Please note the following errors in the original version.

ABSTRACT The erupted tusk of the narwhal exhibits sensory ability. The hypothesized sensory pathway begins with ocean water entering through cementum channels to a network of patent dentinal tubules extending from the dentinocementum junction to the inner pulpal wall. Circumpulpal sensory structures then signal pulpal nerves terminating near the base of the tusk. The maxillary division of the fifth cranial nerve then transmits this sensory information to the brain. This sensory pathway was first described in published results of patent dentinal tubules, and evidence from dissection of tusk nerve connection via the maxillary division of the fifth cranial nerve to the brain. New evidence presented here indicates that the patent dentinal tubules communicate with open channels through a porous cementum from the ocean environment. The ability of pulpal tissue to react to external stimuli is supported by immunohistochemical detection of neuronal markers in the pulp and gene expression of pulpal sensory nerve tissue. Final confirmation of sensory ability is demonstrated by significant changes in heart rate when alternating solutions of high‐salt and fresh water are exposed to the external tusk surface. Additional supporting information for function includes new observations of dentinal tubule networks evident in unerupted tusks, female erupted tusks, and vestigial teeth. New findings of sexual foraging divergence documented by stable isotope and fatty acid results add to the discussion of the functional significance of the narwhal tusk. The combined evidence suggests multiple tusk functions may have driven the tooth organ system's evolutionary development and persistence. Anat Rec, 297:599–617, 2014. © 2014 Wiley Periodicals, Inc.

Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.

Narwhal tusks, although well described and characterized within publications, are clouded by contradictory references, which refer to them as both incisors and canines. Vestigial teeth are briefly mentioned in the scientific literature with limited descriptions and no image renderings. This study first examines narwhal maxillary osteoanatomy to determine whether the erupted tusks are best described as incisiform or caniniform teeth. The study also offers evidence to support the evolutionary obsolescence of the vestigial teeth through anatomic, morphologic, and histologic descriptions. Examination of 131 skull samples, including 110 museum skull specimens and 21 harvested skulls, revealed the erupted tusks surrounded by maxillary bone over the entire length of their bone socket insertion, and are thus more accurately termed caniniform or canine teeth. The anatomy, morphology, and development of vestigial teeth in five skull samples are more fully described and documented. Vestigial tooth samples included 14 embedded pairs or individual teeth that were partially exposed or removed from the maxillary bone. Their location was posterior, ventral, and lateral to the tusks, although male vestigial teeth often exfoliate in the mouth lodging between the palatal tissue and underlying maxillary bone. Their myriad morphologies, sizes, and eruption patterns suggest that these teeth are no longer guided by function but rather by random germ cell differentiation and may eventually cease expression entirely. The conclusions reached are that the narwhal tusks are the expression of canine teeth and that vestigial teeth have no apparent functional characteristics and are following a pattern consistent with evolutionary obsolescence. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Musculoskeletal diseases are the most commonly reported health conditions in the United States.1 These diseases include various forms of arthritis, congenital deformities and anomalies, fractures, and pain associated with the back, neck, or intervertebral disks.1 Millions of surgeries are performed every year to correct musculoskeletal diseases, yet a high percentage fail to obtain a satisfactory outcome. 2–5 For example, only 64% of patients treated surgically for lumbar spinal stenosis reported good-to-excellent outcomes.4 Treatments and lost wages due to musculoskeletal diseases are a significant burden on society, representing 7.7% gross domestic product (~$849,000,000,000) for 2002 to 2004.1 Therefore, new therapies that reduce the cost and morbidity of musculoskeletal diseases are in great demand. MSCs have been the focus of widespread attention in recent years, and are being studied in over one hundred clinical trials world-wide.6 In addition to their obvious potential for treatment of musculoskeletal diseases, MSCs have demonstrated promise as a cell therapy in many pre-clinical and early-stage clinical trials for a range of diseases, including diabetes, cardiac disease, bone marrow transplant-associated GVHD, and osteogenesis imperfecta.6,7 Although proven to be safe, efficacy in late stage clinical trials has not met expectations.8,9 This has led some to develop strategies that can enhance the potency of MSCs before infusion, including activation or transfection of cells before infusion, and cell surface engineering.10,11 A critical challenge in the development of these enhancement strategies is quantification of in vivo MSC homing and therapeutic efficiency. To address this challenge, we applied in vivo confocal and multi-photon microscopy, a powerful singe-cell detection and evaluation technique. MSC use in pre-clinical and clinical models began with their discovery in the 1960’s during bone marrow transplant experiments that led to the hypothesis that a cell type existed within the bone marrow that could differentiate into osteoblasts, aid in the development of sinusoidal structures, and support hematopoiesis in ectopic sites.12 These skeletal stem cells were first purified from bone marrow based on adherence to tissue culture plastic and were re-named MSCs for their ability to differentiate into adipocytes, chondrocytes, and osteoblasts.13,14 MSCs have been well characterized in vitro, and are defined as being tissue culture plastic adherent; positive for CD105, CD73, and CD90 antigens; negative for HLA-DR, and CD45, CD34, CD14, CD19 antigens; and able to differentiate into osteoblasts, adipocytes, and chondrocytes.15 Recently, it has been suggested that MSCs are perivascular cells in vivo. This has raised many interesting questions about how pericytes and adventitial cells respond to acute bone injury and if perivascular cells in non-bone tissues are also MSCs.16–18 Our research describes the ability of in vivo confocal and multi-photon microscopy to quantify the behavior of exogenous engineered MSCs and evaluate endogenous perivascular cell response to bone injury. Our results contribute to our understanding of how exogenous MSCs interact with diseased tissue and how endogenous perivascular cells respond to musculoskeletal injury. These results may help improve current cell therapies and lead to the development of novel therapeutics.

Recent research has shown that adipose tissues contain abundant MSCs (mesenchymal stem cells). The origin and location of the adipose stem cells, however, remain unknown, presenting an obstacle to the further purification and study of these cells. In the present study, we aimed at investigating the origins of adipose stem cells. α-SMA (α-smooth muscle actin) is one of the markers of pericytes. We harvested ASCs (adipose stromal cells) from α-SMA-GFP (green fluorescent protein) transgenic mice and sorted them into GFP-positive and GFP-negative cells by FACS. Multilineage differentiation tests were applied to examine the pluripotent ability of the α-SMA-GFP-positive and -negative cells. Immunofluorescent staining for α-SMA and PDGF-Rβ (platelet-derived growth factor receptor β) were applied to identify the α-SMA-GFP-positive cells. Then α-SMA-GFP-positive cells were loaded on a collagen-fibronectin gel with endothelial cells to test their vascularization ability both in vitro and in vivo. Results show that, in adipose tissue, all of the α-SMA-GFP-positive cells congregate around the blood vessels. Only the α-SMA-GFP-positive cells have multilineage differentiation ability, while the α-SMA-GFP-negative cells can only differentiate in an adipogenic direction. The α-SMA-GFP-positive cells maintained expression of α-SMA during multilineage differentiation. The α-SMA-GFP-positive cells can promote the vascularization of endothelial cells in three-dimensional culture both in vitro and in vivo. We conclude that the adipose stem cells originate from perivascular cells and congregate around blood vessels.

Breast cancer preferentially metastasizes to bone. We therefore addressed the role of Notch signaling in osteoblast-cancer cell interactions and in bone metastasis. Human bone marrow osteoblasts selectively enhanced the expression of Notch3 and its ligand Jagged1 in human breast cancer cell lines. Osteoblasts also stimulated cancer cell colony formation in soft agar, which was reduced by a chemical inhibitor of Notch signaling and anti-transforming growth factor beta1 (TGFbeta1) antibody. TGFbeta1, a major prometastatic product of osteoblasts, also stimulated cancer cell Notch3 expression. Notch3 knockdown in the cancer cells by stable short hairpin RNA interference decreased the osteoblast- and TGFbeta1-stimulated colony formation as well as TGFbeta1-mediated Smad3/Smad2 phosphorylation; Jagged1 level was coordinately reduced. In addition, expression of snail, a regulator of epithelial-mesenchymal transition, and the mesenchymal markers fibronectin and vimentin was attenuated by reducing Notch3 levels. To study the role of Notch3 signaling in bone metastasis, cancer cells were inoculated into athymic mice, either into femoral bone marrow cavities or into the systemic circulation via the left ventricle. Compared with robust osteolysis in mice receiving control cells, osteolytic lesions were significantly reduced following inoculation of cells with constitutively reduced Notch3 expression. Taken together, our results suggest that enhanced Notch3 expression in breast cancer cells, triggered by osteoblasts and their secretion of TGFbeta1 in the bone marrow niche, may stand as a novel mechanism for promoting bone metastasis.

Formation of multinucleated bone-resorbing osteoclasts results from activation of the receptor activated NF-kappa B ligand (RANKL)-receptor activated NF-kappa B (RANK) signaling pathway in primary bone marrow macrophages and a macrophage cell line (RAW 264.7). Osteoclasts, through bone remodeling, are key participants in the homeostatic regulation of calcium and phosphate levels within the body. Microarray analysis using Gene Expression Dynamic Inspector (GEDI) clustering software indicated that osteoclast differentiation is correlated with an increase in xenotropic and polytropic virus receptor 1 (XPR1) mRNA transcripts. XPR1 is a receptor of the xenotropic and polytropic murine leukemia virus and homolog of yeast Syg1 and plant Pi transporter PHO1. Quantitative PCR was used to validate the up-regulation of XPR1 message following RANKL stimulation in both primary bone marrow cells and a macrophage cell line. Immunostaining for the XPR1 protein showed that there is translocation of XPR1 to the membranes of the sealing zone in mature osteoclasts. This study is the first to demonstrate that the expression of retro-viral receptor, XPR1, is regulated by RANKL-RANK signaling. (c) 2010 Elsevier Inc. All rights reserved.

In the bone marrow (BM), osteoblasts and endothelium constitute functional niches providing positive or negative signals for hematopoietic stem cell (HSC) self-renewal. In addition to hematopoietic cells, endothelial cells, and osteoblasts, adult BM contains numerous adipocytes. Interestingly, the number of adipocytes correlates inversely with the gross hematopoietic activity of the marrow. Whether adipocytes have a direct effect on hematopoietic progenitors or whether they act as mere space-fillers in this context remains unclear. To determine the potential role of bone marrow adipocytes in hematopoiesis, we induced bone marrow-derived OP9 mesenchymal cells to differentiate into either osteoblastic or adipocytic stroma, and then tested their capacity to serve as surrogate HSC niches during in vitro hematopoietic culture in the absence of exogenous growth factors. We found that the presence of BM-derived adipocytes suppresses the expansion of short-term hematopoietic progenitors by at least two fold, as measured by the number of CD45+ cells expanded, the number of colony forming unit cells (CFU-Cs) and the competitive repopulation units (CRUs) during the first two months post-transplant. As an in vivo correlate, we compared the hematopoietic activity within the BM of the adipocyte-poor thoracic vertebrae and the adipocyte-rich proximal tail vertebrae. Indeed, we found that the percentage of HSCs (ckit+Lin-Sca1+), CMPs, GMPs and MEPs was decreased by two fold in the adipocyte-rich BM as determined phenotypically by FACS and functionally by short-term and long-term competitive repopulation assays. Mechanistically, oligonucleotide expression microarrays and conditioned media experiments on the OP9 co-cultures suggest that the inhibition of the progenitor compartment in adipocyte-rich environments is due to the loss of supportive factors (Notch ligands, N-cadherin, Angiopoietin-1, SCF, BMPs and Wnt5a) in addition to the presence of an active inhibitor. Finally, we found bone marrow adipocytes to accumulate in great numbers upon bone marrow ablation, a process that is hindered in genetically adipocyte-deficient mice. Since early BM transplant survival depends on the rapid accumulation of short term hematopoietic progenitors, whose replication we found hindered in adipocyte-rich BM, we were interested to explore whether the absence of adipocytes in the context of BM transplantation would foster faster recovery of lethally irradiated mice. As predicted, circulating leukocyte counts on the third week post-transplant were 3–5 times higher on the recovering fatless mice. Accordingly, the percentage of CFU-Cs, HSCs, CMPs, GMPs and MEPs on day 17 post-transplant was doubled on the adipocyte-depleted mice as compared to their wildtype littermates. We therefore conclude that, as seen in vitro, the presence of adipocytes in the recovering HSC niche is detrimental to the rapid hematopoietic expansion required to reconstitute blood production. We are currently examining the pharmacologic modulation of adipocyte formation for its effects in BM transplantation.

Osteoclasts degrade bone matrix by demineralization followed by degradation of type I collagen through secretion of the cysteine protease, cathepsin K. Current imaging modalities are insufficient for sensitive observation of osteoclast activity, and in vivo live imaging of osteoclast resorption of bone has yet to be demonstrated. Here, we describe a near-infrared fluorescence reporter probe whose activation by cathepsin K is shown in live osteoclast cells and in mouse models of development and osteoclast upregulation. Cathepsin K probe activity was monitored in live osteoclast cultures and correlates with cathepsin K gene expression. In ovariectomized mice, cathepsin K probe upregulation precedes detection of bone loss by micro-computed tomography. These results are the first to demonstrate non-invasive visualization of bone degrading enzymes in models of accelerated bone loss, and may provide a means for early diagnosis of upregulated resorption and rapid feedback on efficacy of treatment protocols prior to significant loss of bone in the patient.

Introduction: Postnatal human dental pulp is a potentially promising source of progenitor cells. Sustaining and amplifying progenitor cell populations would be beneficial for basic science research with application in pulpal regeneration. Hypoxia has been observed to promote the undifferentiated cell state in various stem cell populations. The purpose of this study was to examine human dental pulp cells (DPCs) proliferation in normoxia and hypoxia. Methods: Dental pulp cells were obtained from third molars of adult patients and cultured in alpha modification of Eagle's medium culture medium with 10% fetal bovine serum. For cell proliferation, DPCs were divided into two groups: (1) DPCs incubated in normoxic conditions (20% oxygen tension) and (2) DPC incubated in hypoxic conditions (3% oxygen tension). Cell proliferation assays were performed every 2 to 3 days from day 3 to day 14 by trypsinization and quantification of cells with a hemacytometer. Fluorescence-activated cell sorting analysis was completed to investigate stem cell markers, CD133, and STRO-1. Results: DPCs proliferated significantly more in hypoxia than in normoxia (ie, two-fold throughout the experiment, p < 0.0001). The primitive stem cell marker, CD133, decreased in hypoxia, whereas the osteoprogenitor marker, STRO-1, increased by 8.5-fold. Conclusions: This study suggested that hypoxia is an effective treatment to amplify numbers of progenitor cells from human dental pulp. (J Endod 2009;35:818-823)

We find anomalously high gadolinium (Gd) concentrations in the femoral head bones of patients exposed to chelated Gd, commonly used as a contrast agent for medical imaging. Gd is introduced in chelated form to protect patients from exposure to toxic free Gd3+, a calciumantagonist which disrupts cellular processes. Recent studies suggest Gd chelates break down in vivo, and Gd accumulation in tissue is linked to medical conditions such as nephrogenic systemic fibrosis (NSF), acute kidney failure, and in some cases death. We measure Gd and other rare earth element (REE) concentrations in 35 femoral heads by solution based ICP-MS. Gd concentrations in patients with documented exposure to Gd-based contrast agents (n = 13: Gd DTPA-BMA (Omniscan) n = 6; Gd HP-DO3A (Prohance) n = 5; unknown type n = 4) are significantly higher (p < 0.001) than the control group (n = 17). We use our control group to establish the ‘natural’ background level of Gd in human bone (cortical 95% CI: 0.023, 0.041 nmol/g; trabecular 95% CI: 0.054, 0.107 nmol/g). A control group outlier reveals the occurrence of individuals with high concentrations of all REEs, including Gd. Because of this, we calculate Gd anomalies from the concentrations of adjacent REEs and normalize to the control group mean to isolate Gd input from contrast agents. Normalized Gd anomalies, (Gd/Gd*)N, for exposed patients range up to >800 times the ‘natural’ level (95% CI: 124, 460). Our data confirm that Gd, introduced in chelated form, incorporates into bone and is retained for more than 8 years. No difference was observed in bone Gd concentrations and anomalies between patients dosed with Gd DTPA-BMA (Omniscan; n = 6) and Gd HP-DO3A (Prohance; n = 5). Osteoporotic fracture patients exposed to Gd have significantly lower Gd concentrations than osteoarthritis patients (p < 0.001). This indicates different mechanisms of metal incorporation and/or retention in osteoporotic bone tissues, and may signal an increased risk of endogenous Gd release for patients with increased rates of bone resorption (e.g. osteoporosis patients and menopausal, pregnant, and lactating women) who are exposed to Gd-based contrast agents.

Interdisciplinary studies of narwhal cranial and tooth anatomy are combined with Inuit traditional knowledge to render a more complete description of tooth-related structures and to propose a new hypothesis for tusk function in the adult mate. Gross anatomy findings from computed tomography (CT) and magnetic resonance (MR) imaging and dissections of an adult male and female and one fetus, four to six months in development, were documented. Computed tomography scans rendered images of the tusks and vestigial teeth and their shared sources of innervation at the base of the tusks. Paired and asymmetrical tusks and vestigial teeth were observed in all three samples, and their relative positions reversed during development. Vestigial teeth shifted anteriorly during growth, and the developing tusks moved posteriorly as they developed. Examination of tusk micro-anatomy revealed the presence of a dentinal tubule network with lumena approximately 2 micrometers in diameter and 10-20 micrometers apart over the pulpal and erupted tusk surfaces. Orifices were present on the cementum surface indicating direct communication and sensory capability from the environment to the inner pulpal wall. Flexural strength of 95 MPa at mid tusk and 165 MPa at the base indicated resistance to high flexural stresses. Inuit knowledge describes a tusk with remarkable and combined strength and flexibility. Elder observations of anatomy are described by variable phenotypes and classified by skin coloration, sex, and tusk expression. Behavioral observations of males leading seasonal migration groups, nonaggressive tusk encounters, and frequent sightings of smaller groups separated by sex add to the discussion of tusk function.

We demonstrated recently that CNT scaffolds support proliferation of mature osteoblasts as well as production of mineralized bone in vitro. More specifically, we showed that osteoblasts grown on single-walled (SW) CNTs retain plasma membrane electrical functions involved in secretory activities. Here we studied osteoinductive properties of SWCNT scaffolds as they support the growth of osteoblastic precursors, their differentiation into mature osteoblasts, and expression of membrane proteins involved in secretory processes. We used electrically neutral ("as prepared", AP-) SWCNTs, and SWCNTs chemically modified with carboxyl (-COOH, net negative electric charge), polyethylene glycol (PEG, electrically neutral), and poly-(m-amino-benzene sulfonic acid, PABS, net positive and negative electric charges) functional groups. We found that PEG-SWCNT and PABS-SWCNT showed the highest upregulation of ALP activity as a measure of osteoinduction. In addition, we found that AP-SWCNTs induced the expression of voltage-gated chloride channels ClC-3 and ClC-5 involved in secretory activities in hFOB osteoblasts. SWCNT preparations might be seen as osteoinductive materials with great potential for use in bone regeneration and repair.

5546 Background: Recent studies support the concept that cancer as a disease is driven by a small subset of cells, cancer stem cells. These cells can initiate tumor growth and re-initiate tumor expansion after therapy. The aim of this study is to generate a cancer stem cell gene expression signature from isolated Side Populations (SP) of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Methods: All patient specimens were collected/studied under protocols approved by the institutional review board of the parent institution. Gene expression profiling using Affymetrix U133 2.0 Plus microarray was performed on SP and Main Populations (MP) isolated by Hoechst staining/FACS method. Paired T-test analyses (p<0.01) identified differentially expressed genes between SP and MP. The microarray data was validated using qRT-PCR. Results: 446 differentially expressed genes (138 up- and 302 down-regulated) were found between all 10 SP/MP pairs. Microarray validation on 19 randomly selected differentially regulated genes, showed 17/19 (89.5%) genes has robust correlations between microarray and qRT-PCR expression data. The gene signature was enriched for genes in GO biological processes of cell cycle, transport, apoptosis, regulation of translation/transcription, signal transduction, and cell proliferation. Further data mining for biologically relevant processes using Pathway Studio 5.0 identified overexpressed genes in SP related to functional cancer stem cell-like phenotypes of cell survival (PAWR), proliferation (EPHB), and apoptosis (AKT). WNT signaling pathway genes (FZD) were downregulated in SP, and genes implicated in normal stem cell function (NUP, ST3GAL, LTBP) were upregulated in SP. These results highlight the ‘stemness function‘ of the ovarian cancer SP gene signature. Conclusions: We generated an expression profile from SP enriched for cancer stem cells from ascites from ovarian cancer patients. The ‘stemness‘ nature of the SP gene signature was revealed by the identification of several stem cell-related genes and genes involved in the WNT -signaling pathway. These genes may be key players involved in the mechanisms controlling ovarian cancer stem cell functions, and may represent potential therapeutic targets. No significant financial relationships to disclose.

VEGF dependent angiogenesis is required for normal bone development and has been implicated in cancer metastasis to bone. These processes, while dependent on osteoclastic bone resorption, are reportedly mediated by endothelial cells, stromal osteoblasts, chondrocytes, and/or tumor cells. We demonstrate here that VEGF treatment of purified murine bone marrow osteoclast precursors directly enhances their survival, differentiation into mature osteoclasts, and resorptive activity. The actions of VEGF on mature osteoclasts principally involve the receptor VEGFR2 (Flk1, KDR), and the receptor signaling utilizes both the P13-kinase -> Akt and MEK -> ERK pathways. Increased osteoclast survival and resorptive activity is correlated with VEGF-dependent phosphorylation of multiple downstream targets of activated Akt [glycogen synthase kinase, GSK-3 beta; forkhead transcription factor, FKHR; and the Bcl-2 antagonist of cell death, Bad (Ser136)] and activated ERK1/2 [ribosomal S6 kinase, p90RSK; and Bad (Ser112)]. Expression of the VEGFR2 gene increases 20-fold during the 6 day in vitro differentiation of mature osteoclasts from mononuclear precursors, while alternate receptors VEGFR1 and neuropilin-1, decrease 30- and 3-fold respectively. Additionally, VEGF enhancement of osteoclast survival is diminished in cells prepared from beta 3 integrin-deficient mice, thus associating VEGF signaling in osteoclasts with their attachment to extracellular matrix. Our results indicate that VEGF directly targets osteoclasts, thereby playing a novel role in bone development, angiogenesis, and tumor metastasis. (C) 2008 Elsevier B.V All rights reserved.

Metal ions originating from mechanical debris and corrosive wear of prosthetic implant alloys accumulate in peri-implant soft tissues, bone mineral, and body fluids. Eventually, metal ions such as cobalt (II) (Co2+), which is a major component of cobalt–chromium-based implant alloys and a known activator of osteolysis, are incorporated into the mineral phase of bone. We hypothesize that the accumulation of Co2+ in the mineral could directly activate osteolysis by targeting osteoclasts. To test this hypothesis, we coated tissue culture plastic with a thin layer of calcium phosphate (CaP) containing added traces of Co2+, thereby mimicking the bone mineral accumulation of Co2+. Murine bone marrow osteoclasts formed in the presence of M-CSF and RANKL were cultured on these surfaces to examine the effects of Co2+ on osteoclast formation and resorptive activity. Treatment conditions with Co2+ involved incorporation into the CaP layer, adsorption to the mineral surface, or addition to culture media. Micromolar concentrations of Co2+ delivered to developing osteoclast precursors by all 3 routes increased both osteoclast differentiation and resorptive function. Compared to CaP layers without Co2+, we observed a maximal 75% increase in osteoclast numbers and a 2.3- to 2.7-fold increase in mineral resorption from the tissue culture wells containing 0.1μm Co2+ and 0.1–10μm Co2+, respectively. These concentrations are well within the range found in peri-implant tissues in vivo. This direct effect of Co2+ on osteoclasts appears to act independently of the particulate phagocytosis/inflammation-mediated pathways, thus enhancing osteolysis and aseptic implant loosening.

Estrogen deficiency in menopause is a major cause of osteoporosis in women. Estrogen acts to maintain the appropriate ratio between bone-forming osteoblasts and bone-resorbing osteoclasts in part through the induction of osteoclast apoptosis. Recent studies have suggested a role for Fas ligand (FasL) in estrogen-induced osteoclast apoptosis by an autocrine mechanism involving osteoclasts alone. In contrast, we describe a paracrine mechanism in which estrogen affects osteoclast survival through the upregulation of FasL in osteoblasts (and not osteoclasts) leading to the apoptosis of pre-osteoclasts. We have characterized a cell-type-specific hormone-inducible enhancer located 86 kb downstream of the FasL gene as the target of estrogen receptor-alpha induction of FasL expression in osteoblasts. In addition, tamoxifen and raloxifene, two selective estrogen receptor modulators that have protective effects in bone, induce apoptosis in pre-osteoclasts by the same osteoblast-dependent mechanism. These results demonstrate that estrogen protects bone by inducing a paracrine signal originating in osteoblasts leading to the death of pre-osteoclasts and offer an important new target for the prevention and treatment of osteoporosis.

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 5005 Recent studies support the concept that cancer as a disease is driven by cancer stem cells and these are the cells that can both initiate tumor growth and then re-initiate tumor expansion after therapy. Hence an understanding of gene expression profiles of cancer stem cells may lead to improved therapies as well as novel insights into the cellular mechanisms of carcinogenesis in ovary. The aim of this study is to generate a cancer stem cell gene expression signature from isolated Side Populations of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma at the time of cytoreductive surgery at Brigham and Women's Hospital, Boston, MA. All patients had been diagnosed with ovarian cancer for the first time. All patient specimens were collected and studied under protocols approved by the institutional review board of the parent institution. Gene expression profiling using Affymetrix Human Genome U133 2.0 Plus microarray platform was performed on Side Populations (SP) and Main Populations (MP) isolated by Hoechst staining/FACS method. Array data was analyzed using BRB-ArrayTools. Paired T-test analyses (p<0.01) was utilized to identify differentially expressed genes between SP and MP. We identified 446 genes (138 up-regulated and 302 down-regulated) that were differentially expressed between all 10 SP and MP pairs. The microarray data was validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) on 19 randomly selected differentially regulated genes. 17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The gene signature was enriched for genes in Gene Ontology biological processes of cell cycle, transport, apoptosis, regulation of translation/transcription, signal transduction, and cell proliferation. Further data mining for biologically relevant processes using Pathway Studio 5.0 identified genes overexpressed in SP that were related to functional cancer stem cell-like phenotypes of cell survival (PAWR), proliferation (EPHB), and apoptosis (AKT). WNT signaling pathway genes (FZD) were downregulated in SP, and genes implicated in normal stem cell (NUP, ST3GAL, LTBP) were upregulated in SP. Taken together these results highlight the function of the ovarian cancer SP gene signature. In conclusion, we generated an expression profile from SP enriched for cancer stem cells from ascites from ovarian cancer patients. The nature of the stemness of the SP gene signature was revealed by the identification of several stem cell-related genes and genes involved in the WNT -signaling pathway. These genes may be key players involved in the mechanisms controlling ovarian cancer stem cell functions, and may represent potential therapeutic targets.