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Canada's federal, provincial, and territorial governments gave Canadian Blood Services a mandate for organ and tissue donation and transplantation, including system performance, data and analytics. In 2012 Canadian Blood Services facilitated an eye and tissue banking workshop focused on standardized specifications and practices. At the workshop, the Canadian tissue community directed Canadian Blood Services to facilitate the development and implementation of a national data stream and analytics. Prior to this no national data was prospectively collected or collated on tissue donation, production or distribution activity. An eye and tissue data committee was formed with representation from eye and tissue banks in all Canadian jurisdictions. A minimum data set, standardized definitions, a data submission form and a quality assurance process was developed. Training was provided to data personal identified by each eye and tissue bank. Data collection was initiated January 1, 2013; with quarterly data submitted to Canadian Blood Services via excel spreadsheet. Data was submitted by sixteen Canadian eye and tissue banks, located in eight of Canada's thirteen provinces and territories, representing a census of activity. Annual data reports, with trend analysis, are generated and distributed to the tissue community to inform operational strategy and system performance improvement. This report provides an overview of the data process and provides visibility to the Canadian tissue donation, production and distribution activities for 3 years; January 1, 2013 to December 31, 2015.

OBJECTIVES Lanthanum (La) and gadolinium (Gd) are known to deposit in bone of exposed populations, namely those who are orally administered lanthanum carbonate (LaC, La2(CO3)3) or are injected with Gd-based contrast agents, respectively. In this work, bone La and Gd concentrations from the environment and diet were measured using x-ray fluorescence in ten post-mortem human tibiae. As a secondary objective, bone barium (Ba) and iodine concentrations were estimated. APPROACH Two calibration lines were produced for La and Gd and the minimum detection limits (MDLs) of the system were determined using a 180° irradiation-detection geometry. MAIN RESULTS The MDLs of the system were 0.4 µg La g-1 bone mineral and 0.5 µg Gd g-1 bone mineral. The mean concentrations were -0.02 ± 0.1 µg La g-1 bone mineral and 0.1 ± 0.2 µg Gd g-1 bone mineral in tibiae. The average Ba and iodine concentrations estimated from the experimental La calibration line and Monte-Carlo derived sensitivity factors were determined to be 3.4 ± 0.8 µg Ba g-1 bone mineral and -0.5 ± 0.3 µg iodine g-1 bone mineral. Since it was discovered that four donors previously received an iodine-based contrast agent, the mean concentrations in these donors was 27.8 ± 28.4 µg iodine g-1 bone mineral. SIGNIFICANCE The XRF system has determined baseline concentrations of these four heavy metals in trace quantities from natural exposure pathways (with the exception of iodine in four donors). This indicates that the system can measure low levels in ex vivo tibiae samples and can potentially be further developed for in vivo studies involving live subjects who are directly exposed to these metals.

412 Background: NMIBC has a highly variable clinical behavior not adequately predicted by histological grade or clinical parameters. Some are indolent; others quickly progress to MIBC. Discrepancies between phenotype and genotype is compounded further by interobserver variability in pathological grading. There is an unmet need to improve the prediction of NMIBC. Methods: Whole transcriptomic analysis of 178 bladder tumors (158 NMIBC, 20 MIBC/metastatic) was performed from FFPE tissue incorporating messenger RNA expression, splice variants, gene fusion, mutation detection and immune checkpoint inhibitor cascades. CTLA, PD-1, LAG3, TIM3, TIGIT and B7 were compiled as an index including all major cascade genes. Data were integrated and tested for correlations with pathological grading and clinical outcomes. Conventional pathological grading for WHO 1973 (Grade 1, 2, 3) and 2004 (LG vs HG) classifications was reviewed by 3 expert uro-pathologists. Kappa statistic for interobserver variability was calculated. For validation we used an independent RNA-seq dataset (n = 209, Hedegaard et al. 2016 Cancer Cell). Results: Unsupervised clustering of RNA-Seq data distinguished 3 molecular subtypes of NMIBC; Molecular Grade Related Index (MGRI) 1, MGRI2, MGRI3. MGRI1 comprised of almost exclusively LG tumors. MGRI3 clustered with HG MIBC. Kappa for interobserver variability of expert pathologists was 0.40 and 0.78 in 1973 and 2004 WHO classification, respectively. FGFR3 mutations, FGFR3::TACC3 fusion events and MGRI1 genes were associated with components of xenobiotic metabolism (p = 2.51x10-09) signalling systems, in particular, GTPase regulation (p = 0.002), respiratory cycle genes (p = 0.004), HOX cluster (p = 0.005). MGRI independently predicted progression to MIBC (n = 138, HR = 2.96, 95%CI = 1.70-5.13, p = 1.20x10-04). 5-year PFS in a combined data set (n = 347) differed significantly for MGRI1 (100%) vs MGRI2 (92.2%) vs MGRI3 (73.5%, p = 1.99x10-05, Gray’s test). PD-1 ICC independently predicted progression (OR = 2.85, p < 0.05). Conclusions: RNA-seq delineates 3 molecular classes of NMIBC with different risks of progression to MIBC compared to conventional histologic grading.

Background: Little is known about the mechanisms and treatment of radiation-induced inhibition of craniofacial bone growth. In an earlier study, the radio-protector amifostine (WR-2721) administered to rabbits before irradiation radioprotected cultured orbitozygomatic complex periosteal osteoblast-like cells. This study assessed the effects of amifostine and its active metabolite on the radiation survival, function, and phenotype of mouse calvarial osteoblast-like cells in a cell culture model. Methods: MC3T3-E1 newborn mouse calvarial osteoblast-like cells underwent γ-radiation (0 to 10 Gy) in the presence or absence of either WR-2721 or WR-1065, its active metabolite (10 -7 to 10 -3 M). The effects of radiation with and without drugs were assessed using endpoints of colony-forming ability, cell viability, alkaline phosphatase activity, and expression of osteoblastic phenotype genes (alkaline phosphatase, collagen type I, osteocalcin, and osteopontin). All experiments were replicated at least in triplicate. Results: Irradiation resulted in a dose-dependent inhibition of clonogenic cell survival. Pretreatment with WR-1065, but not WR-2721, resulted in a significant improvement of osteoblast-like cell survival. Specifically, maximum radioprotection was observed with 10 -4 M WR-1065 at a clinically relevant 2-Gy dose of irradiation. No significant radioprotection was observed at the lower (5 X 10 -7 M) concentration of WR-1065. Furthermore, radiation seemed to suppress the expression of ostecblastic phenotype-related genes in a dose-dependent manner. Conclusions: This study reveals improved survival after irradiation in osteoblast-like cells treated with WR-1065 in vitro and corroborates previous findings from animal models. Further studies using this agent and similar drugs are important for devising strategies to prevent radiation-induced inhibition of craniofacial bone growth.

Golgi beta1,6-N-acetylglucosaminyltransferase V (Mgat5) produces beta1,6GlcNAc-branched N-glycans on glycoproteins, which increases their affinity for galectins and opposes loss from the cell surface to constitutive endocytosis. Oncogenic transformation increases Mgat5 expression, increases beta1,6GlcNAc-branched N-glycans on epidermal growth factor and transforming growth factor-beta receptors, and enhances sensitivities to ligands, cell motility, and tumor metastasis. Here, we demonstrate that Mgat5(-/-) mouse embryonic fibroblasts (MEFs) display reduced sensitivity to anabolic cytokines and reduced glucose uptake and proliferation. Mgat5(-/-) mice are also hypoglycemic, resistant to weight gain on a calorie-enriched diet, hypersensitive to fasting, and display increased oxidative respiration and reduced fecundity. Serum-dependent activation of the extracellular response kinase (growth) and Smad2/3 (arrest) pathways in Mgat5(-/-) MEFs and bone marrow cells reveals an imbalance favoring arrest. Mgat5(-/-) mice have fewer muscle satellite cells, less osteogenic activity in bone marrow, and accelerated loss of muscle and bone mass with aging. Our results suggest that beta1,6GlcNAc-branched N-glycans promote sensitivity to anabolic cytokines, and increase fat stores, tissue renewal, and longevity.

Molecular profiling of individual cancers is key to personalised medicine. While sequencing technologies have required stringent sample collection and handling, recent technical advances offer sequencing from tissues collected in routine practice and tissues already stored in archives. In this paper, we establish methods for whole-transcriptome RNA sequencing (RNA-seq) from formalin-fixed paraffin-embedded tissues. We obtain average RNA-seq reads of >100 million per sample using the Illumina HiSeq2000 platform. We find high concordance with results from matching fresh frozen samples (>0.8 Spearman correlation). For validation, we compared low- and high-grade bladder cancer transcriptomes in 49 tumour samples after transurethral resection of bladder tumour. We found 947 differentially expressed protein-coding genes. While high-grade lesions exhibited distinct intertumour transcriptome heterogeneity, the transcriptome of low-grade tumours was homogeneous. Patient summary In this report, we show that it is now possible to use universally available bladder cancer samples that have been fixed in formalin to perform high-quality transcriptome analysis. This ability will facilitate the development of transcriptome-wide tests based on gene expression correlated with clinical outcome.

The serum glycoprotein fetuin is expressed during embryogenesis in multiple tissues including limb buds and has been shown to promote bone remodeling and stimulate cell proliferation in vitro. In this report, we demonstrate that fetuin antagonizes the antiproliferative action of transforming growth factor-beta1 (TGF-beta1) in cell cultures. Surface plasmon resonance measurements show that fetuin binds directly to TGF-beta1 and TGF-beta2 and with greater affinity to the TGF-beta-related bone morphogenetic proteins (BMP-2, BMP-4, and BMP-6). In a competitive enzyme-linked immunosorbent assay, fetuin blocked binding of TGF-beta1 to the extracellular domain of TGF-beta receptor type II (TbetaRII), one of the primary TGF-beta-binding receptors. A comparison of fetuin and TbetaRII shows homology in an 18-19-amino acid sequence, which we have designated TGF-beta receptor II homology 1 domain (TRH1). Since the TRH1 sequence is known to form a disulfide loop in fetuin, cyclized TRH1 peptides from both fetuin and TbetaRII were chemically synthesized and tested for cytokine binding activity. Cyclized TRH1 peptide from TbetaRII bound to TGF-beta1 with greater affinity than to BMP-2, while the cyclized TRH1 peptide from fetuin bound preferentially to BMP-2. Finally, fetuin or neutralizing anti-TGF-beta antibodies blocked osteogenesis and deposition of calcium-containing matrix in cultures of dexamethasone-treated rat bone marrow cells. In summary, these experiments define the TRH1 peptide loop as a cytokine-binding domain in both TbetaRII and fetuin and suggest that fetuin is a natural antagonist of TGF-beta and BMP activities.

Background Tissue non-specific alkaline phosphatase (TN-AP) is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic activity. This enzyme is distributed virtually in all mammalian tissues during embryonic development (it can be demonstrated as early as the 2-cell stage) where its expression is stage specific. The expression of TN-AP is frequently associated with cell differentiation and as such it has been used as a marker for this process. By employing a stable gene transfer and forced gene expression technique, previous findings suggested that TN-AP expression might influence cellular proliferation and morphological differentiation. The focus of this study was to determine whether this was a cell-specific effect or not. Methods The effects of TN-AP on various aspects of cellular activity were assessed by transferring and expressing the gene for this enzyme into three target populations; 1) CHO, 2) R1610, and 3) Rat-2. The parameters of cellular activity studied included cellular proliferation, cell cycle, cell migration, and tumorigenic potential (in the nude mouse). Cell cycle and cell proliferation analyses were accomplished, in part, through the use of fluorescence activated cell sorting (FACS) as were determinations of cell-associated TN-AP activity and amount. Northern and southern blot analyses were used to estimate gene copy number and to evaluate gene expression respectively in transfected cell lines. Results Our data indicate that the TN-AP gene under control of various gene promoters was stably integrated into three fibroblast-like cell lines (CHO, R1610, and Rat-2). TN-AP activity and TN-AP protein levels were correlated to the strength of the various gene promoters, but not to inserted gene copy numbers. The expression of the TN-AP gene in these three cell types further suggests cell-specific effects as demonstrated by the following findings. The expression of TN-AP under control of a weak gene promoter in CHO and Rat-2 cells clearly decreased cell proliferation and cell migration. However, the expression of TN-AP under control of either a weak or even a strong gene promoter in R1610 cells did not induce any changes in that cell line's behaviour (apart from expression of TN-AP). Such changes in CHO cells were also associated with a 2.2-fold increase in tubulin transcription as well as suppressed tumorigenic potential. Conclusion Taken together, these findings suggest that the inhibitory effects of TN-AP expression on proliferation and cell migration are not non-specific and that high expression of TN-AP may induce changes in cell behaviour which may be consistent with or at least related to induction of terminal differentiation. © 1996 Wiley-Liss, Inc.

Glucocorticoids and sex-steroids can modulate osteogenesis in vivo and in vitro. Although the effects of glucocorticoids on bone cells in vitro have been described in detail, the role of sex-steroids is not as well defined. We examined whether sex-steroids influence bone metabolism indirectly by regulating glucocorticoid effects on bone. Interactions of the sex-steroid progesterone or its analog RU38486 with the glucocorticoid dexamethasone (dex) were studied in functional assays of osteogenesis. Three osteoblastic models were evaluated: (1) the rat bone marrow stromal cell (RBMC) nodule system; (2) the chick periosteal osteogenesis (CPO) model; and (3) ROS 17/2.8 cells. RU38486, progesterone, and unlabelled dex competitively inhibited 3H-dex uptake by ROS 17/2.8 cells as well as its (3H-dex) binding to cytosol preps. Both RU38486 and progesterone inhibited dex-induced increases in alkaline phosphatase in CPO cultures, in RBMC cultures, and in ROS 17/2.8 cells. Dex-induced decreases in cell proliferation in ROS 17/2.8 cells were reversed by RU38486 but dex-induced increases in proliferation in the CPO model were not affected. In CPO cultures, dex-induced increases in collagen synthesis were inhibited completely by RU38486 and progesterone. Dex-dependent nodule formation in the RBMC was blocked by RU38486. Both RU38486 and dex mediated reduction of calcium uptake in the CPO model but did not affect mineralized tissue area. The data indicate that RU38486 and progesterone competitively inhibit dex-mediated stimulation of osteogenesis in vitro; this inhibition is exerted on early but not late stage differentiation events of osteoprogenitor cells.

467 Background: There is an unmet need for a comprehensive genomic characterization of non muscle invasive bladder cancer (NMIBC). NMIBC comprise over 70% of all bladder cancers at presentation. They have highly variable clinical behavior that is not always adequately predicted on the basis of their histological grade (2004 World Health Organization low and high grade, LG-HG). The discrepancy between phenotype and genotype is compounded further by interobserver variability in pathological grading. We have previously established methods for whole transcriptome RNAseq from formalin fixed paraffin embedded tissues (FFPE). Methods: Whole transcriptomic analysis of 110 NMI FFPE BC both LG and HG was performed incorporating messenger RNA expression, splice variants, gene fusion, and pathway perturbation. We used a discovery (n = 40) and a validation cohort (n = 70). These data were integrated and tested for correlation with both pathological grading and clinical outcomes. Grade Risk Index (GRI) score quantifying how closely a patient's transcriptome is related to a reference set of LG NMIBC samples was established. Conventional pathological grading was reviewed by 3 different expert uro-pathologists and interobserver variability calculated. Results: Unsupervised clustering of data from RNA sequencing uncovered classification of three robust - - nonoverlapping, prognostically significant subtypes of NMIBC with distinct GRIs and signatures. When applied by expert pathologists, interobserver variability in histological grading was observed in 17.5%. In the intermediate group (GRI 0.13 to 0.19), pathologists disagreed in 37.5% whether BC was LG or HG. HG NMIBC clustered with MIBC. LG NMIBC in the intermediate GRI group included either very bulky tumors or extremely rare metastatic LG BC (n = 4). HG disease was associated with a shift in BMP signaling and a germ stem cell-like phenotype. Multiple components of the centromere complex and APOBEC3B were upregulated in HG BC. FGFR3::TACC3 fusion events were observed in LG NMIBC only (11.5%). Conclusions: Whole transcriptomic sequencing data delineated three molecular classes of NMIBC.

It is well documented that oral microorganisms play a significant role in the initiation and progression of periodontal disease. By using various in vitro models, it has been shown that some bacteria considered periodontal pathogens or their products can stimulate bone resorption and some other parameters of osteoblast-like cell activity. However, the effects of these organisms and their products on osteogenesis itself are not known. This study was undertaken to determine the direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis on bone formation in the chick periosteal osteogenesis model. Cultures of P. gingivalis 2561 were grown under standard anaerobic culture conditions. The spent medium was collected, and following centrifugation, sonicated bacterial extracts were prepared from the bacterial pellet. These were added in various proportions to the chick periosteal osteogenesis cultures. Sonicated extracts were further fractionated into five molecular-size ranges and similarly tested. Parameters of osteogenesis, including alkaline phosphatase activity, calcium and Pi accumulation, and collagen synthesis, were measured on 6-day-old cultures. Compared with controls devoid of bacterial products, osteogenesis was inhibited significantly in cultures treated with either conditioned medium or extracts obtained from P. gingivalis. Various amounts of inhibitory activity were observed in the different ultrafiltration molecular-size fractions, with very profound inhibitory effects observed in the < 5-kDa range. Histological observations indicated the presence of cells, some bone, and/or new fibrous connective tissue at all concentrations, indicating that toxicity was not a factor. These results suggest that periodontal pathogens such as P. gingivalis might contribute to the bone loss in periodontal diseases not only by stimulating resorption but, possibly, by inhibiting bone formation directly.

Individuals and families carrying mutations in BRCA1 and BRCA2 (BRCA1/2) have a markedly elevated risk of developing breast and ovarian cancers. The first-generation of BRCA1/2 mutation analysis targeted only the coding exons and has implicated protein-truncating mutations (indel, nonsense) in BRCA1/2 inactivation. Recently, heritable breast cancers have also been attributed to other exonic mutations (missense, silent) and mutations in introns and untranslated regions. However, analysis of these alterations has been prohibitively laborious and cost intensive, and the proportion of cases carrying mutations in unscreened regions of BRCA1/2 and other predisposition genes is unknown. We have developed and validated a next-generation sequencing (NGS) approach for BRCA1/2 mutation analysis by applying long-range PCR and deep sequencing. Genomic DNA from familial breast cancer patients (N = 12) were screened and NGS successfully identified all 19 distinct (51 total) BRCA1 and 35 distinct (63 total) BRCA2 sequence alterations detectable by the Sanger sequencing, with no false-negative or positive results. In addition, we report the robust detection of variants from introns and untranslated regions. These results illustrate that NGS can provide comprehensive genetic information more quickly, accurately, and at a lower cost than conventional approaches, and we propose NGS to be a more effective method for BRCA1/2 mutational analysis. Advances in NGS will play an important role in enabling molecular diagnostics and personalized treatment of breast and ovarian cancers.

BACKGROUND: Radiotherapy for the management of head and neck cancer in pediatric patients results in severe inhibition of craniofacial bone growth. Previously, the infant rabbit orbitozygomatic complex was established as an experimental model. Amifostine, a cytoprotective agent, was found effective in preventing radiation-induced bone growth inhibition. This study was designed to investigate the effects radiation on osteogenic cells from infant rabbit orbitozygomatic complex periostea and to assess the effects of cytoprotection in vitro. METHODS: Infant New Zealand White rabbits (n = 18) were randomized into three groups and received radiation (0, 10, or 15 Gy) to both orbitozygomatic complexes. Cell cultures were developed from orbitozygomatic complex periostea, and cell numbers, proliferation, alkaline phosphatase, and collagen type I expression and mineralization were assessed. Subsequently, rabbits (n = 18) were randomized into three groups to receive either radiation at the effective dose, pretreatment with amifostine (300 mg/kg, intravenously, 20 minutes before irradiation) with the effective radiation dose, or no treatment. Cell cultures were developed and tested for proliferation and alkaline phosphatase expression. RESULTS: Irradiation resulted in a significant inhibition of cell numbers (p < 0.001) and proliferation (p < 0.01) at the 15-Gy dose and no statistically significant changes in alkaline phosphatase activity. Collagen type I expression and mineralization were also significantly reduced at the 15-Gy dose. Pretreatment with amifostine significantly (p < 0.05) enhanced the number of surviving cells. CONCLUSIONS: Amifostine is capable of protecting orbitozygomatic complex periosteum-derived osteogenic cells from the deleterious effects of radiation. This study provides the basis for understanding the cellular mechanisms of radiation-induced craniofacial bone growth inhibition and cytoprotection by amifostine.

Previously, we demonstrated that through binding a novel intracellular receptor of microM affinity (HIC), histamine mediates, and the HIC antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine. HCl (DPPE) inhibits, platelet aggregation and serotonin granule secretion; the latter response is dependent upon the same processes that mediate histamine release from mast cell granules. We now show that, as for platelet serotonin release, DPPE blocks concanavalin A-stimulated mast cell histamine release with a potency (IC50 = 30 microM) greater than the H1-antagonist, pyrilamine (IC50 = 150 microM) or the H2-antagonist cimetidine (IC50 = 5 mM), correlating with rank order of potency to inhibit 3H-histamine binding in rat brain membranes and liver microsomes. We postulate that histamine release from mast cells is mediated at HIC by second messenger intracellular histamine. However, unlike platelets, mast cells do not appear to rely on newly synthesized histamine. Rather, as for calcium, histamine may be mobilized from bound stores to mediate histamine secretion.

Soluble transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP)-binding proteins are widely distributed in mammalian tissues and control cytokine access to membrane signaling receptors. The serum and bone-resident glycoprotein alpha2-HS-glycoprotein/fetuin (ASHG) binds to TGF-beta/BMP cytokines and blocks TGF-beta1 binding to cell surface receptors. Therefore, we examined bone growth and remodeling phenotypes in ASHG-deficient mice. The skeletal structure of Ahsg(-/-) mice appeared normal at birth, but abnormalities were observed in adult Ahsg(-/-) mice. Maturation of growth plate chondrocytes was impaired, and femurs lengthened more slowly between 3 and 18 months of age in Ahsg(-/-) mice. However, bone formation was increased in Ahsg(-/-) mice as indicated by greater cortical thickness, accelerated trabecular bone remodeling, and increased osteoblast numbers on bone surfaces. The normal age-related increase in cortical thickness and bone mineral density was accelerated in Ahsg(-/-) mice and was associated with increased energy required to fracture. Bone formation in response to implanted BMP cytokine extended further from the implant in Ahsg(-/-) compared with Ahsg(+/+) mice, confirming the interaction between ASHG and TGF-beta/BMP cytokines in vivo. Our results demonstrate that ASHG blocks TGF-beta-dependent signaling in osteoblastic cells, and mice lacking ASHG display growth plate defects, increased bone formation with age, and enhanced cytokine-dependent osteogenesis.

Aryl hydrocarbon receptor (AhR) ligands are environmental contaminants found in cigarette smoke and other sources of air pollution. The prototypical compound is TCDD (2,3,7, 8-tetrachlorodibenzo-p-dioxin), also known as dioxin. There is an increasing body of knowledge linking cigarette smoking to osteoporosis and periodontal disease, but the direct effects of smoke-associated aryl hydrocarbons on bone are not well understood. Through the use of resveratrol (3,5,4'-trihydroxystilbene), a plant antifungal compound that we have recently demonstrated to be a pure AhR antagonist, we have investigated the effects of TCDD on osteogenesis. It was postulated that TCDD would inhibit osteogenesis in bone-forming cultures and that this inhibition would be antagonized by resveratrol. We employed the chicken periosteal osteogenesis (CPO) model, which has been shown to form bone in vitro in a pattern morphologically and biochemically similar to that seen in vivo, as well as a rat stromal cell bone nodule formation model. In the CPO model, alkaline phosphatase (AP) activity was reduced by up to 50% (P

Osteoporosis is a common problem of aging and results from a failure of homeostatic mechanisms to regulate osteogenesis and mineralization. Bovine and human forms of fetuin glycoprotein bind to the transforming growth factor (TGF)-beta/BMP (bone morphogenic protein) cytokines and block their osteogenic activity in cell culture assays (Demetriou, M., Binkert, C., Sukhu, B., Tenenbaum, H. C., and Dennis, J. W. (1996) J. Biol. Chem. 271, 12755-12761). Fetuin is a prominent serum glycoprotein and a major noncollagenous component of mineralized bone in mammals. In this study, we show that recombinant fetuin and native serum protein have similar potency as inhibitors of osteogenesis in dexamethasone-treated rat bone marrow cell cultures (dex-RBMC). Recombinant bovine fetuin also bound to TGF-beta1 and BMP-2 in vitro with kinetics similar to native fetuin. Although TGF-beta1 is required for osteogenesis in dex-RBMC, the cytokine also inhibited osteogenesis at concentrations >/=10 pM. Titration of fetuin or anti-TGF-beta1 antibodies into the bone marrow cultures in the presence of 10 pM TGF-beta1 restored osteogenesis, whereas titrations of the same reagents into cultures with 0.3 pM added TGF-beta1 were inhibitory, confirming the biphasic nature of the TGF-beta1 response. Suppression of osteogenesis by both TGF-beta1 and the antagonist proteins required their presence within the first 6 days of culture, well before mineralization at 10-12 days. Northern analysis showed that both fetuin and high dose TGF-beta1 suppressed expression of the bone-associated transcripts alkaline phosphatase, osteopontin, collagen type I, and bone sialoprotein. The suppression of osteogenesis by fetuin and by high dose TGF-beta1 was accompanied by the differentiation of an alternate cell lineage with adipocyte characteristics. In summary, the biphasic osteogenic response to TGF-beta1 suggests that overlapping gradients of TGF-beta/BMP cytokines and fetuin regulate osteogenesis in remodeling bone.