610C) ;Lrp5(+/+) littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2(+/p.G610C) mice with a monoclonal antibody that inhibits sclerostin activity (Scl-Ab). We found that antibody-treated mice had significantly increased bone mass and strength compared to vehicle-treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. © 2014 American Society for Bone and Mineral Research.

  8. En bloc resection of primary malignant bone tumors of the cervical spine.

    PubMed

    Mattei, Tobias A; Mendel, Ehud

    2014-11-01

    Due to the poor response of primary malignant bone tumors to adjuvant therapies, surgical resection performed in an en bloc fashion with free margins remains the best option for long-term recurrence-free survival of patients harboring such lesions. In this article the authors provide a stepwise review of the technical details involved in the performance of en bloc resections of tumoral lesions in the cervical spine. Due to the anatomical peculiarities of the cervical spine related to the presence of functional nerve roots as well as the vertebral arteries, en bloc resections in this region remains a challenging surgical procedure.

  9. Primary epiphyseal arteriopathy in a mouse model of steroid-induced osteonecrosis.

    PubMed

    Janke, Laura J; Liu, Chengcheng; Vogel, Peter; Kawedia, Jitesh; Boyd, Kelli L; Funk, Amy J; Relling, Mary V

    2013-07-01

    Patients undergoing glucocorticoid therapy for a variety of disorders, including autoimmune diseases and hematological malignancies, are at risk of developing osteonecrosis. Despite extensive research in both patients and animal models, the underlying pathogenesis remains unclear. Proposed inciting mechanisms include intravascular thrombotic occlusion, marrow fat hypertrophy, osteocyte and/or endothelial cell apoptosis, hypercoagulability, and vasoconstriction of specific arteries and arterioles supplying bone. Our laboratory has developed a model of steroid-induced osteonecrosis in BALBcJ mice which reflects clinically relevant exposures to glucocorticoids in which treated mice develop osteonecrosis of the distal femoral epiphysis when administered 4 to 8 mg/L dexamethasone in drinking water for 6 weeks. We identified lesions in arterioles supplying this area, with the mildest occurring in knees without any evidence of osteonecrosis. However, arteriopathy was more common among mice that did versus did not develop osteonecrosis (P < 0.0001); in mice with osteonecrosis, the associated vessels showed transmural necrosis and thickening of the vessel wall progressing to the point of luminal obstruction. In the most severe cases of osteonecrosis, end-stage lesions consisted of fully occluded vessels with marrow and bone necrosis involving the entire epiphysis. We propose that a primary arteriopathy is the initiating event in the genesis of steroid-induced osteonecrosis and provides a basis for future investigation of this disease process.

  10. Inducible protective processes in animal systems: adaptive response to a low dose of methyl methanesulfonate in mouse bone marrow cells.

    PubMed

    Mahmood, R; Vasudev, V; Harish, S K; Guruprasad, K P

    1996-06-01

    To investigate the induction of adaptive response (inducible protective processes) in mitotic cells of Swiss albino mouse, a monofunctional alkylating agent methyl methanesulfonate (MMS) was employed. When the animals treated with a low dose of 50 mg/kg body weight were challenged with a subsequent high (challenging) dose of 150 mg/kg body weight, after different time lags (2,5,8 or 10 hr), the yield of chromosomal aberrations in bone marrow cells was found to be significantly reduced compared to the additive effects of both conditioning and challenging doses. It seems, therefore, that the low dose of MMS employed has made the cells less sensitive against further clastogenic effect of challenge dose of MMS. The data clearly suggest that the phenomenon of adaptive response to methylating agents can be encountered in in vivo mammalian cells. Furthermore, it is also observed that ethylating agent EMS is a poor inducer of adaptive response than its corresponding methylating agent MMS in the bone marrow cells of mouse.

  11. Characterization of xenogeneic mouse-to-rat bone marrow chimeras. I. Examination of hematologic and immunologic function

    SciTech Connect

    Wade, A.C.; Luckert, P.H.; Tazume, S.; Niedbalski, J.L.; Pollard, M.

    1987-07-01

    Eighteen xenogeneic chimeric rats (survival: greater than 100 days) were established by transplanting bone marrow cells from femurs of 10 gnotobiotic CFW mice into each germfree Sprague-Dawley or Wistar rat. The erythrocytes circulating in the rats were of mouse origin as determined by hemagglutination. Hemoglobin electrophoresis, radial immunodiffusion for IgG, and assay of granulocytic neutrophils for leukocyte alkaline phosphatase verified that true chimerism was achieved. The extent of hematological and immunological reconstitution varied. In general, hematocrit levels were low to normal, white blood cell counts and differentials were within normal limits, and serum protein levels were normal. Levels of circulating IgG of each species were comparable to those of germfree rat and mouse controls. Natural killer (NK) activity was depressed, a phenomenon that may be attributable to the radiation treatment of recipients, or to failure to transfer NK cells or precursors. Mitogenic stimulation reactions were varied, but most chimeric rats demonstrated moderately depressed responses. Reactions as a whole suggested that gnotobiotic rats with xenogeneic bone marrow are incompletely reconstituted, both hematologically and immunologically. No acute graft-versus-host reaction was seen.

  12. Targeting the LRP5 pathway improves bone properties in a mouse model of Osteogenesis Imperfecta

    PubMed Central

    Jacobsen, Christina M.; Barber, Lauren A.; Ayturk, Ugur M.; Roberts, Heather J.; Deal, Lauren E.; Schwartz, Marissa A.; Weis, MaryAnn; Eyre, David; Zurakowski, David; Robling, Alexander G.; Warman, Matthew L.

    2014-01-01

    The cell surface receptor low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5p.A214V) that is orthologous to a human HBM-causing mutation have increased bone mass and strength. Osteogenesis Imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5+/p.A214V mice to Col1a2+/p.G610C mice, which model human type IV OI. We found that Col1a2+/p.G610C;Lrp5+/p.A214V offspring had significantly increased bone mass and strength compared to Col1a2+/p.G610C;Lrp5+/+ littermates. The improved bone properties were not due to altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2+/p.G610C mice with a monoclonal antibody that inhibits sclerostin activity (Scl-Ab). We found that antibody treated mice had significantly increased bone mass and strength compared to vehicle treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. PMID:24677211

  13. Microribbon-based hydrogels accelerate stem cell-based bone regeneration in a mouse critical-size cranial defect model

    PubMed Central

    Han, Li-Hsin; Conrad, Bogdan; Chung, Michael T.; Deveza, Lorenzo; Jiang, Xinyi; Wang, Andrew; Butte, Manish J.; Longaker, Michael T.; Wan, Derrick; Yang, Fan

    2016-01-01

    Stem cell-based therapies hold great promise for enhancing tissue regeneration. However, the majority of cells die shortly after transplantation, which greatly diminishes the efficacy of stem cell-based therapies. Poor cell engraftment and survival remain a major bottleneck to fully exploiting the power of stem cells for regenerative medicine. Biomaterials such as hydrogels can serve as artificial matrices to protect cells during delivery and guide desirable cell fates. However, conventional hydrogels often lack macroporosity, which restricts cell proliferation and delays matrix deposition. Here we report the use of injectable, macroporous microribbon (µRB) hydrogels as stem cell carriers for bone repair, which supports direct cell encapsulation into a macroporous scaffold with rapid spreading. When transplanted in a criticalsized, mouse cranial defect model, µRB-based hydrogels significantly enhanced the survival of transplanted adipose-derived stromal cells (ADSCs) (81%) and enabled up to three-fold cell proliferation after 7 days. In contrast, conventional hydrogels only led to 27% cell survival, which continued to decrease over time. MicroCT imaging showed µRBs enhanced and accelerated mineralized bone repair compared to hydrogels (61% vs. 34% by week 6), and stem cells were required for bone repair to occur. These results suggest that paracrine signaling of transplanted stem cells are responsible for the observed bone repair, and enhancing cell survival and proliferation using µRBs further promoted the paracrine-signaling effects of ADSCs for stimulating endogenous bone repair. We envision µRB-based scaffolds can be broadly useful as a novel scaffold for enhancing stem cell survival and regeneration of other tissue types. PMID:26991141

  14. Femoral diaphyseal endoprosthetic reconstruction after segmental resection of primary bone tumours.

    PubMed

    Hanna, S A; Sewell, M D; Aston, W J S; Pollock, R C; Skinner, J A; Cannon, S R; Briggs, T W R

    2010-06-01

    Segmental resection of malignant bone disease in the femoral diaphysis with subsequent limb reconstruction is a major undertaking. This is a retrospective review of 23 patients who had undergone limb salvage by endoprosthetic replacement of the femoral diaphysis for a primary bone tumour between 1989 and 2005. There were 16 males and seven females, with a mean age of 41.3 years (10 to 68). The mean overall follow-up was for 97 months (3 to 240), and 120 months (42 to 240) for the living patients. The cumulative patient survival was 77% (95% confidence interval 63% to 95%) at ten years. Survival of the implant, with failure of the endoprosthesis as an endpoint, was 85% at five years and 68% (95% confidence interval 42% to 92%) at ten years. The revision rate was 22% and the overall rate of re-operation was 26%. Complications included deep infection (4%), breakage of the prosthesis (8%), periprosthetic fracture (4%), aseptic loosening (4%), local recurrence (4%) and metastases (17%). The 16 patients who retained their diaphyseal endoprosthesis had a mean Musculoskeletal Tumour Society score of 87% (67% to 93%). They were all able to comfortably perform most activities of daily living. Femoral diaphyseal endoprosthetic replacement is a viable option for reconstruction following segmental resection of malignant bone disease. It allows immediate weight-bearing, is associated with a good long-term functional outcome, has an acceptable complication and revision rate and, most importantly, does not appear to compromise patient survival.

  15. Mouse Genome Informatics (MGI): Resources for Mining Mouse Genetic, Genomic, and Biological Data in Support of Primary and Translational Research.

    PubMed

    Eppig, Janan T; Smith, Cynthia L; Blake, Judith A; Ringwald, Martin; Kadin, James A; Richardson, Joel E; Bult, Carol J

    2017-01-01

    The Mouse Genome Informatics (MGI), resource ( www.informatics.jax.org ) has existed for over 25 years, and over this time its data content, informatics infrastructure, and user interfaces and tools have undergone dramatic changes (Eppig et al., Mamm Genome 26:272-284, 2015). Change has been driven by scientific methodological advances, rapid improvements in computational software, growth in computer hardware capacity, and the ongoing collaborative nature of the mouse genomics community in building resources and sharing data. Here we present an overview of the current data content of MGI, describe its general organization, and provide examples using simple and complex searches, and tools for mining and retrieving sets of data.

  16. Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells.

    PubMed

    Kassmer, Susannah H; Bruscia, Emanuela M; Zhang, Ping-Xia; Krause, Diane S

    2012-03-01

    Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.

  17. Nonhematopoietic Cells are the Primary Source of Bone Marrow-Derived Lung Epithelial Cells

    PubMed Central

    Kassmer, Susannah H.; Bruscia, Emanuela M.; Zhang, Ping-Xia; Krause, Diane S.

    2013-01-01

    Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM. PMID:22162244

  18. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

    PubMed

    Sampaziotis, Fotios; Justin, Alexander W; Tysoe, Olivia C; Sawiak, Stephen; Godfrey, Edmund M; Upponi, Sara S; Gieseck, Richard L; de Brito, Miguel Cardoso; Berntsen, Natalie Lie; Gómez-Vázquez, María J; Ortmann, Daniel; Yiangou, Loukia; Ross, Alexander; Bargehr, Johannes; Bertero, Alessandro; Zonneveld, Mariëlle C F; Pedersen, Marianne T; Pawlowski, Matthias; Valestrand, Laura; Madrigal, Pedro; Georgakopoulos, Nikitas; Pirmadjid, Negar; Skeldon, Gregor M; Casey, John; Shu, Wenmiao; Materek, Paulina M; Snijders, Kirsten E; Brown, Stephanie E; Rimland, Casey A; Simonic, Ingrid; Davies, Susan E; Jensen, Kim B; Zilbauer, Matthias; Gelson, William T H; Alexander, Graeme J; Sinha, Sanjay; Hannan, Nicholas R F; Wynn, Thomas A; Karlsen, Tom H; Melum, Espen; Markaki, Athina E; Saeb-Parsy, Kourosh; Vallier, Ludovic

    2017-08-01

    The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

  19. Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

    PubMed

    Orsini, Malina; Sperber, Saskia; Noor, Fozia; Hoffmann, Esther; Weber, Susanne N; Hall, Rabea A; Lammert, Frank; Heinzle, Elmar

    2017-06-08

    Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 9999: 1-8, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  20. Hydrocortisone effect of arylsulfatase A in primary mouse brain cell cultures

    SciTech Connect

    Marcelo, A.; Pieringer, R.A.

    1986-05-01

    The primary goal of this study was to study the mechanism of action of hydrocortisone (HC) on arylsulfatase A (ASA) in primary cultures of cells that were dissociated from the brains of embryonic mice. Cells were cultured in a defined medium in the absence or in the presence of 3 ..mu..M HC. The specific activity of ASA in nontreated cells was 1.297 U/mg (U = ..mu..mol/hr) while the value for the HC-treated cells was 0.783 U/mg. The authors data shows that HC inhibits ASA activity in these cultures cells (p < 0.001). The determination of the ASA enzyme activity was assayed primarily with the artificial substrate p-nitrocatechol sulfate. However, the natural substrate (cerebroside /sup 35/S-sulfate) also as active and correlated linearly with the activity of p-nitrocatechol sulfate. Purified ASA was isolated from calf brains and used to generate an antibody (Ab) against ASA. The specificity of the Ab for the ASA protein of cell cultures was tested in Ouchterlony double immunodiffusion studies. The Ab was used in a competitive enzyme-linked immunosorbent assay to quantify the number of ASA molecules in the cell extracts from the embryonic mouse cell cultures. Preliminary data suggest that HC decreases the number of ASA molecules.

  1. Cerium oxide nanoparticles stimulate proliferation of primary mouse embryonic fibroblasts in vitro.

    PubMed

    Popov, Anton L; Popova, Nelly R; Selezneva, Irina I; Akkizov, Azamat Y; Ivanov, Vladimir K

    2016-11-01

    The increasing application of cell therapy technologies in the treatment of various diseases requires the development of new effective methods for culturing primary cells. The major limitation for the efficient use of autologous cell material is the low rate of cell proliferation. Successful cell therapy requires sufficient amounts of cell material over a short period of time with the preservation of their differentiation and proliferative potential. In this regard, the development of novel, highly efficient stimulators of proliferative activity in stem cells is a truly urgent task. In this paper we have demonstrated that citrate-stabilized cerium oxide nanoparticles (nanoceria) enhance the proliferative activity of primary mouse embryonic fibroblasts in vitro. Cerium oxide nanoparticles stimulate cell proliferation in a wide range of concentrations (10(-3)М-10(-9)M) through reduction of intracellular levels of reactive oxygen species (ROS) during the lag phase of cell growth and by modulating the expression level of the major antioxidant enzymes. We found the optimal concentration of nanoceria, which provides the greatest acceleration of cell proliferation in vitro, while maintaining the levels of intracellular ROS and mRNA of antioxidant enzymes in the physiological range. Our results confirm that nanocrystalline ceria can be considered as a basis for effective and inexpensive supplements in cell culturing.

  2. Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes.

    PubMed

    Zheng, Jinying; Peng, Chuan; Ai, Yanbiao; Wang, Heng; Xiao, Xiaoqiu; Li, Jibin

    2016-01-20

    The increase in fructose consumption is considered to be a risk factor for developing nonalcoholic fatty liver disease (NAFLD). We investigated the effects of docosahexaenoic acid (DHA) on hepatic lipid metabolism in fructose-treated primary mouse hepatocytes, and the changes of Endoplasmic reticulum (ER) stress pathways in response to DHA treatment. The hepatocytes were treated with fructose, DHA, fructose plus DHA, tunicamycin (TM) or fructose plus 4-phenylbutyric acid (PBA) for 24 h. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining. The mRNA expression levels and protein levels related to lipid metabolism and ER stress response were determined by real-time PCR and Western blot. Fructose treatment led to obvious TG accumulation in primary hepatocytes through increasing expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two key enzymes in hepatic de novo lipogenesis. DHA ameliorates fructose-induced TG accumulation by upregulating the expression of carnitine palmitoyltransferase 1A (CPT-1α) and acyl-CoA oxidase 1 (ACOX1). DHA treatment or pretreatment with the ER stress inhibitor PBA significantly decreased TG accumulation and reduced the expression of glucose-regulated protein 78 (GRP78), total inositol-requiring kinase 1 (IRE1α) and p-IRE1α. The present results suggest that DHA protects against high fructose-induced hepatocellular lipid accumulation. The current findings also suggest that alleviating the ER stress response seems to play a role in the prevention of fructose-induced hepatic steatosis by DHA.

  3. IL-32-induced Inflammatory Cytokines Are Selectively Suppressed by α1-antitrypsin in Mouse Bone Marrow Cells.

    PubMed

    Lee, Siyoung; Choi, Dong-Ki; Kwak, Areum; Kim, Sinae; Nguyen, Tam Thanh; Gil, Gaae; Kim, Eunhye; Yoo, Kwang Ha; Kim, In Ae; Lee, Youngmin; Jhun, Hyunjhung; Chan, Edward D; Bai, Xiyuan; Kim, Hyunwoo; Kim, Yong-Sung; Kim, Soohyun

    2017-04-01

    The induction of interleukin (IL)-32 in bone marrow (BM) inflammation is crucial in graft versus host disease (GvHD) that is a common side effect of allogeneic BM transplantation. Clinical trials on α-1 antitrypsin (AAT) in patients with GvHD are based on the preliminary human and mouse studies on AAT reducing the severity of GvHD. Proteinase 3 (PR3) is an IL-32-binding protein that was isolated from human urine. IL-32 primarily induces inflammatory cytokines in myeloid cells, probably due to PR3 expression on the membrane of the myeloid lineage cells. The inhibitory activity of AAT on serine proteinases may explain the anti-inflammatory effect of AAT on GvHD. However, the anti-inflammatory activity of AAT on BM cells remains unclear. Mouse BM cells were treated with IL-32γ and different inflammatory stimuli to investigate the anti-inflammatory activity of AAT. Recombinant AAT-Fc fusion protein inhibited IL-32γ-induced IL-6 expression in BM cells, but failed to suppress that induced by other stimuli. In addition, the binding of IL-32γ to PR3 was abrogated by AAT-Fc. The data suggest that the specific anti-inflammatory effect of AAT in mouse BM cells is due to the blocking of IL-32 binding to membrane PR3.

  4. Bone Marrow-Derived Cells May Not Be the Original Cells for Carcinogen-Induced Mouse Gastrointestinal Carcinomas

    PubMed Central

    Yang, Chen; Gu, Liankun; Deng, Dajun

    2013-01-01

    Aim It has been reported that bone marrow-derived cells (BMDC) can be original cells of mouse gastric cancers induced by Helicobacter felis (H. felis) infection. However, it is unknown whether BMDCs are also the original cells of mouse gastrointestinal cancers induced by gastric carcinogens N-nitroso-N-methylurea (NMU) and H. felis infection. Methods C57BL/6 recipient mice were initially irradiated with 10Gy X-ray, reconstituted with bone marrow cells from the C57BL/6-Tg (CAG-EGFP) donor mice to label BMDCs with green fluorescence protein (GFP). After 4 weeks of recovery, the bone marrow-transplanted mice were given NMU in drinking water (240 ppm) and subsequently infected with H. felis by gavage. Eighty weeks later, all mice were euthanized for pathological examination. The BMDCs expressing GFP were detected in tissues using direct GFP fluorescence confocal microscopy analysis and immunohistochemistry staining (IHC) assays. Results Neoplastic lesions were induced by NMU treatment and/or H. felis infection at the antrum of the glandular stomach and small intestine. In the direct GFP fluorescence confocal assay, GFP(+) epithelial cell cluster or glands were not observed in these gastrointestinal tumors, however, most GFP(+) BMDCs sporadically located in the tumor stromal tissues. Some of these GFP(+) stromal BMDCs co-expressed the hematopoietic marker CD45 or myofibroblasts markers αSMA and SRF. In the indirect GFP IHC assay, similar results were observed among 11 gastric intraepithelial neoplasia lesions and 2 small intestine tumors. Conclusion These results demonstrated that BMDCs might not be the source of gastrointestinal tumor cells induced by NMU and/or H. felis infection. PMID:24260263

  5. A method to establish a mouse model of bone marrow microenvironment injury.

    PubMed

    Cheng, Wenzhe; Ge, Quanhu; Wan, Longfei; Wang, Xiaoyi; Chen, Xueling; Wu, Xiangwei

    2017-06-13

    A normal bone marrow microenvironment plays a very important role in the normal functioning of hematopoietic stem cells. Once disturbed, this microenvironment can become favorable for the occurrence of blood disorders, cancers, and other diseases. Therefore, further studies on the bone marrow microenvironment should be performed to reveal regulatory and stem cell fate determination mechanisms and promote the development of bone marrow transplantation, tissue repair and regenerative medicine, and other fields. A small animal model for further research is also urgently needed. In this study, an electric shock device was designed to elicit a femur bone marrow microenvironment injury in mice. A wire was inserted into the distal femur but not into the proximal femur, and the bone marrow microenvironment was evidently damaged by application of 100 ± 10 V for 1.5 ± 0.5 min; mortality, however, was low in the mice. Gross observation, hematoxylin and eosin staining, immunohistochemistry, bright-field microscopy, and micro-CT scanning were also conducted. A large number of new blood capillaries and sinusoids appeared in the injured distal femur after 2 weeks. The capillaries in the injured femur disappeared after 4 weeks, and mature blood vessels were scattered throughout the injured area. Red blood cells disappeared, and the cellular structure and trabecular bone were better than those observed 2 weeks previously. Thus, we developed a simply operated, accurate, reliable, and easily controlled small animal model as a good technical platform to examine angiogenesis and segmentation damage in the bone marrow microenvironment.

  6. Benefit of Consolidative Radiation Therapy for Primary Bone Diffuse Large B-Cell Lymphoma

    SciTech Connect

    Tao, Randa; Allen, Pamela K.; Rodriguez, Alma; Shihadeh, Ferial; Pinnix, Chelsea C.; Arzu, Isadora; Reed, Valerie K.; Oki, Yasuhiro; Westin, Jason R.; Fayad, Luis E.; Medeiros, L. Jeffrey; Dabaja, Bouthaina

    2015-05-01

    Purpose: Outcomes for patients with diffuse large B-cell lymphoma (DLBCL) differ according to the site of presentation. With effective chemotherapy, the need for consolidative radiation therapy (RT) is controversial. We investigated the influence of primary bone presentation and receipt of consolidative RT on progression-free survival (PFS) and overall survival (OS) in patients with DLBCL. Methods and Materials: We identified 102 patients with primary bone DLBCL treated consecutively from 1988 through 2013 and extracted clinical, pathologic, and treatment characteristics from the medical records. Survival outcomes were calculated by the Kaplan-Meier method, with factors affecting survival determined by log-rank tests. Univariate and multivariate analyses were done with a Cox regression model. Results: The median age was 55 years (range, 16-87 years). The most common site of presentation was in the long bones. Sixty-five patients (63%) received R-CHOP–based chemotherapy, and 74 (72%) received rituximab. RT was given to 67 patients (66%), 47 with stage I to II and 20 with stage III to IV disease. The median RT dose was 44 Gy (range, 24.5-50 Gy). At a median follow-up time of 82 months, the 5-year PFS and OS rates were 80% and 82%, respectively. Receipt of RT was associated with improved 5-year PFS (88% RT vs 63% no RT, P=.0069) and OS (91% vs 68%, P=.0064). On multivariate analysis, the addition of RT significantly improved PFS (hazard ratio [HR] = 0.14, P=.014) with a trend toward an OS benefit (HR=0.30, P=.053). No significant difference in PFS or OS was found between patients treated with 30 to 35 Gy versus ≥36 Gy (P=.71 PFS and P=.31 OS). Conclusion: Patients with primary bone lymphoma treated with standard chemotherapy followed by RT can have excellent outcomes. The use of consolidative RT was associated with significant benefits in both PFS and OS.

  7. From Breast to Bone: Tracking Gene Expression Changes Responsible for Breast Cancer Metastasis in a Humanized Mouse Model with Molecular Imaging

    DTIC Science & Technology

    2015-11-01

    AWARD NUMBER: W81XWH-11-1-0616 TITLE: From Breast to Bone: Tracking Gene Expression Changes Responsible for Breast Cancer Metastasis in a... Breast to Bone: Tracking Gene Expression Changes Responsible for Breast Cancer Metastasis in a Humanized Mouse Model with Molecular Imaging 5b. GRANT... Breast cancer is the leading cause of cancer -related death in women worldwide, and metastasis is responsible for the majority of these deaths. Triple

  8. The impact of vitamin D status on hungry bone syndrome after surgery for primary hyperparathyroidism.

    PubMed

    Kaderli, Reto Martin; Riss, Philipp; Dunkler, Daniela; Pietschmann, Peter; Selberherr, Andreas; Scheuba, Christian; Niederle, Bruno

    2017-09-06

    Prolonged hypocalcaemia but normal intact parathyroid hormone (iPTH) levels after surgery for primary hyperparathyroidism (PHPT) are referred to as "hungry bone syndrome" (HBS). The aim was to evaluate preoperative risk factors for HBS with a focus on the impact of 25-hydroxyvitamin D (25(OH)D) deficiency. Patients having undergone initial successful surgery for sporadic PHPT within 6 years were considered for retrospective analysis. A total of 385 patients were evaluated, of whom 33 (8.6%) developed HBS influencing negatively the postoperative bone metabolism. All patients underwent biochemical evaluations two days before parathyroid surgery and were followed biochemically on a daily basis in the first postoperative week and thereafter at 8 weeks and 6 months. Univariate analysis of preoperative 25(OH)D levels revealed no impact on HBS (c-index = 0.587). Univariate logistic models adjusted for osteocalcin, bone mineral density of the left femoral neck (BMDneck) and 25(OH)D showed a significant relationship between iPTH and HBS (OR 2.09, 95% CI, 1.47-2.97; p < 0.001). iPTH had the highest predictive value (c-index = 0.785, 95% CI, 0.693-0.877), followed by osteocalcin (c-index = 0.685, 95% CI, 0.567-0.804), BMDneck (c-index = 0.648, 95% CI, 0.550-0.745) and 25(OH)D (c-index = 0.576, 95% CI, 0.466-0.687). No relationship was established between preoperative 25(OH)D deficiency and HBS. The only significant risk factor for HBS in multivariable analysis was high levels of preoperative iPTH. As HBS therefore cannot be predicted preoperatively, we recommend a consistent postoperative calcium and vitamin D supplementation to improve the bone metabolism.

  9. Primary cilia expression in bone marrow in response to mechanical stimulation in explant bioreactor culture.

    PubMed

    Coughlin, T R; Schiavi, J; Alyssa Varsanik, M; Voisin, M; Birmingham, E; Haugh, M G; McNamara, L M; Niebur, G L

    2016-07-19

    Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent from CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function.

  10. TUMOR CONTAMINATION IN THE BIOPSY PATH OF PRIMARY MALIGNANT BONE TUMORS

    PubMed Central

    Oliveira, Marcelo Parente; Lima, Pablo Moura de Andrade; de Mello, Roberto José Vieira

    2015-01-01

    Objective: To study factors possibly associated with tumor contamination in the biopsy path of primary malignant bone tumors. Method: Thirty-five patients who underwent surgical treatment with diagnoses of osteosarcoma, Ewing's tumor and chondrosarcoma were studied retrospectively. The sample was analyzed to characterize the biopsy technique used, histological type of the tumor, neoadjuvant chemotherapy used, local recurrences and tumor contamination in the biopsy path. Results: Among the 35 patients studied, four cases of contamination occurred (11.43%): one from osteosarcoma, two from Ewing's tumor and one from chondrosarcoma. There was no association between the type of tumor and presence of tumor contamination in the biopsy path (p = 0.65). There was also no association between the presence of tumor contamination and the biopsy technique (p = 0.06). On the other hand, there were associations between the presence of tumor contamination and local recurrence (p = 0.01) and between tumor contamination and absence of neoadjuvant chemotherapy (p = 0.02). Conclusion: Tumor contamination in the biopsy path of primary malignant bone tumors was associated with local recurrence. On the other hand, the histological type of the tumor and the type of biopsy did not have an influence on tumor contamination. Neoadjuvant chemotherapy had a protective effect against this complication. Despite these findings, tumor contamination is a complication that should always be taken into consideration, and removal of the biopsy path is recommended in tumor resection surgery. PMID:27047877

  11. Pathological femoral fracture caused by primary bone tumour: a population-based study.

    PubMed

    Godley, K; Watts, A C; Robb, J E

    2011-02-01

    This population-based study aimed to analyse the demographic, clinical and histological features of patients with a malignant primary bone tumour of the femur presenting with a pathological fracture. Eighty-four patients were identified from a prospectively gathered national tumour database between 1960 and 2004. Demographic data, presenting features, tumour location, histological diagnosis, treatment, local recurrence, metastasis and survival data were gathered. An estimate of the annual incidence was obtained using population data from the General Register Office and was 0.4 per million population per annum. The mean age was 56 years (range 4-87 years) with a bimodal distribution and 46% were men or boys. Forty-one percent of patients presented with a history of trauma. The average duration of symptoms before presentation was 1-3 months. The most common histological diagnoses were osteosarcoma (14 patients) and Paget's sarcoma (12 patients). The local recurrence rate was 38% and the overall five-year survival was 22%. The prognosis was made worse by local tumour recurrence, the development of metastasis and age at diagnosis greater than 21 years. Limb salvage surgery did not alter the prognosis. Patients who present with pathological fracture of a primary malignant bone tumour, carry a poor prognosis in all tumour types and no improvement in survival was identified over the period of the study.

  12. On the relation between surface roughness of metallic substrates and adhesion of human primary bone cells.

    PubMed

    Anselme, K; Bigerelle, M

    2014-01-01

    Surface characteristics of materials, whether their topography, chemistry, or surface energy, play an essential part in osteoblast adhesion on biomaterials. Thus, the quality of cell adhesion will influence the cell's capacity to proliferate and differentiate in contact with a biomaterial. We have developed for more than ten years numerous studies on the influence of topography and chemistry of metallic substrates on the response of primary human bone cells. The originality of our approach is that contrary to most of other authors, we quantified the adhesion of primary human bone cells on metallic substrates with perfectly characterized surface topography after some hours but also over 21 days. Moreover, we have developed original statistical approaches for characterizing the relation between surface roughness and cell-adhesion parameters. In this article, we will illustrate different studies we did these last ten years concerning the development of a new adhesion parameter, the adhesion power; the correlation between short-term adhesion, long-term adhesion, and proliferation; the influence of roughness organization on cell adhesion and the development of the order parameter; our modeling approach of cell adhesion on surface topography; the relative influence of surface chemistry and topography on cell adhesion and contact angle; the relation between surface features dimensions and cell adhesion. Further, some considerations will be given on the methods for scanning surface topography for cell-adhesion studies. Finally, perspectives will be given to elucidate these intracellular mechanotransduction mechanisms induced by the deformation of cells on model sinusoidal peaks-or-valleys surfaces.

  13. Clinical characterization and outcome of primary bone lymphoma: a retrospective study of 61 Chinese patients

    PubMed Central

    Zhang, XuanYe; Zhu, Jun; Song, YuQin; Ping, LingYan; Zheng, Wen

    2016-01-01

    Primary bone lymphoma(PBL) is a rare disease. To assess the clinical characteristics, outcome, and prognostic factors of this entity in Chinese population, we retrospectively analyzed 61 PBL patients initially treated in our institution between 1997 and 2014. The median age was 45 years. The most common histological subtype was diffuse large B-cell lymphoma (DLBCL) (55.7%), followed by T-cell lymphoma (18.0%). All patients underwent systemic chemotherapy as initial treatment while 24 patients (39.3%) were additionally treated with radiotherapy. The 5-year overall survival (OS) and the 5-year progression-free survival (PFS) rates of 57 cases with completed follow-up were 52.3% and 40.1%, respectively. In further analysis of the primary bone DLBCL (PB-DLBCL) subgroup, the 5-year OS and PFS rates were 53.0% and 47.0%, and a multivariable analysis revealed that baseline Eastern Cooperative Oncology Group (ECOG) score and response to initial treatment (complete remission versus no complete remission) were independent prognostic factors for both OS and PFS. The proportion of T-cell lymphoma is higher in China than in western populations. High baseline ECOG scores (≥2) and unachieved CR in initial therapy were factors for poor PB-DLBCL prognosis. The role of radiotherapy and rituximab in PLB therapy remains to be confirmed in further investigation. PMID:27357354

  14. Infrared imaging microscopy of bone: Illustrations from a mouse model of Fabry disease

    PubMed Central

    Boskey, Adele L.; Goldberg, Michel; Kulkarni, Ashok; Gomez, Santiago

    2006-01-01

    Bone is a complex tissue whose composition and properties vary with age, sex, diet, tissue type, health and disease. In this review, we demonstrate how infrared spectroscopy and infrared spectroscopic imaging can be applied to the study of these variations. A specific example of mice with Fabry disease (a lipid storage disease) is presented in which it is demonstrated that the bones of these young animals, while showing typical spatial variation in mineral content, mineral crystal size, and collagen maturity, do not differ from the bones of age- and sex-matched wild type animals. PMID:16697974

  15. In vivo 4-dimensional tracking of hematopoietic stem and progenitor cells in adult mouse calvarial bone marrow.

    PubMed

    Scott, Mark K; Akinduro, Olufolake; Lo Celso, Cristina

    2014-09-04

    Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood([1-2]). We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells. Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

  16. In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

    PubMed Central

    Scott, Mark K.; Akinduro, Olufolake; Lo Celso, Cristina

    2014-01-01

    Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2]. We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells. Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space. PMID:25225854

  17. The flow field along the entire length of mouse aorta and primary branches.

    PubMed

    Huo, Yunlong; Guo, Xiaomei; Kassab, Ghassan S

    2008-05-01

    There is a spatial disposition to atherosclerosis along the aorta corresponding to regions of flow disturbances. The objective of the present study is to investigate the detailed distribution of hemodynamic parameters (wall shear stress (WSS), spatial gradient of wall shear stress (WSSG), and oscillatory shear index (OSI)) in the entire length of C57BL/6 mouse aorta with all primary branches (from ascending aorta to common iliac bifurcation). The detailed geometrical parameters (e.g., diameter and length of the vessels) were obtained from casts of entire aorta and primary branches of mice. The flow velocity was measured at the inlet of ascending aorta using Doppler flowprobe in mice. The outlet pressure boundary condition was estimated based on scaling law. The continuity and Navier-Stokes equations were solved using three-dimensional finite element method (FEM). The model prediction was tested by comparing the computed flow rate with the flow rate measured just before the common iliac bifurcation, and good agreement was found. It was also found that complex flow patterns occur at bifurcations between main trunk and branches. The major branches of terminal aorta, with the highest proportion of atherosclerosis, have the lowest WSS, and the relatively atherosclerotic-prone aortic arch has much more complex WSS distribution and higher OSI value than other sites. The low WSS coincides with the high OSI, which approximately obeys a power law relationship. Furthermore, the scaling law between flow and diameter holds in the entire aorta and primary branches of mice under pulsatile blood flow conditions. This model will eventually serve to elucidate the causal relation between hemodynamic patterns and atherogenesis in KO mice.

  18. Duration sensitivity of neurons in the primary auditory cortex of albino mouse.

    PubMed

    Wang, Xin; Qi, Qiaozhen; Huang, Caifei; Chomiak, Taylor; Luo, Feng

    2016-02-01

    Many neurons in the central auditory system of a number of species have been found to be sensitive to the duration of sound stimuli. While previous studies have shown that γ-aminobutyric acid (GABA)-ergic inhibitory input is important for duration sensitivity in the inferior colliculus (IC), it is still unknown whether (GABA)-ergic inhibitory input plays an important role in generating duration sensitivity in the cortex. Using free-field sound stimulation and in vivo extracellular recording, we investigated duration sensitivity in primary auditory cortical (AI) neurons of the Nembutal anesthetized albino mouse (Mus musculus, Km) and examined the effect of the GABAA receptor antagonist bicuculline on AI neuron duration sensitivity. A total of 63 duration tuning curves were measured in AI neurons. Of these, 44% (28/63) exhibited duration sensitive responses, while 43% (27/63) lacked duration sensitivity. The remaining 13% (8/63) exhibited long-pass properties likely reflecting both duration sensitive and insensitive features. We found that duration sensitive neurons had shorter first spike latency (FSL) and longer firing duration (FD) when stimulated with best duration (p < 0.05), while duration insensitive neurons had invariable FSL and FD at different sound durations (p>0.05). Furthermore, 60% (6/10) of duration sensitive neurons and 75% (3/4) long-pass neurons lost duration sensitivity following bicuculline application. Taken together, our results show that cortical neurons in the albino mouse are sensitive to sound duration, and that GABAergic inhibition may play an important role in the formation of de novo duration sensitivity in AI. The possible mechanism and behavioral significance of duration sensitivity in AI neurons is discussed.

  19. Voluntary physical exercise promotes ocular dominance plasticity in adult mouse primary visual cortex.

    PubMed

    Kalogeraki, Evgenia; Greifzu, Franziska; Haack, Franziska; Löwel, Siegrid

    2014-11-12

    Ocular dominance (OD) plasticity in the mouse primary visual cortex (V1) declines during aging and is absent beyond postnatal day (P) 110 when mice are raised in standard cages (SCs; Lehmann and Löwel, 2008). In contrast, raising mice in an enriched environment (EE) preserved a juvenile-like OD plasticity into late adulthood (Greifzu et al., 2014). EE raising provides the mice with more social interactions, voluntary physical exercise, and cognitive stimulation compared with SC, raising the question whether all components are needed or whether one of them is already sufficient to prolong plasticity. To test whether voluntary physical exercise alone already prolongs the sensitive phase for OD plasticity, we raised mice from 7 d before birth to adulthood in slightly larger than normal SCs with or without a running wheel (RW). When the mice were older than P135, we visualized V1 activity before and after monocular deprivation (MD) using intrinsic signal optical imaging. Adult RW-raised mice continued to show an OD shift toward the open eye after 7 d of MD, while age-matched SC mice without a RW did not show OD plasticity. Notably, running just during the 7 d MD period restored OD plasticity in adult SC-raised mice. In addition, the OD shift of the RW mice was mediated by a decrease of deprived-eye responses in V1, a signature of "juvenile-like" plasticity. We conclude that voluntary physical exercise alone is sufficient to promote plasticity in adult mouse V1.

  20. Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation.

    PubMed

    Bollag, W B; Ducote, J; Harmon, C S

    1995-05-01

    1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has been proposed as a physiologic regulator of keratinocyte growth and differentiation. Utilizing a proliferative serum-free culture system, we have found that a physiologic (picomolar) concentrations this hormone stimulated proliferation of primary mouse epidermal keratinocytes; at higher (nanomolar to micromolar) doses, growth was inhibited by 1,25(OH)2D3. We investigated the nature of the signal transduction mechanism underlying the response to 1,25(OH)2D3 and observed little or no effect of either low or high concentrations of the hormone on cytosolic calcium levels or Fos expression. Furthermore, the protein kinase C inhibitor, Ro 31-7549, had very little effect on the growth inhibition induced by a high dose (1 microM) of 1,25(OH)2D3. This lack of rapid signal transduction events was consistent with the inability of a short (4-hour) exposure to 1,25(OH)2D3 to initiate a complete growth-inhibitory response as measured using [3H]thymidine incorporation. Our results indicate that physiologic concentrations of 1,25(OH)2D3 are required for optimal keratinocyte growth. Furthermore, we found no evidence of rapid effects of 1,25(OH)2D3 and suggest that in mouse epidermal keratinocytes, the response to this hormone is mediated by a slow transduction pathway, such as that activated by the intracellular 1,25(OH)2D3 receptor (VDR).

  1. Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes.

    PubMed

    Jeon, Yu Jin; Kim, Hyoung Seok; Song, Kyung-Sik; Han, Ho Jae; Park, Soo Hyun; Chang, Woochul; Lee, Min Young

    2015-04-01

    Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl2), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl2 significantly attenuated the CoCl2-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl2-induced decrease of Bcl-2 expression and attenuated the CoCl2-induced increase in the expression of Bax and caspase-3. Treatment with CoCl2 resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl2-induced decrease of Bcl-2 expression and decreased the CoCl2-induced increase of Bax and caspase-3 expressions. CoCl2-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl2-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl2-induced COX-2 expression. Additionally, CoCl2-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl2-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.

  2. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes

    SciTech Connect

    Ruch, R.J.; Klaunig, J.E.; Schultz, N.E.; Askari, A.B.; Lacher, D.A.; Pereira, M.A.; Goldblatt, P.J.

    1986-11-01

    Mechanisms of chloroform (CHCl/sub 3/) and carbon tetrachloride (CCl/sub 4/) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl/sub 3/ and CCl/sub 4/ was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl/sub 3/ (5 mM) and CCl/sub 4/ (2.5 mM) used and with the longest duration of treatment (20 hr). CCl/sub 4/ was approximately 16 times more toxic than CHCl/sub 3/ to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25..mu..M) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl/sub 3/ and CCl/sub 4/ toxicity. The toxicity of CHCl/sub 3/ and CCl/sub 4/ could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25..mu..M), ..cap alpha..-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl/sub 3/ and CCl/sub 4/ are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl/sub 3/ and CCl/sub 4/; and free radicals may be important mediators of the toxicity of these two halomethanes.

  3. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes.

    PubMed Central

    Ruch, R J; Klaunig, J E; Schultz, N E; Askari, A B; Lacher, D A; Pereira, M A; Goldblatt, P J

    1986-01-01

    Mechanisms of chloroform (CHCl3) and carbon tetrachloride (CCl4) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl3 and CCl4 was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl3 (5 mM) and CCl4 (2.5 mM) used and with the longest duration of treatment (20 hr). CCl4 was approximately 16 times more toxic than CHCl3 to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25 microM) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl3 and CCl4 toxicity. The toxicity of CHCl3 and CCl4 could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25 microM), alpha-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl3 and CCl4 are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl3 and CCl4; and free radicals may be important mediators of the toxicity of these two halomethanes. PMID:3816733

  4. Generation of mouse and human induced pluripotent stem cells (iPSC) from primary somatic cells.

    PubMed

    Lorenzo, I M; Fleischer, A; Bachiller, D

    2013-08-01

    Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663-676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotency-associated genes Okita et al. (Science 322(5903):949-953, 2008), lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543-549, 2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85(8):348-362, 2009), removal of the reprogramming vectors by 'piggyBac' transposition Woltjen et al. (Nature 458(7239):766-770, 2009); Kaji et al. (Nature 458(7239):771-775, 2009), Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964-977, 2009), episomal vectors Yu et al. (Science 324(5928):797-801, 2009), cell-penetrating reprogramming proteins Zhou et al. (Stem Cells 4(5):381-384, 2009), mammalian artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011) synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012), miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376-388, 2009); however, although some of these methods are commercially available, in general they still need to attain the reproducibility and reprogramming efficiency required for routine applications Mochiduki and Okita (Biotechnol Journal 7(6):789-797, 2012). Herein we explain, in four detailed protocols, the isolation of mouse and human

  5. Bone marrow micronucleus assay: a review of the mouse stocks used and their published mean spontaneous micronucleus frequencies.

    PubMed

    Salamone, M F; Mavournin, K H

    1994-01-01

    We have examined published negative control data from 581 papers on micronucleated bone marrow polychromatic erythrocytes (mnPCE) for differences in mean frequency and the frequency distribution profile among the mouse stocks used with the bone marrow micronucleus assay. For the 55 mouse stocks with published micronucleus assay data, the overall mean frequency is 1.95 mnPCE/1,000 PCE (1.95 mnPCE/1,000); for the 13 stocks most commonly used in the assay, it is 1.88 mnPCE/1,000. During the last 5 years, the mnPCE rate for these 13 major stocks has been 1.74 mnPCE/1,000. This current mean frequency is a substantial decrease from the mean of 3.07 mnPCE/1,000 observed for these 13 stocks for data published prior to 1981. Of the major stocks, the highest mean mnPCE negative control frequencies were observed for MS/Ae > BALB/c > C57Bl/6, and the lowest for CD-1 < Swiss Webster. We note that hybrid mouse stocks appear to have lower and less variable negative control frequencies than either of their parent strains and that the negative control frequency for some progeny stocks have diverged significantly from that of the parent stocks. Overall mean negative control frequencies appear to be correlated with breadth of the frequency distribution profile of published mean negative control values. Furthermore, a possible correlation between negative control frequency in the micronucleus assay and sensitivity to clastogens of different mouse strains may be indicated. The databases generated here allow us to define a range of norms for both the historical mean frequency and individual experimental mean frequencies for most stocks, but in particular, for the more commonly used mouse stocks. Our analysis, for the most part, bears out the recommendation of the first Gene-Tox Report on the micronucleus assay that the historical negative control frequency for a mouse stock should fall between 1 and 3 mnPCE/1,000. Eighty-six percent of the most commonly used mouse stocks have historical

  6. The protocol for the isolation and cryopreservation of osteoclast precursors from mouse bone marrow and spleen.

    PubMed

    Boraschi-Diaz, Iris; Komarova, Svetlana V

    2016-01-01

    Osteoclasts are responsible for physiological bone remodeling as well as pathological bone destruction in osteoporosis, periodontitis and rheumatoid arthritis, and thus represent a pharmacological target for drug development. We aimed to characterize and compare the cytokine-induced osteoclastogenesis of bone marrow and spleen precursors. Established protocols used to generate osteoclasts from bone marrow were modified to examine osteoclastogenesis of the spleen cells of healthy mice. Osteoclast formation was successfully induced from spleen precursors using receptor activator of nuclear factor κB ligand (50 ng/ml) and macrophage colony stimulating factor (50 ng/ml). Compared to bone marrow cultures, differentiation from spleen required a longer cultivation time (9 days for spleen, as compared to 5 days for marrow cultures) and a higher plating density of non-adherent cells (75,000/cm(2) for spleen, as compared to 50,000/cm(2) for bone marrow). Osteoclasts generated from spleen precursors expressed osteoclast marker genes calcitonin receptor, cathepsin K and matrix metalloproteinase 9 and were capable of resorbing hydroxyapatite. The differentiation capacity of spleen and bone marrow precursors was comparable for BALB/c, C57BL/6 and FVB mice. We also developed and tested a cryopreservation protocol for the osteoclast precursors. While 70-80 % of cells were lost during the first week of freezing, during the subsequent 5 weeks the losses were within 2-5 % per week. Osteoclastogenesis from the recovered bone marrow precursors was successful up to 5 weeks after freezing. Spleen precursors retained their osteoclastogenic capacity for 1 week after freezing, but not thereafter. The described protocol is useful for the studies of genetically modified animals as well as for screening new osteoclast-targeting therapeutics.

  7. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function

    PubMed Central

    Sato, Kazuya; Feng, Xingmin; Chen, Jichun; Li, Jungang; Muranski, Pawel; Desierto, Marie J.; Keyvanfar, Keyvan; Malide, Daniela; Kajigaya, Sachiko; Young, Neal S.

    2016-01-01

    Acquired aplastic anemia is an immune-mediated disease, in which T cells target hematopoietic cells; at presentation, the bone marrow is replaced by fat. It was reported that bone marrow adipocytes were negative regulators of hematopoietic microenvironment. To examine the role of adipocytes in bone marrow failure, we investigated peroxisomal proliferator-activated receptor gamma, a key transcription factor in adipogenesis, utilizing an antagonist of this factor called bisphenol-A-diglycidyl-ether. While bisphenol-A-diglycidyl-ether inhibited adipogenesis as expected, it also suppressed T cell infiltration of bone marrow, reduced plasma inflammatory cytokines, decreased expression of multiple inflammasome genes, and ameliorated marrow failure. In vitro, bisphenol-A-diglycidyl-ether suppressed activation and proliferation, and reduced phospholipase C gamma 1 and nuclear factor of activated T-cells 1 expression, as well as inhibiting calcium flux in T cells. The in vivo effect of bisphenol-A-diglycidyl-ether on T cells was confirmed in a second immune-mediated bone marrow failure model, using different strains and non-major histocompatibility antigen mismatched: bisphenol-A-diglycidyl-ether ameliorated marrow failure by inhibition of T cell infiltration of bone marrow. Our data indicate that peroxisomal proliferator-activated receptor gamma antagonists may attenuate murine immune-mediated bone marrow failure, at least in part, by suppression of T cell activation, which might hold implications in the application of peroxisomal proliferator-activated receptor gamma antagonists in immune-mediated pathophysiologies, both in the laboratory and in the clinic. Genetically “fatless” mice developed bone marrow failure with accumulation of marrow adipocytes in our model, even in the absence of body fat, suggesting different mechanisms of systematic and marrow adipogenesis and physiologic versus pathophysiologic fat accumulation. PMID:26589913

  8. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function.

    PubMed

    Sato, Kazuya; Feng, Xingmin; Chen, Jichun; Li, Jungang; Muranski, Pawel; Desierto, Marie J; Keyvanfar, Keyvan; Malide, Daniela; Kajigaya, Sachiko; Young, Neal S

    2016-01-01

    Acquired aplastic anemia is an immune-mediated disease, in which T cells target hematopoietic cells; at presentation, the bone marrow is replaced by fat. It was reported that bone marrow adipocytes were negative regulators of hematopoietic microenvironment. To examine the role of adipocytes in bone marrow failure, we investigated peroxisomal proliferator-activated receptor gamma, a key transcription factor in adipogenesis, utilizing an antagonist of this factor called bisphenol-A-diglycidyl-ether. While bisphenol-A-diglycidyl-ether inhibited adipogenesis as expected, it also suppressed T cell infiltration of bone marrow, reduced plasma inflammatory cytokines, decreased expression of multiple inflammasome genes, and ameliorated marrow failure. In vitro, bisphenol-A-diglycidyl-ether suppressed activation and proliferation, and reduced phospholipase C gamma 1 and nuclear factor of activated T-cells 1 expression, as well as inhibiting calcium flux in T cells. The in vivo effect of bisphenol-A-diglycidyl-ether on T cells was confirmed in a second immune-mediated bone marrow failure model, using different strains and non-major histocompatibility antigen mismatched: bisphenol-A-diglycidyl-ether ameliorated marrow failure by inhibition of T cell infiltration of bone marrow. Our data indicate that peroxisomal proliferator-activated receptor gamma antagonists may attenuate murine immune-mediated bone marrow failure, at least in part, by suppression of T cell activation, which might hold implications in the application of peroxisomal proliferator-activated receptor gamma antagonists in immune-mediated pathophysiologies, both in the laboratory and in the clinic. Genetically "fatless" mice developed bone marrow failure with accumulation of marrow adipocytes in our model, even in the absence of body fat, suggesting different mechanisms of systematic and marrow adipogenesis and physiologic versus pathophysiologic fat accumulation.

  9. A review of mouse critical size defect models in weight bearing bones.

    PubMed

    Harris, Jonathan S; Bemenderfer, Thomas B; Wessel, Alexander R; Kacena, Melissa A

    2013-07-01

    Current and future advances in orthopedic treatment are aimed at altering biological interactions to enhance bone healing. Currently, several clinical scenarios exist for which there is no definitive treatment, specifically segmental bone loss from high-energy trauma or surgical resection - and it is here that many are aiming to find effective solutions. To test experimental interventions and better understand bone healing, researchers employ critical size defect (CSD) models in animal studies. Here, an overview of CSDs is given that includes the specifications of varying models, a discussion of current scaffold and bone graft designs, and current outcome measures used to determine the extent of bone healing. Many promising graft designs have been discovered along with promising adjunctive treatments, yet a graft that offers biomechanical support while allowing for neovascularization with eventual complete resorption and remodeling remains to be developed. An overview of this important topic is needed to highlight current advances and provide a clear understanding of the ultimate goal in CSD research--develop a graft for clinical use that effectively treats the orthopedic conundrum of segmental bone loss. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Change in Mouse Bone Turnover in Response to Microgravity on RR-1

    NASA Technical Reports Server (NTRS)

    Cheng-Campbell, Margareth A.; Blaber, Elizabeth A.; Almeida, Eduardo A. C.

    2016-01-01

    Mechanical unloading during spaceflight is known to adversely affect mammalian physiology. Our previous studies using the Animal Enclosure Module on short duration Shuttle missions enabled us to identify a deficit in stem cell based-tissue regeneration as being a significant concern for long-duration spaceflight. Specifically, we found that mechanical unloading in microgravity resulted in inhibition of differentiation of mesenchymal and hematopoietic stem cells in the bone marrow compartment. Also, we observed overexpression of a cell cycle arrest molecule, CDKN1ap21, in osteoprecursor cells on the bone surface, chondroprogenitors in the articular cartilage, and in myofibers attached to bone tissue. Specifically in bone tissue during both short (15-day) and long (30-day) microgravity experiments, we observed significant loss of bone tissue and structure in both the pelvis and the femur. After 15-days of microgravity on STS-131, pelvic ischium displayed a 6.23 decrease in bone fraction (p0.005) and 11.91 decrease in bone thickness (p0.002). Furthermore, during long-duration spaceflight we observed onset of an accelerated aging-like phenotype and osteoarthritic disease state indicating that stem cells within the bone tissue fail to repair and regenerate tissues in a normal manner, leading to drastic tissue alterations in response to microgravity. The Rodent Research Hardware System provides the capability to investigate these effects during long-duration experiments on the International Space Station. During the Rodent Research-1 mission 10 16-week-old female C57Bl6J mice were exposed to 37-days of microgravity. All flight animals were euthanized and frozen on orbit for future dissection. Ground (n10) and vivarium controls (n10) were housed and processed to match the flight animal timeline. During this study we collected pelvis, femur, and tibia from all animal groups to test the hypothesis that stem cell-based tissue regeneration is significantly altered after 37

  11. Relief of Preintegration Inhibition and Characterization of Additional Blocks for HIV Replication in Primary Mouse T Cells

    PubMed Central

    Zhang, Jing-xin; Diehl, Gretchen E.; Littman, Dan R.

    2008-01-01

    Development of a small animal model to study HIV replication and pathogenesis has been hampered by the failure of the virus to replicate in non-primate cells. Most studies aimed at achieving replication in murine cells have been limited to fibroblast cell lines, but generating an appropriate model requires overcoming blocks to viral replication in primary T cells. We have studied HIV-1 replication in CD4+ T cells from human CD4/ CCR5/Cyclin T1 transgenic mice. Expression of hCD4 and hCCR5 in mouse CD4+ T cells enabled efficient entry of R5 strain HIV-1. In mouse T cells, HIV-1 underwent reverse transcription and nuclear import as efficiently as in human T cells. In contrast, chromosomal integration of HIV-1 proviral DNA was inefficient in activated mouse T cells. This process was greatly enhanced by providing a secondary T cell receptor (TCR) signal after HIV-1 infection, especially between 12 to 24 h post infection. This effect was specific for primary mouse T cells. The pathways involved in HIV replication appear to be PKCθ−, CARMA1-, and WASp-independent. Treatment with Cyclosporin A (CsA) further relieved the pre-integration block. However, transcription of HIV-1 RNA was still reduced in mouse CD4+ T cells despite expression of the hCyclin T1 transgene. Additional post-transcriptional defects were observed at the levels of Gag expression, Gag processing, Gag release and virus infectivity. Together, these post-integration defects resulted in a dramatically reduced yield of infectious virus (300–500 fold) after a single cycle of HIV-1 replication. This study implies the existence of host factors, in addition to those already identified, that are critical for HIV-1 replication in mouse cells. This study also highlights the differences between primary T cells and cell lines regarding pre-integration steps in the HIV-1 replication cycle. PMID:18446227

  12. Establishment of a primary culture method for mouse intestinal epithelial cells by organ culture of fetal small intestine.

    PubMed

    Yamada, Kiyoshi; Sato, Kanako; Morishita, Satoru; Kaminogawa, Shuichi; Totsuka, Mamoru

    2009-08-01

    Studies of the physiological functions of intestinal epithelial cells (IECs) have been limited by the difficulty of primary culture of IEC. We established a method for primary culture of mouse IEC by culturing fragments of fetal small intestines pretreated with EDTA. This method reproducibly resulted in the expansion of cytokeratin-positive epithelial cells, and vigorous expansion of the epithelial cells was observed only from intestinal fragments of embryonic days 15-16. These cells expressed alkaline phosphatase activity and major histocompatibility complex (MHC) class II molecules, indicating the mature phenotype of IEC in a small intestine. The cells also presented antigens to CD4(+) T cells. Furthermore, the cells expressed various cytokines and chemokines, and the expression was enhanced by bacterial stimulation. These results indicate that the primary-cultured mouse IEC prepared by the method established here can be a beneficial tool in study of the functions of IECs, especially in mucosal immunity.

  13. Basement membrane of mouse bone marrow sinusoids shows distinctive structure and proteoglycan composition: a high resolution ultrastructural study.

    PubMed

    Inoue, S; Osmond, D G

    2001-11-01

    Venous sinusoids in bone marrow are the site of a large-scale traffic of cells between the extravascular hemopoietic compartment and the blood stream. The wall of the sinusoids consists solely of a basement membrane interposed between a layer of endothelial cells and an incomplete covering of adventitial cells. To examine its possible structural specialization, the basement membrane of bone marrow sinusoids has now been examined by high resolution electron microscopy of perfusion-fixed mouse bone marrow. The basement membrane layer was discontinuous, consisting of irregular masses of amorphous material within a uniform 60-nm-wide space between apposing endothelial cells and adventitial cell processes. At maximal magnifications, the material was resolved as a random arrangement of components lacking the "cord network" formation seen in basement membranes elsewhere. Individual components exhibited distinctive ultrastructural features whose molecular identity has previously been established. By these morphological criteria, the basement membrane contained unusually abundant chondroitin sulfate proteoglycan (CSPG) revealed by 3-nm-wide "double tracks," and moderate amounts of both laminin as dense irregular coils and type IV collagen as 1-1.5-nm-wide filaments, together with less conspicuous amounts of amyloid P forming pentagonal frames. In contrast, 4.5-5-nm-wide "double tracks" characteristic of heparan sulfate proteoglycan (HSPG) were absent. The findings demonstrate that, in comparison with "typical" basement membranes in other tissues, the bone marrow sinusoidal basement membrane is uniquely specialized in several respects. Its discontinuous nature, lack of network organization, and absence of HSPG, a molecule that normally helps to maintain membrane integrity, may facilitate disassembly and reassembly of basement membrane material in concert with movements of adventitial cell processes as maturing hemopoietic cells pass through the sinusoidal wall: the

  14. Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors

    PubMed Central

    2012-01-01

    Introduction Ankylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression. Methods PGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling. Results Inflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated. Conclusions This study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process. PMID:23171658

  15. Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors.

    PubMed

    Haynes, Katelin R; Pettit, Allison R; Duan, Ran; Tseng, Hsu-Wen; Glant, Tibor T; Brown, Matthew A; Thomas, Gethin P

    2012-11-22

    Ankylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression. PGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling. Inflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated. This study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process.

  16. Stromal Gene Expression and Function in Primary Breast Tumors that Metastasize to Bone Cancer

    DTIC Science & Technology

    2006-07-01

    Tumour and Matched Spine Metastases (300306) 0 100 200 300 400 500 600 700 1 2 4 5 Mouse R TA Primary Tumour Spine Figure...the tumour lines (pBABE, Lrch2 Flag Fwd, EST Flag Rev, and EST) was examined by their ability to invade through a Matrigel barrier in response to 5 ... tumour weight 0 .1 .2 .3 .4 . 5 .6 .7 .8 4T1.2pBABE 4T1.2 Lrch2Flag F4T1.2 ESTFlag R Pr im ar y tu m ou r w ei gh t ( g) Pr im ar y tu m ou r vo lu

  17. Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages.

    PubMed

    Ishida, Momoko; Ose, Saya; Nishi, Kosuke; Sugahara, Takuya

    2016-07-01

    We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.

  18. Effects of GSM-modulated radiofrequency electromagnetic fields on mouse bone marrow cells.

    PubMed

    Prisco, Maria Grazia; Nasta, Francesca; Rosado, Maria Manuela; Lovisolo, Giorgio Alfonso; Marino, Carmela; Pioli, Claudio

    2008-12-01

    We examined the effects of in vivo exposure to a GSM-modulated 900 MHz RF field on the ability of bone marrow cells to differentiate, colonize lymphatic organs, and rescue lethally X-irradiated mice from death. X-irradiated mice were injected with medium alone or containing bone marrow cells from either RF-field-exposed (SAR 2 W/kg, 2 h/day, 5 days/ week, 4 weeks) or sham-exposed or cage control donor mice. Whereas all mice injected with medium alone died, mice that received bone marrow cells survived. Three and 6 weeks after bone marrow cell transplantation, no differences in thymus cellularity and in the frequencies of differentiating cell subpopulations (identified by CD4/CD8 expression) were observed among the three transplanted groups. Mitogen-induced thymocyte proliferation yielded comparable levels in all transplanted groups. As to the spleen, no effects of the RF-field exposure on cell number, percentages of B and T (CD4 and CD8) cells, B- and T-cell proliferation, and IFN-gamma production were found in transplanted mice. In conclusion, our results show no effect of in vivo exposure to GSM-modulated RF fields on the ability of bone marrow precursor cells to home and colonize lymphoid organs and differentiate in phenotypically and functionally mature T and B lymphocytes.

  19. Characterization of wavefront errors in mouse cranial bone using second-harmonic generation

    NASA Astrophysics Data System (ADS)

    Tehrani, Kayvan Forouhesh; Kner, Peter; Mortensen, Luke J.

    2017-03-01

    Optical aberrations significantly affect the resolution and signal-to-noise ratio of deep tissue microscopy. As multiphoton microscopy is applied deeper into tissue, the loss of resolution and signal due to propagation of light in a medium with heterogeneous refractive index becomes more serious. Efforts in imaging through the intact skull of mice cannot typically reach past the bone marrow (˜150 μm of depth) and have limited resolution and penetration depth. Mechanical bone thinning or optical ablation of bone enables deeper imaging, but these methods are highly invasive and may impact tissue biology. Adaptive optics is a promising noninvasive alternative for restoring optical resolution. We characterize the aberrations present in bone using second-harmonic generation imaging of collagen. We simulate light propagation through highly scattering bone and evaluate the effect of aberrations on the point spread function. We then calculate the wavefront and expand it in Zernike orthogonal polynomials to determine the strength of different optical aberrations. We further compare the corrected wavefront and the residual wavefront error, and suggest a correction element with high number of elements or multiconjugate wavefront correction for this highly scattering environment.

  20. Cytotoxic effect of ZnS nanoparticles on primary mouse retinal pigment epithelial cells.

    PubMed

    Bose, Karthikeyan; Lakshminarasimhan, Harini; Sundar, Krishnan; Kathiresan, Thandavarayan

    2016-11-01

    The multiple properties of zinc sulphide nanoparticles (ZnS-NPs) are attracting great attention in the field of chemical and biological research. ZnS-NPs also find their application in biosensor and photocatalysis. Zinc is an important metal ion in retina and its deficiency leads to age-related macular degeneration. As of now, not much research is available on bio-interaction of ZnS as nanoform with retinal pigment epithelial (RPE) cells. RPE cells in the retina help in maintaining normal photoreceptor function and vision. To begin with, ZnS-NPs were synthesized and characterized using UV-visible spectra, X-ray diffraction, Fourier transform infrared spectrum, transmission electron microscopy and dynamic light scattering. Followed by the confirmation of nanoparticles, our study extended to investigate the impact of ZnS-NPs in primary mouse RPE (MRPE) cells at different concentrations. ZnS-NPs showed dose-dependent cytotoxicity in MRPE cells and no changes were observed in cells' tight intactness at minimal concentration. In addition, exposure to ZnS-NPs increased cellular permeability in dose- and time-dependent manner in MRPE cells. The findings from DCFH-DA analysis revealed that ZnS-NPs-treated cells had elevated level of reactive oxygen species and partial activation of cell apoptosis was identified after exposure to ZnS-NPs at higher concentration. Furthermore, pre-treatment of the primary MRPE cells with ZnS-NPs led to phosphorylation of Akt (Ser 473), which indicates the crucial role of ZnS-NPs in regulating cell survival at minimal concentration. Altogether, this study enumerates requisite dose of using ZnS-NPs to maintain healthy RPE cells and contributes to future studies in development of therapeutic drug and drug carrier for ocular-related disorders.

  1. Impaired mitochondrial functions contribute to 3-bromopyruvate toxicity in primary rat and mouse hepatocytes.

    PubMed

    Sobotka, Ondřej; Endlicher, René; Drahota, Zdeněk; Kučera, Otto; Rychtrmoc, David; Raad, Marjan; Hakeem, Khurum; Červinková, Zuzana

    2016-08-01

    A compound with promising anticancer properties, 3-bromopyruvate (3-BP) is a synthetic derivative of a pyruvate molecule; however, its toxicity in non-malignant cells has not yet been fully elucidated. Therefore, we elected to study the effects of 3-BP on primary hepatocytes in monolayer cultures, permeabilized hepatocytes and isolated mitochondria. After a 1-h treatment with 100 μM 3-BP cell viability of rat hepatocytes was decreased by 30 % as measured by the WST-1 test (p < 0.001); after 3-h exposure to ≥200 μM 3-BP lactate dehydrogenase leakage was increased (p < 0.001). Reactive oxygen species production was increased in the cell cultures after a 1-h treatment at concentrations ≥100 μmol/l (p < 0.01), and caspase 3 activity was increased after a 20-h incubation with 150 μM and 200 μM 3-BP (p < 0.001). This toxic effect of 3-BP was also proved using primary mouse hepatocytes. In isolated mitochondria, 3-BP induced a dose- and time-dependent decrease of mitochondrial membrane potential during a 10-min incubation both with Complex I substrates glutamate + malate or Complex II substrate succinate, although this decrease was more pronounced with the latter. We also measured the effect of 3-BP on respiration of isolated mitochondria. ADP-activated respiration was inhibited by 20 μM 3-BP within 10 min. Similar effects were also found in permeabilized hepatocytes of both species.

  2. Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes

    PubMed Central

    Zheng, Jinying; Peng, Chuan; Ai, Yanbiao; Wang, Heng; Xiao, Xiaoqiu; Li, Jibin

    2016-01-01

    The increase in fructose consumption is considered to be a risk factor for developing nonalcoholic fatty liver disease (NAFLD). We investigated the effects of docosahexaenoic acid (DHA) on hepatic lipid metabolism in fructose-treated primary mouse hepatocytes, and the changes of Endoplasmic reticulum (ER) stress pathways in response to DHA treatment. The hepatocytes were treated with fructose, DHA, fructose plus DHA, tunicamycin (TM) or fructose plus 4-phenylbutyric acid (PBA) for 24 h. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining. The mRNA expression levels and protein levels related to lipid metabolism and ER stress response were determined by real-time PCR and Western blot. Fructose treatment led to obvious TG accumulation in primary hepatocytes through increasing expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two key enzymes in hepatic de novo lipogenesis. DHA ameliorates fructose-induced TG accumulation by upregulating the expression of carnitine palmitoyltransferase 1A (CPT-1α) and acyl-CoA oxidase 1 (ACOX1). DHA treatment or pretreatment with the ER stress inhibitor PBA significantly decreased TG accumulation and reduced the expression of glucose-regulated protein 78 (GRP78), total inositol-requiring kinase 1 (IRE1α) and p-IRE1α. The present results suggest that DHA protects against high fructose-induced hepatocellular lipid accumulation. The current findings also suggest that alleviating the ER stress response seems to play a role in the prevention of fructose-induced hepatic steatosis by DHA. PMID:26805874

  3. Behavior of Primary Cilia and Tricellular Tight Junction Proteins during Differentiation in Temperature-Sensitive Mouse Cochlear Precursor Hair Cells.

    PubMed

    Kakuki, Takuya; Kaneko, Yakuto; Takano, Kenichi; Ninomiya, Takafumi; Kohno, Takayuki; Kojima, Takashi; Himi, Tetsuo

    2016-01-01

    In the sensory hair cells of the mammalian cochlea, the primary cilia in the planar cell polarity as well as the tight junctions in the epithelial cell polarity and the barrier are important to maintain normal hearing. Temperature-sensitive mouse cochlear precursor hair cells were used to investigate the behavior of primary cilia and tricellular tight junction proteins during the differentiation of sensory hair cells. In undifferentiated cells (incubated at 33°C), many acetylated tubulin-positive primary cilia were observed, and each was accompanied with an x03B3;-tubulin-positive basal body. The primary cilia had a '9 + 0' architecture with nine outer microtubule doublets but lacking a central pair of microtubules. In differentiated cells (incubated at 39°C), acetylated tubulin-positive primary cilia as well as acetylated tubulin-positive cilia-like structures were partially observed on the cell surface. In differentiated cells, the number of primary cilia was markedly reduced compared with undifferentiated cells, and innumerable cilia-like structures with no ciliary pockets were partially observed on the cell surface. In undifferentiated cells, few tricellulin molecules and lipolysis-stimulated lipoprotein receptors (LSRs) were observed in the cytoplasm. In differentiated cells, many tricellulin molecules and LSRs were observed on the membranes and within the cytoplasm. Conditional immortalized mouse cochlear precursor hair cells may be useful to investigate the roles of primary cilia and tricellular tight junctions during cellular differentiation and degeneration such as apoptosis.

  4. Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells

    SciTech Connect

    Jeng, A.Y.; Lichti, U.; Strickland, J.E.; Blumberg, P.M.

    1985-11-01

    Interaction of tumor promoting phorbol esters with specific high affinity receptors is probably essential for many of the biological responses elicited by these agents. Since diacylglycerols which can be produced enzymatically from phospholipids by phospholipase C are postulated to be the physiological ligands for the phorbol ester receptor, the authors have examined primary cultures of mouse epidermal basal cells exposed to phospholipase C (Clostridium perfringens) for several biological and biochemical responses characteristic of treatment with 12-O-tetradecanoyl-phorbol-13-acetate, the most potent phorbol ester tumor promoter. Formation of diacylglycerols by treatment with phospholipase C was demonstrated by the dose-dependent release of radioactive diacylglycerols in cells prelabeled with (TH)arachidonic acid. Treatment with phospholipase C led to the morphological changes and to the reduction in epidermal growth factor binding (90%) associated with 12-O-tetradecanoylphorbol-13-acetate treatment. Continuous treatment at the same dose led to the induction of the enzymes ornithine decarboxylase and transglutaminase with a time course and extent similar to the inductions by 12-O-tetradecanoylphorbol-13-acetate. Treatment with phospholipase C yielded substantial suppression of the binding affinity of phorbol-12,13-dibutyrate for its receptors without reduction in total number of binding sites, consistent with the production by phospholipase C of a competitive inhibitor of phorbol ester binding.

  5. Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures.

    PubMed

    Cardenas-Aguayo, Maria del Carmen; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Iqbal, Khalid

    2013-01-01

    The level of brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is down regulated in Alzheimer's disease (AD), Parkinson's disease (PD), depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5) corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18) primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706) of the TrkB receptor, which could be blocked by the Trk's inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2)O(2)-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

  6. Spatial profile of excitatory and inhibitory synaptic connectivity in mouse primary auditory cortex

    PubMed Central

    Levy, Robert B.; Reyes, Alex D.

    2012-01-01

    The role of local cortical activity in shaping neuronal responses is controversial. Among other questions, it is unknown how the diverse response patterns reported in vivo - lateral inhibition in some cases, approximately balanced excitation and inhibition (co-tuning) in others - compare to the local spread of synaptic connectivity. Excitatory and inhibitory activity might cancel each other out, or, if one outweighs the other, receptive field (RF) properties might be substantially affected. As a step toward addressing this question, we used multiple intracellular recording in mouse primary auditory cortical slices to map synaptic connectivity among excitatory pyramidal (P) cells and the two broad classes of inhibitory cells, fast-spiking (FS) and non-FS cells in the principal input layer. Connection probability was distance-dependent; the spread of connectivity, parameterized by Gaussian fits to the data, was comparable for all cell types, ranging from 85 to 114 μm. With brief stimulus trains, unitary synapses formed by FS interneurons were stronger than other classes of synapses; synapse strength did not correlate with distance between cells. The physiological data were qualitatively consistent with predictions derived from anatomical reconstruction. We also analyzed the truncation of neuronal processes due to slicing; overall connectivity was reduced but the spatial pattern was unaffected. The comparable spatial patterns of connectivity and relatively strong excitatory-inhibitory interconnectivity are consistent with a theoretical model where either lateral inhibition or co-tuning can predominate, depending on the structure of the input. PMID:22514322

  7. Spatial organization of excitatory synaptic inputs to layer 4 neurons in mouse primary auditory cortex

    PubMed Central

    Kratz, Megan B.; Manis, Paul B.

    2015-01-01

    Layer 4 (L4) of primary auditory cortex (A1) receives a tonotopically organized projection from the medial geniculate nucleus of the thalamus. However, individual neurons in A1 respond to a wider range of sound frequencies than would be predicted by their thalamic input, which suggests the existence of cross-frequency intracortical networks. We used laser scanning photostimulation and uncaging of glutamate in brain slices of mouse A1 to characterize the spatial organization of intracortical inputs to L4 neurons. Slices were prepared to include the entire tonotopic extent of A1. We find that L4 neurons receive local vertically organized (columnar) excitation from layers 2 through 6 (L6) and horizontally organized excitation primarily from L4 and L6 neurons in regions centered ~300–500 μm caudal and/or rostral to the cell. Excitatory horizontal synaptic connections from layers 2 and 3 were sparse. The origins of horizontal projections from L4 and L6 correspond to regions in the tonotopic map that are approximately an octave away from the target cell location. Such spatially organized lateral connections may contribute to the detection and processing of auditory objects with specific spectral structures. PMID:25972787

  8. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

    NASA Astrophysics Data System (ADS)

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Dayem, Ahmed Abdal; Kwon, Deug-Nam; Kim, Jin-Hoi

    2013-09-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a `green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.

  9. Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization

    PubMed Central

    Ficarro, Scott B.; Hutchinson, John N.; Csepanyi-Komi, Roland; Nguyen, Phi T.; Wisniewski, Eva; Sullivan, Jessica; Hofmann, Oliver; Ligeti, Erzsebet; Marto, Jarrod A.; Wagers, Amy J.

    2016-01-01

    Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization. PMID:27365422

  10. Efficacy of Phosphatidylinositol-3 Kinase Inhibitors in a Primary Mouse Model of Undifferentiated Pleomorphic Sarcoma

    PubMed Central

    Kim, Suzy; Dodd, Rebecca D.; Mito, Jeffrey K.; Ma, Yan; Kim, Yongbaek; Riedel, Richard F.; Kirsch, David G.

    2012-01-01

    Recent advances in sarcoma genomics have identified novel mutations in the PI3K pathway in human sarcomas. Here, we use a mouse model of primary soft-tissue sarcoma for preclinical testing of doxorubicin and inhibitors of the PI3K pathway: BKM120 (PI3K inhibitor) and BEZ235 (a dual PI3K/mTOR inhibitor). Doxorubicin-treated tumors (n = 15) showed a partial response rate of 6.6%, just as the majority of human sarcomas do not respond to doxorubicin. Treatment with BKM120 elicited a partial response in 50% of tumors (n = 10), which was also seen in combination with doxorubicin (n = 10). Additionally, BKM120 treatment produced a robust delay in tumor growth kinetics. BEZ235-treated tumors (n = 9) showed a complete response rate of 11.1%. Combining BEZ235 with doxorubicin (n = 10) increased the complete response rate to 50% (P = 0.035). These studies demonstrate that PI3K pathway inhibition is a viable and attractive target for soft-tissue sarcomas. PMID:22619567

  11. Oxidative damage induced by copper in mouse primary hepatocytes by single-cell analysis.

    PubMed

    Jing, Mingyang; Liu, Yang; Song, Wei; Yan, Yunxing; Yan, Wenbao; Liu, Rutao

    2016-01-01

    Copper can disturb the intracellular redox balance, induce oxidative stress, and subsequently cause irreversible damage, leading to a variety of diseases. In the present study, mouse primary hepatocytes were chosen to elucidate the in vitro oxidative damage of short-term copper exposure (10-200 μM) by single-cell analysis. We evaluated the toxicity of copper by reactive oxygen species (ROS), glutathione (GSH), and oxidative DNA damage at the single-cell level. Oxidative damage induced by copper was verified by the morphological changes, persistent elevations of excessive ROS and malondialdehyde (MDA), a decrease in GSH level, and the oxidative DNA damage. Furthermore, the average ROS generation, GSH consumption, and the indicators in DNA damage did not significantly change at relatively low concentrations (10 or 50 μM), but we can find the alterations of parameters in some single cells clearly. Emphasis on the analysis of single cells is conducive to gain a better understanding on the toxicity of copper. This study will also complement studies on the environmental risk assessment of copper pollution.

  12. Heterotaxy and complex structural heart defects in a mutant mouse model of primary ciliary dyskinesia

    PubMed Central

    Tan, Serena Y.; Rosenthal, Julie; Zhao, Xiao-Qing; Francis, Richard J.; Chatterjee, Bishwanath; Sabol, Steven L.; Linask, Kaari L.; Bracero, Luciann; Connelly, Patricia S.; Daniels, Mathew P.; Yu, Qing; Omran, Heymut; Leatherbury, Linda; Lo, Cecilia W.

    2007-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD. Analysis of homozygous mutant embryos from 18 litters yielded 25% with normal organ situs, 35% with situs inversus totalis, and 40% with heterotaxy. Embryos with heterotaxy had complex structural heart defects that included discordant atrioventricular and ventricular outflow situs and atrial/pulmonary isomerisms. Variable combinations of a distinct set of cardiovascular anomalies were observed, including superior-inferior ventricles, great artery alignment defects, and interrupted inferior vena cava with azygos continuation. The surprisingly high incidence of heterotaxy led us to evaluate the diagnosis of PCD. PCD was confirmed by EM, which revealed missing outer dynein arms in the respiratory cilia. Ciliary dyskinesia was observed by videomicroscopy. These findings show that Dnahc5 is required for the specification of left-right asymmetry and suggest that the PCD-causing Dnahc5 mutation may also be associated with heterotaxy. PMID:18037990

  13. Biocompatibility of microbially reduced graphene oxide in primary mouse embryonic fibroblast cells.

    PubMed

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Kim, Jin-Hoi

    2013-05-01

    Graphene nanosheet is a one-atom thick planar sheet of sp(2)-bonded carbon atoms, which are densely packed in a honeycomb crystal lattice, attracting tremendous attention from both fundamental research and industrial applications. The synthesis of graphene from graphene oxide (GO) using a biological method is one of the important topics in the areas of nanotechnology, because graphene-based nanomaterials have potential applications. A green, simple and non-toxic method for preparing graphene using biomass of Pseudomonas aeruginosa as the reducing reagent is proposed. The resulting microbially reduced graphene oxide (M-rGO) was characterized using a range of analytical techniques. UV-visible spectroscopy confirms the transition of graphene oxide to graphene. Fourier transform infrared spectroscopy (FT-IR) was used to study the changes in surface functionalities. X-ray diffraction (XRD) and high resolution scanning electron microscopy (SEM) were used to investigate the crystalline nature and the morphologies of prepared graphene respectively. Furthermore, the biocompatibility of the M-rGO was investigated using primary mouse embryonic fibroblast (PMEF) cells. The present study suggests that the M-rGO has significant biocompatibility for PMEF cells, even at a high concentration of 100 μg ml(-1). Therefore, the proposed safe and green method confers the M-rGO with a great potential for various biomedical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Scaling of Topologically Similar Functional Modules Defines Mouse Primary Auditory and Somatosensory Microcircuitry

    PubMed Central

    Sadovsky, Alexander J.

    2013-01-01

    Mapping the flow of activity through neocortical microcircuits provides key insights into the underlying circuit architecture. Using a comparative analysis we determined the extent to which the dynamics of microcircuits in mouse primary somatosensory barrel field (S1BF) and auditory (A1) neocortex generalize. We imaged the simultaneous dynamics of up to 1126 neurons spanning multiple columns and layers using high-speed multiphoton imaging. The temporal progression and reliability of reactivation of circuit events in both regions suggested common underlying cortical design features. We used circuit activity flow to generate functional connectivity maps, or graphs, to test the microcircuit hypothesis within a functional framework. S1BF and A1 present a useful test of the postulate as both regions map sensory input anatomically, but each area appears organized according to different design principles. We projected the functional topologies into anatomical space and found benchmarks of organization that had been previously described using physiology and anatomical methods, consistent with a close mapping between anatomy and functional dynamics. By comparing graphs representing activity flow we found that each region is similarly organized as highlighted by hallmarks of small world, scale free, and hierarchical modular topologies. Models of prototypical functional circuits from each area of cortex were sufficient to recapitulate experimentally observed circuit activity. Convergence to common behavior by these models was accomplished using preferential attachment to scale from an auditory up to a somatosensory circuit. These functional data imply that the microcircuit hypothesis be framed as scalable principles of neocortical circuit design. PMID:23986241