SARS-CoV-2 Omicron BA.1 variant breakthrough infections in nursing home residents after an homologous third dose of the Comirnaty® COVID-19 vaccine: Looking for correlates of protection.

We investigated whether peripheral blood levels of SARS-CoV-2 Spike (S) receptor binding domain antibodies (anti-RBD), neutralizing antibodies (NtAb) targeting Omicron S, and S-reactive-interferon (IFN)-γ-producing CD4+ and CD8+ T cells measured after a homologous booster dose (3D) with the Comirnaty® vaccine was associated with the likelihood of subsequent breakthrough infections due to the Omicron variant. An observational study including 146 nursing home residents (median age, 80 years; range, 66-99; 109 female) evaluated for an immunological response after 3D (at a median of 16 days). Anti-RBD total antibodies were measured by chemiluminescent immunoassay. NtAb were quantified by an Omicron S pseudotyped virus neutralization assay. SARS-CoV-2-S specific-IFNγ-producing CD4+ and CD8+ T cells were enumerated by whole-blood flow cytometry for intracellular cytokine staining. In total, 33/146 participants contracted breakthrough Omicron infection (symptomatic in 30/33) within 4 months after 3D. Anti-RBD antibody levels were comparable in infected and uninfected participants (21 123 vs. 24 723 BAU/ml; p = 0.34). Likewise, NtAb titers (reciprocal IC50 titer, 157 vs. 95; p = 0.32) and frequency of virus-reactive CD4+ (p = 0.82) and CD8+ (p = 0.91) T cells were similar across participants in both groups. anti-RBD antibody levels and NtAb titers estimated at around the time of infection were also comparable (3445 vs. 4345 BAU/ml; p = 0.59 and 188.5 vs. 88.9; p = 0.70, respectively). Having detectable NtAb against Omicron or SARS-CoV-2-S-reactive-IFNγ-producing CD4+ or CD8+ T cells after 3D was not correlated with increased protection from breakthrough infection (OR, 1.50; p = 0.54; OR, 0.0; p = 0.99 and OR 3.70; p = 0.23, respectively). None of the immune parameters evaluated herein, including NtAb titers against the Omicron variant, may reliably predict at the individual level the risk of contracting COVID-19 due to the Omicron variant in nursing home residents.

Macrophage proteomic analysis of covalent immobilization of hyaluronic acid and graphene oxide on CoCr alloy in a tribocorrosive environment.

In this work, a sequential covalent immobilization of graphene oxide (GO) and hyaluronic acid (HA) is performed to obtain a biocompatible wear-resistant nanocoating on the surface of the biomedical grade cobalt-chrome (CoCr) alloy. Nanocoated CoCr surfaces were characterized by Raman spectroscopy and electrochemical impedance spectroscopy (EIS) in 3 g/L HA electrolyte. Tribocorrosion tests of the nanocoated CoCr surfaces were carried out in a pin on flat tribometer. The biological response of covalently HA/GO biofunctionalized CoCr surfaces with and without wear-corrosion processes was studied through the analysis of the proteome of macrophages. Raman spectra revealed characteristic bands of GO and HA on the functionalized CoCr surfaces. The electrochemical response by EIS showed a stable and protective behavior over 23 days in the simulated biological environment. HA/GO covalently immobilized on CoCr alloy is able to protect the surface and reduce the wear volume released under tribocorrosion tests. Unsupervised classification analysis of the macrophage proteome via hierarchical clustering and principal component analysis (PCA) revealed that the covalent functionalization on CoCr enhances the macrophage biocompatibility in vitro. On the other hand, disruption of the HA/GO nanocoating by tribocorrosion processes induced a macrophage proteome which was differently clustered and was distantly located in the PCA space. In addition, tribocorrosion induced an increase in the percentage of upregulated and downregulated proteins in the macrophage proteome, revealing that disruption of the covalent nanocoating impacts the macrophage proteome. Although macrophage inflammation induced by tribocorrosion of HA/GO/CoCr surfaces is observed, it is ameliorated by the covalently grafting of HA, which provides immunomodulation by eliciting downregulations in characteristic pro-inflammatory signaling involved in inflammation and aseptic loosening of CoCr joint arthroplasties. Covalent HA/GO nanocoating on CoCr provides potential applications for in vivo joint prostheses led a reduced metal-induced inflammation and degradation by wear-corrosion.

Myths and beliefs about insulin therapy in patients with diabetes mellitus and their family caregivers from a hospital in northern Peru, 2020. / Mitos y creencias sobre la insulinoterapia en pacientes con diabetes mellitus y sus familiares de un hospital del norte del Perú, 2020.

OBJECTIVE.: To analyze and explore the myths and beliefs about insulin therapy in patients with diabetes mellitus and their family caregivers from a general hospital in northern Peru in 2020. MATERIALS AND METHODS.: This qualitative study used a thematic analysis model, following the interpretative paradigm. Sociodemographic and clinical data were obtained from medical records. Patients with diabetes that used some type of insulin for at least three months prior to the study were interviewed, as well as their family caregivers. Patients participated in a focus group and in-depth interviews; family caregivers participated only in in-depth interviews. RESULTS.: Twelve patients with diabetes (11 with type 2 diabetes mellitus) were included; six in the focus group and six in the in-depth interviews. Seven family caregivers were included. After analysis, we obtained four categories: 1) beliefs related to starting insulin treatment: treatment of choice after failure of other drugs, cures diabetes, regulates sugar, fear of injectables; 2) beliefs related to treatment adherence: decompensation for not using insulin, insulin is necessary to live; 3) beliefs related to alternative therapies and cost: use of alternative therapies, high cost of insulin; and 4) myths related to the use of insulin: generates dependence, dependence for insulin administration, negative effects of insulin. CONCLUSIONS.: The beliefs and myths of patients treated with insulin arise from the beginning of treatment, remain throughout the course of treatment, and are often reinforced by the worldview of family members.

OBJETIVO.: Analizar y explorar los mitos y creencias sobre la insulinoterapia en pacientes con diabetes mellitus y sus familiares cuidadores de un hospital general del norte peruano en el 2020. MATERIALES Y MÉTODOS.: Se realizo un estudio con enfoque cualitativo, paradigma interpretativo y tipo de análisis temático. Se obtuvieron datos sociodemográficos y clínicos de las historias clínicas y se entrevistaron pacientes con diabetes, con uso de algún tipo de insulina por lo menos tres meses antes del estudio, y a sus familiares cuidadores. Los pacientes participaron de un grupo focal y de entrevistas a profundidad; los familiares participaron solo en entrevistas a profundidad. RESULTADOS.: Participaron 12 pacientes con diabetes (11 con diabetes mellitus tipo 2); seis en el grupo focal y seis en las entrevistas a profundidad y siete familiares. Luego del análisis se obtuvieron cuatro categorías: 1) creencias relacionadas al inicio de tratamiento con insulina: tratamiento de elección después del fracaso con otros fármacos, cura la diabetes, regula el azúcar, temor a los inyectables; 2) creencias relacionadas al mantenimiento del tratamiento: descompensación por no usar insulina, la insulina es necesaria para vivir; 3) creencias relacionadas a terapias alternativas y costo: uso de terapias alternativas, costo elevado de la insulina; y 4) mitos relacionados al uso de insulina: genera dependencia, dependencia para la administración de insulina, efectos negativos de la insulina. CONCLUSIONES.: Las creencias y mitos de los pacientes, en tratamiento con insulina, emergen desde el inicio del tratamiento y se mantienen con la evolución de este, siendo en muchas ocasiones reforzados por la cosmovisión de los familiares.

Metastable FeMg particles for controlling degradation rate, mechanical properties, and biocompatibility of Poly(l-lactic) acid (PLLA) for orthopedic applications.

Poly(l-lactic) acid (PLLA) is commonly used in bioabsorbable medical implants, but it suffers from slow degradation rate and rapid decline in mechanical properties for orthopedic applications. To address this drawback, recent research has explored the use of Mg as a filler for PLLA, resulting in composites with improved degradation rate and cytocompatibility compared to neat PLLA. In this study, FeMg powder particles were proposed as fillers for PLLA to investigate the potential of PLLA/FeMg composites for bioabsorbable implants. Cylinder specimens of PLLA, PLLA/Fe, PLLA/Mg and PLLA/FeMg were prepared using solvent casting followed by thermo-molding. The microstructure, thermal behavior, in vitro degradation behavior in simulated body fluid, mechanical properties and cytocompatibility of these composites were examined. The results indicate that the presence of FeMg particles prevents the deterioration of the composite mechanical properties, at least up to 14 days. Once a certain amount of degradation of the composite is reached, the degradation is faster than that of PLLA. Direct cytotoxicity assays revealed that pre-osteoblast MC3T3-E1 cells successfully adhered to and proliferated on the PLLA/FeMg surface. The inclusion of a low percentage of Mg into the Fe lattice not only accelerated the degradation rate of Fe but also improved its cytocompatibility. The enhanced degradation rate, mechanical properties, and osteoconductive properties of this composite make it a promising option for temporary orthopedic biomedical devices.

Wettability, Corrosion Resistance, and Osteoblast Response to Reduced Graphene Oxide on CoCr Functionalized with Hyaluronic Acid.

The durability of metal-metal prostheses depends on achieving a higher degree of lubrication. The beneficial effect of hyaluronic acid (HA) on the friction and wear of both natural and artificial joints has been reported. For this purpose, graphene oxide layers have been electrochemically reduced on CoCr surfaces (CoCrErGO) and subsequently functionalized with HA (CoCrErGOHA). These layers have been evaluated from the point of view of wettability and corrosion resistance in a physiological medium containing HA. The wettability was analyzed by contact angle measurements in phosphate buffer saline-hyaluronic acid (PBS-HA) solution. The corrosion behavior of functionalized CoCr surfaces was studied with electrochemical measurements. Biocompatibility, cytotoxicity, and expression of proteins related to wound healing and repair were studied in osteoblast-like MC3T3-E1 cell cultures. All of the reported results suggest that HA-functionalized CoCr surfaces, through ErGO layers in HA-containing media, exhibit higher hydrophilicity and better corrosion resistance. Related to this increase in wettability was the increase in the expressions of vimentin and ICAM-1, which favored the growth and adhesion of osteoblasts. Therefore, it is a promising material for consideration in trauma applications, with improved properties in terms of wettability for promoting the adhesion and growth of osteoblasts, which is desirable in implanted materials used for bone repair.

Gentisic acid, a compound associated with plant defense and a metabolite of aspirin, heads a new class of in vivo fibroblast growth factor inhibitors.

Fibroblast growth factors are key proteins in many intercellular signaling networks. They normally remain attached to the extracellular matrix, which confers on them a considerable stability. The unrestrained accumulation of fibroblast growth factors in the extracellular milieu, either due to uncontrolled synthesis or enzymatic release, contributes to the pathology of many diseases. Consequently, the neutralization of improperly mobilized fibroblast growth factors is of clear therapeutic interest. In pursuing described rules to identify potential inhibitors of these proteins, gentisic acid, a plant pest-controlling compound, an aspirin and vegetarian diet common catabolite, and a component of many traditional liquors and herbal remedies, was singled out as a powerful inhibitor of fibroblast growth factors. Gentisic acid was used as a lead to identify additional compounds with better inhibitory characteristics generating a new chemical class of fibroblast growth factor inhibitors that includes the agent responsible for alkaptonuria. Through low and high resolution approaches, using representative members of the fibroblast growth factor family and their cell receptors, it was shown that this class of inhibitors may employ two different mechanisms to interfere with the assembly of the signaling complexes that trigger fibroblast growth factor-driven mitogenesis. In addition, we obtained evidence from in vivo disease models that this group of inhibitors may be of interest to treat cancer and angiogenesis-dependent diseases.

Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: a bona fide tracer.

Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF-Cys2 mutant was recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group's side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF-Cys2-heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF-Cys2-heparin complex might represent a biologically relevant complex in physiological media.

Immobilization and bioactivity evaluation of FGF-1 and FGF-2 on powdered silicon-doped hydroxyapatite and their scaffolds for bone tissue engineering.

Fibroblast growth factors (FGFs) are polypeptides that control the proliferation and differentiation of various cell types including osteoblasts. FGFs are also strong inducers of angiogenesis, necessary to obtain oxygen and nutrients during tissue repair. With the aim to incorporate these desirable FGF biological properties into bioceramics for bone repair, silicon substituted hydroxyapatites (Si-HA) were used as materials to immobilize bioactive FGF-1 and FGF-2. Thus, the binding of these growth factors to powdered Si-HA and Si-HA scaffolds was carried out efficiently in the present study and both FGFs maintained its biological activity on osteoblasts after its immobilization. The improvement of cell adhesion and proliferation onto Si-HA scaffolds suggests the potential utility of these FGF/scaffolds for bone tissue engineering.

Antimitogenic polymer drugs based on AMPS: monomer distribution-bioactivity relationship of water-soluble macromolecules.

A number of polysulfonated molecules have demonstrated their interaction with fibroblast growth factor (FGF), hampering their binding to its receptors (low affinity heparan sulfate proteoglycans (HSPG) and high affinity tyrosine kinase FGF receptors) and inhibiting the intracellular signaling and mitogenic response in cultured endothelial cells. The aim of this work was the synthesis and characterization of new copolymers based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) with antiproliferative activity for antitumoral applications. N-Vinylpyrrolidone (VP) or butyl acrylate (BA) was copolymerized with the sulfonated monomer to obtain macromolecules with different hydrophilic/hydrophobic balance and distribution of the sulfonated groups within the macromolecules. In vitro cell culture proliferative assays showed that monomer distribution affected the inhibition of the proliferative action of FGF. Reactivity ratios of the systems were determined following the free radical copolymerization by in situ (1)H NMR, and the correlation of the monomer sequence distribution with the bioactivity is discussed.

The Role of Heart Rate on the Associations Between Body Composition and Heart Rate Variability in Children With Overweight/Obesity: The ActiveBrains Project.

BACKGROUND: Heart rate variability (HRV) is negatively associated with body mass index and adiposity in several populations. However, less information is available about this association in children with overweight and obesity, especially severe/morbid obesity, taking into consideration the dependence of HRV on heart rate (HR). OBJECTIVES: (1) to examine associations between body composition measures and HRV, (2) to study differences in HRV between children with overweight and severe/morbid obesity; and (3) to test whether relationships and differences tested in objectives 1 and 2, respectively are explained by the dependency of HRV on HR. METHODS: A total of 107 children with overweight/obesity (58% boys, 10.03 ± 1.13 years) participated in this study. Body composition measures were evaluated by Dual-energy X-ray absorptiometry (DXA). HRV parameters were measured with Polar RS800CX®. RESULTS: Body composition measures were negatively associated with HRV indicators of parasympathetic activity (ß values ranging from -0.207 to -0.307, all p < 0.05). Children with severe/morbid obesity presented lower HRV values with respect to children with overweight/mild obesity in HRV parameters indicators of parasympathetic activity (p = 0.035). All associations disappeared after further adjustment for HR (all p > 0.05). CONCLUSION: All associations between adiposity/obesity and HRV could be explained by HR, suggesting a key confounding role of HR in HRV studies in children with weight disturbances.

Mitos y creencias sobre la insulinoterapia en pacientes con diabetes mellitus y sus familiares de un hospital del norte del Perú, 2020 / Myths and beliefs about insulin therapy in patients with diabetes mellitus and their family caregivers from a hospital in northern Peru, 2020

Objetivo. Analizar y explorar los mitos y creencias sobre la insulinoterapia en pacientes con diabetes mellitus y sus familiares cuidadores de un hospital general del norte peruano en el 2020. Materiales y métodos. Se realizo un estudio con enfoque cualitativo, paradigma interpretativo y tipo de análisis temático. Se obtuvieron datos sociodemográficos y clínicos de las historias clínicas y se entrevistaron pacientes con diabetes, con uso de algún tipo de insulina por lo menos tres meses antes del estudio, y a sus familiares cuidadores. Los pacientes participaron de un grupo focal y de entrevistas a profundidad; los familiares participaron solo en entrevistas a profundidad. Resultados. Participaron 12 pacientes con diabetes (11 con diabetes mellitus tipo 2); seis en el grupo focal y seis en las entrevistas a profundidad y siete familiares. Luego del análisis se obtuvieron cuatro categorías: 1) creencias relacionadas al inicio de tratamiento con insulina: tratamiento de elección después del fracaso con otros fármacos, cura la diabetes, regula el azúcar, temor a los inyectables; 2) creencias relacionadas al mantenimiento del tratamiento: descompensación por no usar insulina, la insulina es necesaria para vivir; 3) creencias relacionadas a terapias alternativas y costo: uso de terapias alternativas, costo elevado de la insulina; y 4) mitos relacionados al uso de insulina: genera dependencia, dependencia para la administración de insulina, efectos negativos de la insulina. Conclusiones. Las creencias y mitos de los pacientes, en tratamiento con insulina, emergen desde el inicio del tratamiento y se mantienen con la evolución de este, siendo en muchas ocasiones reforzados por la cosmovisión de los familiares.

Objective. To analyze and explore the myths and beliefs about insulin therapy in patients with diabetes mellitus and their family caregivers from a general hospital in northern Peru in 2020. Materials and methods. This qualitative study used a thematic analysis model, following the interpretative paradigm. Sociodemographic and clinical data were obtained from medical records. Patients with diabetes that used some type of insulin for at least three months prior to the study were interviewed, as well as their family caregivers. Patients participated in a focus group and in-depth interviews; family caregivers participated only in in-depth interviews. Results. Twelve patients with diabetes (11 with type 2 diabetes mellitus) were included; six in the focus group and six in the in-depth interviews. Seven family caregivers were included. After analysis, we obtained four categories: 1) beliefs related to starting insulin treatment: treatment of choice after failure of other drugs, cures diabetes, regulates sugar, fear of injectables; 2) beliefs related to treatment adherence: decompensation for not using insulin, insulin is necessary to live; 3) beliefs related to alternative therapies and cost: use of alternative therapies, high cost of insulin; and 4) myths related to the use of insulin: generates dependence, dependence for insulin administration, negative effects of insulin. Conclusions. The beliefs and myths of patients treated with insulin arise from the beginning of treatment, remain throughout the course of treatment, and are often reinforced by the worldview of family members.

Macrophage Biocompatibility of CoCr Wear Particles Produced under Polarization in Hyaluronic Acid Aqueous Solution.

Macrophages are the main cells involved in inflammatory processes and in the primary response to debris derived from wear of implanted CoCr alloys. The biocompatibility of wear particles from a high carbon CoCr alloy produced under polarization in hyaluronic acid (HA) aqueous solution was evaluated in J774A.1 mouse macrophages cultures. Polarization was applied to mimic the electrical interactions observed in living tissues. Wear tests were performed in a pin-on-disk tribometer integrating an electrochemical cell in phosphate buffer solution (PBS) and in PBS supplemented with 3 g/L HA, an average concentration that is generally found in synovial fluid, used as lubricant solution. Wear particles produced in 3 g/L HA solution showed a higher biocompatibility in J774A.1 macrophages in comparison to those elicited by particles obtained in PBS. A considerable enhancement in macrophages biocompatibility in the presence of 3 g/L of HA was further observed by the application of polarization at potentials having current densities typical of injured tissues suggesting that polarization produces an effect on the surface of the metallic material that leads to the production of wear particles that seem to be macrophage-biocompatible and less cytotoxic. The results showed the convenience of considering the influence of the electric interactions in the chemical composition of debris detached from metallic surfaces under wear corrosion to get a better understanding of the biological effects caused by the wear products.

Solution NMR structure of a human FGF-1 monomer, activated by a hexasaccharide heparin-analogue.

The 3D structure of a complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed synthetic heparin hexasaccharide has been determined by NMR spectroscopy. This hexasaccharide can substitute natural heparins in FGF-1 mitogenesis assays, in spite of not inducing any apparent dimerization of the growth factor. The use of this well defined synthetic heparin analogue has allowed us to perform a detailed NMR structural analysis of the heparin-FGF interaction, overcoming the limitations of NMR to deal with the high molecular mass and heterogeneity of the FGF-1 oligomers formed in the presence of natural heparin fragments. Our results confirm that glycosaminoglycans induced FGF-1 dimerization either in a cis or trans disposition with respect to the heparin chain is not an absolute requirement for biological activity.

An exercise-based randomized controlled trial on brain, cognition, physical health and mental health in overweight/obese children (ActiveBrains project): Rationale, design and methods.

The new and recent advances in neuroelectric and neuroimaging technologies provide a new era for further exploring and understanding how brain and cognition function can be stimulated by environmental factors, such as exercise, and particularly to study whether physical exercise influences brain development in early ages. The present study, namely the ActiveBrains project, aims to examine the effects of a physical exercise programme on brain and cognition, as well as on selected physical and mental health outcomes in overweight/obese children. A total of 100 participants aged 8 to 11 years are randomized into an exercise group (N=50) or a control group (N=50). The intervention lasts 20-weeks, with 3-5 sessions per week of 90 min each, and is mainly focused on high-intensity aerobic exercise yet also includes muscle-strengthening exercises. The extent to what the intervention effect remains 8-months after the exercise programme finishes is also studied in a subsample. Brain structure and function and cognitive performance are assessed using structural and functional magnetic resonance imaging and electroencephalographic recordings. Secondary outcomes include physical health outcomes (e.g. physical fitness, body fatness, bone mass and lipid-metabolic factors) and mental health outcomes (e.g. chronic stress indicators and overall behavioural and personality measurements such as anxiety or depression). This project will substantially contribute to the existing knowledge and will have an impact on societies, since early stimulation of brain development might have long lasting consequences on cognitive performance, academic achievement and in the prevention of behavioural problems and the promotion of psychological adjustment and mental health. Clinical trials. Gov identifier: NCT02295072.

Activating brown adipose tissue through exercise (ACTIBATE) in young adults: Rationale, design and methodology.

AIMS: The energy expenditure capacity of brown adipose tissue (BAT) makes it an attractive target as a therapy against obesity and type 2 diabetes. BAT activators namely catecholamines, natriuretic peptides and certain myokines, are secreted in response to exercise. ACTIBATE will determine the effect of exercise on BAT activity and mass measured by positron emission tomography/computed tomography (PET/CT, primary outcome) in young adults. ACTIBATE will also investigate the physiological consequences of activating BAT (secondary outcomes). METHODS: ACTIBATE will recruit 150 sedentary, healthy, young adults (50% women) aged 18-25 years. Eligible participants will be randomly assigned to a non-exercise group (n ≈ 50) or one of two exercise groups (n=50 each). Participants in the exercise groups will perform aerobic and strength training 3-4 days/week at a heart rate equivalent to 60% of heart rate reserve (HRres), and at 50% of 1 repetition maximum (RM) for the moderate-intensity group, and at 80% of HRres and 70% RM for the vigorous-intensity group. Laboratory measures completed at baseline and 6 months include BAT activity and mass, resting energy expenditure, meal and cold-induced thermogenesis, body temperature regulation and shivering threshold, body composition and cardiovascular disease risk factors. We will also obtain biopsies from abdominal subcutaneous white adipose tissue and skeletal muscle to analyse the expression of genes encoding proteins involved in the thermogenic machinery. DISCUSSION: Findings from ACTIBATE will have significant implications for our understanding of exercise and its protective effects against the development of type 2 diabetes, obesity and related metabolic diseases. ClinicalTrials.gov ID: NCT02365129.

Response of MC3T3-E1 osteoblasts, L929 fibroblasts, and J774 macrophages to fluoride surface-modified AZ31 magnesium alloy.

The present work evaluates the biocompatibility of a fluoride surface-modified AZ31 magnesium alloy (AZ31HF) with different cell lines that coexist in the implant environment to test its potential use as a biodegradable and absorbable biomaterial for bone repair. A clear stimulation of cell proliferation and an enhancement of the mitochondrial respiratory activity were observed when mouse osteoblasts (MC3T3-E1), fibroblasts (L929), and macrophages (J774) cell lines were cultured with the modified alloy. No significant change in apoptosis or viability rates was observed when osteoblasts and fibroblasts cultures were grown in the presence of this alloy. A proteomic analysis of the MC3T3-E1 cell extracts cultured in the presence of AZ31HF showed an overexpression of proteins related with the mineralization process, which is a necessary step for bone repair. An increase in the lactate dehydrogenase activity was observed in the MC3T3-E1 and J774 cell cultures that could be a response of the oxidative stress produced by the presence of the material. This stress could be related to the increase observed in the respiratory mitochondrial activity or respiratory burst measured in theses cultures that indicate damage in the cell membranes and subsequently some cell death. Results reported here, for and against AZ31HF, should be taken into account when considering the potential use of this modified alloy in bone repair applications.

Signaling pathways of immobilized FGF-2 on silicon-substituted hydroxyapatite.

Therapeutic strategies for bone regeneration involve the selection of suitable biomaterials, growth factors, and cell types to mimic the cellular microenvironment where molecular and mechanical signals control the reconstruction of bone tissue. The immobilization of basic fibroblast growth factor (FGF-2) on powdered silicon-substituted hydroxyapatite (Si-HA) allows to prepare a biofunctional biomaterial able to interact with bone cells in a very specific way. The biological activity of FGF-2/Si-HA, evaluated in Saos-2 osteoblasts and MC3T3-E1 preosteoblasts through the PLCγ and MAPK/ERK signal transduction pathways, shows that FGF-2 immobilized on Si-HA provides the right signals to cells stimulating crucial intracellular mechanisms of osteoblast proliferation and differentiation.

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells.

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.

Dobesilate diminishes activation of the mitogen-activated protein kinase ERK1/2 in glioma cells.

Fibroblast growth factors (FGFs) and their receptors, regularly expressed at high levels in gliomas, are further upregulated during the transition of the tumor from low- to high-grade malignancy, and are essential for glioma progression. FGFs induce upregulation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured glioma cells, which suggests that MAPK pathway participates in the FGF-dependent glioma development. Recently, it has been shown that dobesilate, an inhibitor of FGF mitogenic activity, shows antiproliferative and proapoptotic activities in glioma cell cultures. Accordingly, it should be expected this new synthetic FGF inhibitor to affect the activation levels of MAPK. Here we report that immunocytochemical and Western blot data unequivocally show that treatment of cell cultures with dobesilate causes a significant decrease of the intracellular levels of ERK1/2 activation, one of the components of the MAPK signalling cascade. This finding supports an important role for dobesilate in glioma growth, suggesting that dobesilate should be a treatment to be born in mind for glioma management.

Backbone dynamics of a biologically active human FGF-1 monomer, complexed to a hexasaccharide heparin-analogue, by 15N NMR relaxation methods.

The binding site and backbone dynamics of a bioactive complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed heparin hexasaccharide has been investigated by HSQC and relaxation NMR methods. The comparison of the relaxation data for the free and bound states has allowed showing that the complex is monomeric, and still induces mutagenesis, and that the protein backbone presents reduced motion in different timescale in its bound state, except in certain points that are involved in the interaction with the fibroblast growth factor receptor (FGFR).