Differential Regulation of Human Bone Marrow Mesenchymal Stromal Cell Chondrogenesis by Hypoxia Inducible Factor-1α Hydroxylase Inhibitors.

The transcriptional profile induced by hypoxia plays important roles in the chondrogenic differentiation of marrow stromal/stem cells (MSC) and is mediated by the hypoxia inducible factor (HIF) complex. However, various compounds can also stabilize HIF's oxygen-responsive element, HIF-1α, at normoxia and mimic many hypoxia-induced cellular responses. Such compounds may prove efficacious in cartilage tissue engineering, where microenvironmental cues may mediate functional tissue formation. Here, we investigated three HIF-stabilizing compounds, which each have distinct mechanisms of action, to understand how they differentially influenced the chondrogenesis of human bone marrow-derived MSC (hBM-MSC) in vitro. hBM-MSCs were chondrogenically-induced in transforming growth factor-ß3-containing media in the presence of HIF-stabilizing compounds. HIF-1α stabilization was assessed by HIF-1α immunofluorescence staining, expression of HIF target and articular chondrocyte specific genes by quantitative polymerase chain reaction, and cartilage-like extracellular matrix production by immunofluorescence and histochemical staining. We demonstrate that all three compounds induced similar levels of HIF-1α nuclear localization. However, while the 2-oxoglutarate analog dimethyloxalylglycine (DMOG) promoted upregulation of a selection of HIF target genes, desferrioxamine (DFX) and cobalt chloride (CoCl2 ), compounds that chelate or compete with divalent iron (Fe2+ ), respectively, did not. Moreover, DMOG induced a more chondrogenic transcriptional profile, which was abolished by Acriflavine, an inhibitor of HIF-1α-HIF-ß binding, while the chondrogenic effects of DFX and CoCl2 were more limited. Together, these data suggest that HIF-1α function during hBM-MSC chondrogenesis may be regulated by mechanisms with a greater dependence on 2-oxoglutarate than Fe2+ availability. These results may have important implications for understanding cartilage disease and developing targeted therapies for cartilage repair. Stem Cells 2018;36:1380-1392.

3D printed hybrid scaffolds do not induce adverse inflammation in mice and direct human BM-MSC chondrogenesis in vitro.

Biomaterials that can improve the healing of articular cartilage lesions are needed. To address this unmet need, we developed novel 3D printed silica/poly(tetrahydrofuran)/poly(ε-caprolactone) (SiO2/PTHF/PCL-diCOOH) hybrid scaffolds. Our aim was to carry out essential studies to advance this medical device towards functional validation in pre-clinical trials. First, we show that the chemical composition, microarchitecture and mechanical properties of these scaffolds were not affected by sterilisation with gamma irradiation. To evaluate the systemic and local immunogenic reactivity of the sterilised 3D printed hybrid scaffolds, they were implanted subcutaneously into Balb/c mice. The scaffolds did not trigger a systemic inflammatory response over one week of implantation. The interaction between the host immune system and the implanted scaffold elicited a local physiological reaction with infiltration of mononuclear cells without any signs of a chronic inflammatory response. Then, we investigated how these 3D printed hybrid scaffolds direct chondrogenesis in vitro. Human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs) seeded within the 3D printed hybrid scaffolds were cultured under normoxic or hypoxic conditions, with or without chondrogenic supplements. Chondrogenic differentiation assessed by both gene expression and protein production analyses showed that 3D printed hybrid scaffolds support hBM-MSC chondrogenesis. Articular cartilage-specific extracellular matrix deposition within these scaffolds was enhanced under hypoxic conditions (1.7 or 3.7 fold increase in the median of aggrecan production in basal or chondrogenic differentiation media). Our findings show that 3D printed SiO2/PTHF/PCL-diCOOH hybrid scaffolds have the potential to support the regeneration of cartilage tissue.

Biocompatibility of mannan nanogel--safe interaction with plasma proteins.

BACKGROUND: Self-assembled mannan nanogels are designed to provide a therapeutic or vaccine delivery platform based on the bioactive properties of mannan to target mannose receptor expressed on the surface of antigen-presenting cells, combined with the performance of nanogels as carriers of biologically active agents. METHODS: Proteins in the corona around mannan nanogel formed in human plasma were identified by mass spectrometry after size exclusion chromatography or centrifugation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Structural changes and time dependent binding of human apolipoprotein A-I (apoA-I) and human serum albumin (HSA) to mannan nanogel were studied using intrinsic tryptophan fluorescence and circular dichroism spectroscopy. The mannan nanogel effect on blood coagulation and fibrillation of Alzheimer's disease-associated amyloid ß peptide and hemodialysis-associated amyloidosis ß2 microglobulin was evaluated using thrombin generation assay or thioflavin T fluorescence assay, respectively. RESULTS: The protein corona around mannan nanogel is formed through a slow process, is quite specific comprising apolipoproteins B-100, A-I and E and HSA, evolves over time, and the equilibrium is reached after hours to days. Structural changes and time dependent binding of apoA-I and HSA to mannan nanogel are minor. The mannan nanogel does not affect blood coagulation and retards the fibril formation. CONCLUSIONS: Mannan nanogel has a high biosafety and biocompatibility, which is mandatory for nanomaterials to be used in biomedical applications. GENERAL SIGNIFICANCE: Our research provides a molecular approach to evaluate the safety aspects of nanomaterials, which is of general concern in society and science.

Polymeric nanogels as vaccine delivery systems.

Polymeric nanogels find a relevant field of application in the formulation of a new generation of therapeutic and preventive vaccines, aiming at the fine-tuned modulation of the immune response. Intrinsic properties of polymeric nanogels, such as material chemistry, size and shape, surface charge, and hydrophobicity or hydrophilicity, may be determining factors in shaping the induced immune response. These materials can thus work as synthetic adjuvants, which can also be conjugated with immunostimulants. Polymeric nanogels protect vaccine antigens from degradation in vivo and, surface-conjugated with antibodies or specific ligands, could increase active targeting specificity. This review covers the recent published data concerning the modulation of innate and adaptive immune responses by engineered polymeric nanogels and their potential application as delivery systems in vaccination. FROM THE CLINICAL EDITOR: In this review, the utility of polymeric nanogels is discussed as adjuvants and protective agents for enhanced vaccination with more robust immune response and a more uniform outcome.

Self-assembled dextrin nanogel as protein carrier: controlled release and biological activity of IL-10.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer. Given the potential of IL-10 for application in various medical conditions, it is essential to develop systems for its effective delivery. In previous work, it has been shown that a dextrin nanogel effectively incorporated and stabilized rIL-10, enabling its release over time. In this work, the delivery system based on dextrin nanogels was further analyzed. The biocompatibility of the nanogel was comprehensively analyzed, through cytotoxicity (lactate dehydrogenase (LDH) release, MTS, Live, and Dead) and genotoxicity (comet) assays. The release profile of rIL-10 and its biological activity were evaluated in vivo, using C57BL/6 mice. Although able to maintain a stable concentration of IL-10 for at least 4 h in mice serum, the amount of protein released was rather low. Despite this, the amount of rIL-10 released from the complex was biologically active inhibiting TNF-α production, in vivo, by LPS-challenged mice. In spite of the significant stabilization achieved using the nanogel, rIL-10 still denatures rather quickly. An additional effort is thus necessary to develop an effective delivery system for this cytokine, able to release active protein over longer periods of time. Nevertheless, the good biocompatibility, the protein stabilization effect and the ability to perform as a carrier with controlled release suggest that self-assembled dextrin nanogels may be useful protein delivery systems.

Bioglass/carbonate apatite/collagen composite scaffold dissolution products promote human osteoblast differentiation.

OssiMend® Bioactive (Collagen Matrix Inc., NJ) is a three-component porous composite bone graft device of 45S5 Bioglass/carbonate apatite/collagen. Our in vitro studies showed that conditioned media of the dissolution products of OssiMend Bioactive stimulated primary human osteoblasts to form mineralized bone-like nodules in vitro in one week, in basal culture media (no osteogenic supplements). Osteoblast differentiation was followed by gene expression analysis and a mineralization assay. In contrast, the dissolution products from commercial OssiMend (Bioglass-free carbonate apatite/collagen scaffolds), or from 45S5 Bioglass particulate alone, did not induce the mineralization of the extracellular matrix, but did induce osteoblast differentiation to mature osteoblasts, evidenced by the strong upregulation of BGLAP and IBSP mRNA levels. The calcium ions and soluble silicon species released from 45S5 Bioglass particles and additional phosphorus release from OssiMend mediated the osteostimulatory effects. Medium conditioned with OssiMend Bioactive dissolution had a much higher concentration of phosphorus and silicon than media conditioned with OssiMend and 45S5 Bioglass alone. While OssiMend and OssiMend Bioactive led to calcium precipitation in cell culture media, OssiMend Bioactive produced a higher concentration of soluble silicon than 45S5 Bioglass and higher dissolution of phosphorus than OssiMend. These in vitro results suggest that adding 45S5 Bioglass to OssiMend produces a synergistic osteostimulation effect on primary human osteoblasts. In summary, dissolution products of a Bioglass/carbonate apatite/collagen composite scaffold (OssiMend® Bioactive) stimulate human osteoblast differentiation and mineralization of extracellular matrix in vitro without any osteogenic supplements. The mineralization was faster than for dissolution products of ordinary Bioglass.

Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy.

Growing interest in exploring mechanically mediated biological phenomena has resulted in cell culture substrates and 3D matrices with variable stiffnesses becoming standard tools in biology labs. However, correlating stiffness with biological outcomes and comparing results between research groups is hampered by variability in the methods used to determine Young's (elastic) modulus, E, and by the inaccessibility of relevant mechanical engineering protocols to most biology labs. Here, we describe a protocol for measuring E of soft 2D surfaces and 3D hydrogels using atomic force microscopy (AFM) force spectroscopy. We provide instructions for preparing hydrogels with and without encapsulated live cells, and provide a method for mounting samples within the AFM. We also provide details on how to calibrate the instrument, and give step-by-step instructions for collecting force-displacement curves in both manual and automatic modes (stiffness mapping). We then provide details on how to apply either the Hertz or the Oliver-Pharr model to calculate E, and give additional instructions to aid the user in plotting data distributions and carrying out statistical analyses. We also provide instructions for inferring differential matrix remodeling activity in hydrogels containing encapsulated single cells or organoids. Our protocol is suitable for probing a range of synthetic and naturally derived polymeric hydrogels such as polyethylene glycol, polyacrylamide, hyaluronic acid, collagen, or Matrigel. Although sample preparation timings will vary, a user with introductory training to AFM will be able to use this protocol to characterize the mechanical properties of two to six soft surfaces or 3D hydrogels in a single day.

Self-assembled nanogel made of mannan: synthesis and characterization.

Amphiphilic mannan (mannan-C(16)) was synthesized by the Michael addition of hydrophobic 1-hexadecanethiol (C(16)) to hydroxyethyl methacrylated mannan (mannan-HEMA). Mannan-C(16) formed nanosized aggregates in water by self-assembly via the hydrophobic interaction among C(16) molecules as confirmed by hydrogen nuclear magnetic resonance ((1)H NMR), fluorescence spectroscopy, cryo-field emission scanning electron microscopy (cryo-FESEM), and dynamic light scattering (DLS). The mannan-C(16) critical aggregation concentration (cac), calculated by fluorescence spectroscopy with Nile red and pyrene, ranged between 0.04 and 0.02 mg/mL depending on the polymer degree of substitution of C(16) relative to methacrylated groups. Cryo-FESEM micrographs revealed that mannan-C(16) formed irregular spherical macromolecular micelles, in this work designated as nanogels, with diameters ranging between 100 and 500 nm. The influence of the polymer degree of substitution, DS(HEMA) and DS(C(16)), on the nanogel size and zeta potential was studied by DLS at different pH values and ionic strength and as a function of mannan-C(16) and urea concentrations. Under all tested conditions, the nanogel was negatively charged with a zeta potential close to zero. Mannan-C(16) with higher DS(HEMA) and DS(C(16)) values formed larger nanogels and were also less stable over a 6 month storage period and at concentrations close to the cac. When exposed to solutions of different pH and aggressive conditions of ionic strength and urea concentration, the size of mannan-C(16) varied to some extent but was always in the nanoscale range.

Three-dimensional niche stiffness synergizes with Wnt7a to modulate the extent of satellite cell symmetric self-renewal divisions.

Satellite cells (SCs), the resident adult stem cells of skeletal muscle, are required for tissue repair throughout life. While many signaling pathways are known to control SC self-renewal, less is known about the mechanisms underlying the spatiotemporal control of self-renewal during skeletal muscle repair. Here, we measured biomechanical changes that accompany skeletal muscle regeneration and determined the implications on SC fate. Using atomic force microscopy, we quantified a 2.9-fold stiffening of the SC niche at time-points associated with planar-oriented symmetric self-renewal divisions. Immunohistochemical analysis confirms increased extracellular matrix deposition within the basal lamina. To test whether three-dimensional (3D) niche stiffness can alter SC behavior or fate, we embedded isolated SC-associated muscle fibers within biochemically inert agarose gels tuned to mimic native tissue stiffness. Time-lapse microscopy revealed that a stiff 3D niche significantly increased the proportion of planar-oriented divisions, without effecting SC viability, fibronectin deposition, or fate change. We then found that 3D niche stiffness synergizes with WNT7a, a biomolecule shown to control SC symmetric self-renewal divisions via the noncanonical WNT/planar cell polarity pathway, to modify stem cell pool expansion. Our results provide new insights into the role of 3D niche biomechanics in regulating SC fate choice.

Hypoxia impacts human MSC response to substrate stiffness during chondrogenic differentiation.

Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-ß1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-ß may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167 kPa) or stiff (49.6 kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-ß3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering strategies often aim to drive progenitor cell differentiation by replicating the local environment of the native tissue, including by regulating oxygen concentration and mechanical stiffness. However, the pathways that regulate cellular responses to mechanotransduction and hypoxia may not act independently, but rather also impact one another. Here, we show that on soft, but not stiff surfaces, hypoxia impacts human MSC (hMSC) morphology and colony formation, and inhibition of cytoskeletal tension negates the hypoxia-mediated upregulation of molecular markers of chondrogenesis. These observations suggest that hypoxia's effects during hMSC chondrogenesis are modulated, at least in part, by mechanosensitive pathways, and may impact strategies to develop scaffolds for cartilage tissue engineering, as hypoxia's chondrogenic effects may be enhanced on soft materials.

Neighboring cells override 3D hydrogel matrix cues to drive human MSC quiescence.

Physical properties of modifiable hydrogels can be tuned to direct stem cell differentiation in a role akin to that played by the extracellular matrix in native stem cell niches. However, stem cells do not respond to matrix cues in isolation, but rather integrate soluble and non-soluble signals to balance quiescence, self-renewal and differentiation. Here, we encapsulated single cell suspensions of human mesenchymal stem cells (hMSC) in hyaluronic acid-based hydrogels at high and low densities to unravel the contributions of matrix- and non-matrix-mediated cues in directing stem cell response. We show that in high-density (HD) cultures, hMSC do not rely on hydrogel cues to guide their fate. Instead, they take on characteristics of quiescent cells and secrete a glycoprotein-rich pericellular matrix (PCM) in response to signaling from neighboring cells. Preventing quiescence precluded the formation of a glycoprotein-rich PCM and forced HD cultures to differentiate in response to hydrogel composition. Our observations may have important implications for tissue engineering as neighboring cells may act counter to matrix cues provided by scaffolds. Moreover, as stem cells are most regenerative if activated from a quiescent state, our results suggest that ex vivo native-like niches that incorporate signaling from neighboring cells may enable the production of clinically relevant, highly regenerative cells.

An engineered, quantifiable in vitro model for analysing the effect of proteostasis-targeting drugs on tissue physical properties.

Cellular function depends on the maintenance of protein homeostasis (proteostasis) by regulated protein degradation. Chronic dysregulation of proteostasis is associated with neurodegenerative and age-related diseases, and drugs targeting components of the protein degradation apparatus are increasingly used in cancer therapies. However, as chronic imbalances rather than loss of function mediate their pathogenesis, research models that allow for the study of the complex effects of drugs on tissue properties in proteostasis-associated diseases are almost completely lacking. Here, to determine the functional effects of impaired proteostatic fine-tuning, we applied a combination of materials science characterisation techniques to a cell-derived, in vitro model of bone-like tissue formation in which we pharmacologically perturbed protein degradation. We show that low-level inhibition of VCP/p97 and the proteasome, two major components of the degradation machinery, have remarkably different effects on the bone-like material that human bone-marrow derived mesenchymal stromal cells (hMSC) form in vitro. Specifically, whilst proteasome inhibition mildly enhances tissue formation, Raman spectroscopic, atomic force microscopy-based indentation, and electron microscopy imaging reveal that VCP/p97 inhibition induces the formation of bone-like tissue that is softer, contains less protein, appears to have more crystalline mineral, and may involve aberrant micro- and ultra-structural tissue organisation. These observations contrast with findings from conventional osteogenic assays that failed to identify any effect on mineralisation. Taken together, these data suggest that mild proteostatic impairment in hMSC alters the bone-like material they form in ways that could explain some pathologies associated with VCP/p97-related diseases. They also demonstrate the utility of quantitative materials science approaches for tackling long-standing questions in biology and medicine, and could form the basis for preclinical drug testing platforms to develop therapies for diseases stemming from perturbed proteostasis or for cancer therapies targeting protein degradation. Our findings may also have important implications for the field of tissue engineering, as the manufacture of cell-derived biomaterial scaffolds may need to consider proteostasis to effectively replicate native tissues.

Bi-directional cell-pericellular matrix interactions direct stem cell fate.

Modifiable hydrogels have revealed tremendous insight into how physical characteristics of cells' 3D environment drive stem cell lineage specification. However, in native tissues, cells do not passively receive signals from their niche. Instead they actively probe and modify their pericellular space to suit their needs, yet the dynamics of cells' reciprocal interactions with their pericellular environment when encapsulated within hydrogels remains relatively unexplored. Here, we show that human bone marrow stromal cells (hMSC) encapsulated within hyaluronic acid-based hydrogels modify their surroundings by synthesizing, secreting and arranging proteins pericellularly or by degrading the hydrogel. hMSC's interactions with this local environment have a role in regulating hMSC fate, with a secreted proteinaceous pericellular matrix associated with adipogenesis, and degradation with osteogenesis. Our observations suggest that hMSC participate in a bi-directional interplay between the properties of their 3D milieu and their own secreted pericellular matrix, and that this combination of interactions drives fate.

Author Correction: Bi-directional cell-pericellular matrix interactions direct stem cell fate.

The original version of this Article contained an error in the author affiliations. The affiliation of Marjan Enayati with 'Ludwig Boltzmann Cluster for Cardiovascular Research at the Center for Biomedical Research, Medical University of Vienna, Austria' was inadvertently omitted. This has now been corrected in both the PDF and HTML versions of the Article.

Author Correction: Bi-directional cell-pericellular matrix interactions direct stem cell fate.

In the original version of this Article the dataset identifier in the Data Availability statement was incorrect. The correct dataset identifier is PXD009500. This has been corrected in the HTML and PDF versions of this Article.

Unraveling the uptake mechanisms of mannan nanogel in bone-marrow-derived macrophages.

The mechanisms associated with the cellular internalization of nanomedicines must be carefully considered when designing drug- and vaccine-delivery systems. The cellular fate and effects of nanomedicines depend to a large extent on the cell uptake routes. A self-assembled mannan nanogel is developed as a vaccination platform for antigen and adjuvant delivery. The mannan nanogel uptake by murine bone-marrow-derived macrophages is found to be time-, concentration-, and energy-dependent, involving mannose-receptor-mediated phagocytosis and clathrin-mediated endocytosis. The nanogel is also visualized in the cytosol suggesting endolysosomal escape. These results indicate that mannan nanogel is a promising versatile carrier for intracellular delivery of vaccines or therapeutic agents.

Self-assembled mannan nanogel: cytocompatibility and cell localization.

Amphiphilic mannan, produced by the Michael addition of hydrophobic 1-hexadecanethiol to vinyl methacrylated mannan, self-assembles in aqueous medium through hydrophobic interactions among alkyl chains. Resultant nanogel is stable, spherical, polydisperse, with 50-140 nm mean hydrodynamic diameter depending on the polymer degree of substitution, and nearly neutral negative surface charge. No cytotoxicity of mannan nanogel is detected up to about 0.4 mg/mL in mouse embryo fibroblast cell line 3T3 and mouse bone marrow-derived macrophages (BMDM) using cell proliferation, lactate dehydrogenase and Live/Dead assays. Comet assay, under the tested conditions, reveals no DNA damage in fibroblasts but possible in BMDM. BMDM internalize the mannan nanogel, which is observed in vesicles in the cytoplasm by confocal laser scanning microscopy. Confocal colocalization image analysis denotes that the entrance and exit of nanogel and FM 4-64 might occur by the same processes--endocytosis and exocytosis--in BMDM. Physicochemical characteristics, in vitro cytocompatibility and uptake of self-assembled mannan nanogel by mouse BMDM are great signals of the potential applicability of this nanosystem for macrophages targeted delivery of vaccines or drugs, acting as potential nanomedicines, always with the key goal of preventing and/or treating diseases.

Synthesis and Characterization of Self-Assembled Nanogels Made of Pullulan.

Self-assembled nanogels made of hydrophobized pullulan were obtained using a versatile, simple, reproducible and low-cost method. In a first reaction pullulan was modified with hydroxyethyl methacrylate or vinyl methacrylate, further modified in the second step with hydrophobic 1-hexadecanethiol, resulting as an amphiphilic material, which self-assembles in water via the hydrophobic interaction among alkyl chains. Structural features, size, shape, surface charge and stability of the nanogels were studied using hydrogen nuclear magnetic resonance, fluorescence spectroscopy, cryo-field emission scanning electron microscopy and dynamic light scattering. Above the critical aggregation concentration spherical polydisperse macromolecular micelles revealed long-term colloidal stability in aqueous medium, with a nearly neutral negative surface charge and mean hydrodynamic diameter in the range 100-400 nm, depending on the polymer degree of substitution. Good size stability was observed when nanogels were exposed to potential destabilizing pH conditions. While the size stability of the nanogel made of pullulan with vinyl methacrylate and more hydrophobic chains grafted was affected by the ionic strength and urea, nanogel made of pullulan with hydroxyethyl methacrylate and fewer hydrophobic chains grafted remained stable.

Supramolecular assembled nanogel made of mannan.

The supramolecular assembly of amphiphilic mannan, synthesized by the Michael addition of hydrophobic 1-hexadecanethiol to vinyl methacrylated mannan, originates in aqueous medium the formation of a nanogel, stabilized by hydrophobic interactions among alkyl chains. The critical aggregation concentration, calculated by fluorescence spectroscopy ranged between 0.002 and 0.01 mg/mL, depending on the polymer degree of substitution. The cryo-field emission scanning electron microscopy showed spherical macromolecular micelles with diameters between 100 and 500 nm. The dynamic light scattering analysis revealed a polydisperse colloidal system, with mean hydrodynamic diameter between 50 and 140 nm, depending on the polymer degree of substitution. The nanogel is negatively charged, stable over a 6 months storage period, and stable at pH 3-8, salt or urea solutions. Bovine serum albumin and curcumin were spontaneously incorporated in the nanogel, being stabilized by the hydrophobic domains, opening the possibility for future applications as potential delivery systems for therapeutic molecules. In vitro assays were carried out to characterize the biocompatibility of the nanogel. A toxic effect of mannan-C(16) was observed, specific to mouse macrophage-like cell line J774, not affecting mouse embryo fibroblast cell line 3T3 viability.

A cutoff point for peak oxygen consumption in the prognosis of heart failure patients with ß-blocker therapy.

PURPOSE: Beta-blockers (BB) have shown to improve outcomes among heart failure patients (HF). Adequate risk stratification is still a major concern for HF. The prognostic indexes have been detected, but only few parameters maintain consistently high power in predicting progression of disease and mortality. Peak oxygen consumption (VO(2) peak, ml kg(-1) min(-1)) is traditionally used for risk stratification in HF, however, there is limited evidence regarding predictive value of VO(2) peak in patients taking BB. METHODS: Two hundred twenty nine patients, aged 49 ± 13 years with diagnosed HF for more than 6 months due to ischemic (n=73), idiopathic dilated (n=149) and Chagas disease (n=7) underwent a cardiopulmonary exercise test (CPX). The ejection fraction was 38 ± 10%; clinical stability was defined as no change in the NYHA class or absence of hospitalization for heart failure and stable medical treatment during 3 months prior to CPX. Subjects were tracked for cardiac-related mortality after CPX. RESULTS: The mean follow-up period was 2.5 ± 1.1 years and means value for VO(2) peak was 16.3 ± 4. Current BB therapy included carvedilol (83.4%), metoprolol (7.8%), bisoprolol (3.9%) and others (4.8%). The area under the ROC curve for VO(2) peak was 0.80 (95% CI: 0.69-0.90, optimal threshold: 12.5 and 82% sensitivity/26% specificity, p<0.001). Kaplan-Meier analysis that revealed event-free survival for subjects in < and >12.5 was 28% and 2.8%, respectively (long-rank 34.8; p<0.001). CONCLUSIONS: VO(2) peak seems to maintain prognostic value in HF patients BB therapy. The present study also provides new evidence that optimal threshold value for VO(2) peak in the BB era is 12.5 ml kg(-1) min(-1).