Monitoring of Leptospira in captive turtles by DNA analysis / [Monitoramento de Leptospira em quelônios mantidos em cativeiro pela análise de DNA]

Existem poucos estudos sobre doenças infecciosas em animais silvestres. O objetivo deste estudo foi pesquisar DNA de Leptospira spp. em sangue de tartarugas mantidas em cativeiro, pertencentes ao Bosque Rodrigues Alves (Jardim Zoobotânico da Amazônia). O DNA foi isolado das amostras de sangue coletadas de 148 tartarugas pertencentes a seis espécies diferentes. A reação em cadeia da polimerase (PCR) foi realizada utilizando-se iniciadores específicos para DNA de Leptospira spp. Nenhuma das amostras apresentou resultado positivo para Leptospira spp.(AU)

Magnetic responsive materials modulate the inflammatory profile of IL-1ß conditioned tendon cells.

Tendinopathies represent half of all musculoskeletal injuries worldwide. Inflammatory events contribute to both tendon healing and to tendinopathy conditions but the cellular triggers leading to one or the other are unknown. In previous studies, we showed that magnetic field actuation modulates human tendon cells (hTDCs) behavior in pro-inflammatory environments, and that magnetic responsive membranes could positively influence inflammation responses in a rat ectopic model. Herein, we propose to investigate the potential synergistic action of the magnetic responsive membranes, made of a polymer blend of starch with polycaprolactone incorporating magnetic nanoparticles (magSPCL), and the actuation of pulsed electromagnetic field (PEMF): 5 Hz, 4mT of intensity and 50% of duty cycle, in IL-1ß-treated-hTDCs, and in the immunomodulatory response of macrophages. It was found that the expression of pro-inflammatory (TNFα, IL-6, IL-8, COX-2) and ECM remodeling (MMP-1,-2,-3) markers tend to decrease in cells cultured onto magSPCL membranes under PEMF, while the expression of TIMP-1 and anti-inflammatory genes (IL-4, IL-10) increases. Also, CD16++ and CD206+ macrophages were only found on magSPCL membranes with PEMF application. Magnetic responsive membranes show a modulatory effect on the inflammatory profile of hTDCs favoring anti-inflammatory cues which is also supported by the anti-inflammatory/repair markers expressed in macrophages. These results suggest that magnetic responsive magSPCL membranes can contribute for inflammation resolution acting on both resident cell populations and inflammatory cells, and thus significantly contribute to tendon regenerative strategies. Statement of significance Magnetically-assisted strategies have received great attention in recent years to remotely trigger and guide cell responses. Inflammation plays a key role in tendon healing but persistent pro-inflammatory molecules can contribute to tendon disorders, and therefore provide a therapeutic target for advanced treatments. We have previously reported that magnetic fields modulate the response of human tendon cells (hTDCs) conditioned to pro-inflammatory environments (IL-1ß-treated-hTDCs), and that magnetic responsive membranes positively influence immune responses. In the present work, we combined pulsed electromagnetic field (PEMF) and magnetic responsive membranes to guide the inflammatory profile of IL-1ß-treated-hTDCs and of macrophages. The results showed that the synergistic action of PEMF and magnetic membranes supports the applicability of magnetically actuated systems to regulate inflammatory events and stimulate tendon regeneration.

Exploring Stem Cells and Inflammation in Tendon Repair and Regeneration.

Tendon injuries are frequent and are responsible for substantial morbidity both in sports and in the workplace. Despite the endogenous mechanisms of tendon repair and regeneration, tendon healing upon injury is slow and often insufficient to restore complete biomechanics functionality.Inflammation has a pivotal role in tendon healing and failed healing responses contribute to the progression of tendinopathies. However, the molecular and cellular mechanisms involved are poorly understood requiring further insights.During inflammation, bioactive molecules such as cytokines secreted locally at the injury site, influence resident stem cells that contribute as modulatory agents over the niche towards homeostasis, holding great promise as therapeutic agents for tendon pathological conditions associated to unresolved inflammation and failed healing.This review overviews the role of cytokines and resident cells, focusing on the participation of tendon stem cell population in inflammation and tendon healing upon injury and their potential action in resolution of pathological conditions.

Triggering the activation of Activin A type II receptor in human adipose stem cells towards tenogenic commitment using mechanomagnetic stimulation.

Stem cell therapies hold potential to stimulate tendon regeneration and homeostasis, which is maintained in response to the native mechanical environment. Activins are members of the mechano-responsive TGF-ß superfamily that participates in the regulation of several downstream biological processes. Mechanosensitive membrane receptors such as activin can be activated in different types of stem cells via magnetic nanoparticles (MNPs) through remote magnetic actuation resulting in cell differentiation. In this work, we target the Activin receptor type IIA (ActRIIA) in human adipose stem cells (hASCs), using anti-ActRIIA functionalized MNPs, externally activated through a oscillating magnetic bioreactor. Upon activation, the phosphorylation of Smad2/3 is induced allowing translocation of the complex to the nucleus, regulating tenogenic transcriptional responses. Our study demonstrates the potential remote activation of MNPs tagged hASCs to trigger the Activin receptor leading to tenogenic differentiation. These results may provide insights toward tendon regeneration therapies.

Human adipose tissue-derived tenomodulin positive subpopulation of stem cells: A promising source of tendon progenitor cells.

Cell-based therapies are of particular interest for tendon and ligament regeneration given the low regenerative potential of these tissues. Adipose tissue is an abundant source of stem cells, which may be employed for the healing of tendon lesions. However, human adult multipotent adipose-derived stem cells (hASCs) isolated from the stromal vascular fraction of adipose tissue originate highly heterogeneous cell populations that hinder their use in specific tissue-oriented applications. In this study, distinct subpopulations of hASCs were immunomagnetic separated and their tenogenic differentiation capacity evaluated in the presence of several growth factors (GFs), namely endothelial GF, basic-fibroblast GF, transforming GF-ß1 and platelet-derived GF-BB, which are well-known regulators of tendon development, growth and healing. Among the screened hASCs subpopulations, tenomodulin-positive cells were shown to be more promising for tenogenic applications and therefore this subpopulation was further studied, assessing tendon-related markers (scleraxis, tenomodulin, tenascin C and decorin) both at gene and protein level. Additionally, the ability for depositing collagen type I and III forming extracellular matrix structures were weekly assessed up to 28 days. The results obtained indicated that tenomodulin-positive cells exhibit phenotypical features of tendon progenitor cells and can be biochemically induced towards tenogenic lineage, demonstrating that this subset of hASCs can provide a reliable source of progenitor cells for therapies targeting tendon regeneration.

Potential risk assessment in stream sediments, soils and waters after remediation in an abandoned W>Sn mine (NE Portugal).

The mining complex of Murçós belongs to the Terras de Cavaleiros Geopark, located in Trás-os-Montes region, northeast Portugal. A stockwork of NW-SE-trending W>Sn quartz veins intruded Silurian metamorphic rocks and a Variscan biotite granite. The mineralized veins contain mainly quartz, cassiterite, wolframite, scheelite, arsenopyrite, pyrite, sphalerite, chalcopyrite, galena, rare pyrrhotite, stannite, native bismuth and also later bismuthinite, matildite, joseite, roosveltite, anglesite, scorodite, zavaritskite and covellite. The exploitation produced 335t of a concentrate with 70% of W and 150t of another concentrate with 70% of Sn between 1948 and 1976. The exploitation took place mainly in four open pit mines as well as underground. Three lakes were left in the area. Remediation processes of confination and control of tailings and rejected materials and phytoremediation with macrophytes from three lakes were carried out between 2005 and 2007. Stream sediments, soils and water samples were collected in 2008 and 2009, after the remediation process. Most stream sediments showed deficiency or minimum enrichment for metals. The sequential enrichment factor in stream sediments W>Bi>As>U>Cd>Sn=Ag>Cu>Sb>Pb>Be>Zn is mainly associated with the W>Sn mineralizations. Stream sediments receiving drainage of a mine dump were found to be significantly to extremely enriched with W, while stream sediments and soils were found to be contaminated with As. Two soil samples collected around mine dumps and an open pit lake were also found to be contaminated with U. The waters from the Murçós W>Sn mine area were acidic to neutral. After the remediation, the surface waters were contaminated with F(-), Al, As, Mn and Ni and must not be used for human consumption, while open pit lake waters must also not be used for agriculture because of contamination with F(-), Al, Mn and Ni. In most waters, the As occurred as As (III), which is toxic and is easily mobilized in the drainage system. The remediation promoted a decrease in metals and As concentrations of soils and waters, however the applied processes were not enough to rehabilitate the area.

Indoor radon risk associated to post-tectonic biotite granites from Vila Pouca de Aguiar pluton, northern Portugal.

At Vila Pouca de Aguiar area, northern Portugal, crops out a post-tectonic Variscan granite pluton, related with the Régua-Vila Real-Verín fault zone, comprising three types of biotite granites. Among these granites, PSG granite yield the highest average contents of U, probably due to its enrichment in accessory U-bearing minerals such as zircon. In the proximity of faults and joints, these granites are often affected by different degrees of hydrothermal alteration, forming reddish altered rocks, commonly known as "episyenites". These altered rocks are probably associated to the occurrence of hydrothermal processes, which led to uranium enrichment in the most advanced stages of episyenitization. In these granites, both average gamma absorbed dose rates in outdoor and indoor air are higher than those of the world average. Furthermore, even in the worst usage scenario, all these granites can be used as a building material, since their annual effective doses are similar to the limit defined by the European Commission. The geometric mean of radon activity of 91 dwellings located at the Vila Pouca de Aguiar pluton is 568Bqm(-3), exceeding that of other northern Portuguese granites. Measurements carried out during a winter season, indicate that 62.6% of the analysed dwellings yield higher indoor radon average values than the Portuguese legislation limit (400Bqm(-3)), and annual effective doses due higher than the world's average value (1.2mSvy(-1)). The interaction of geogenic, architectural and anthropogenic features is crucial to explain the variance in the geometric mean of radon activity of dwellings from Vila Pouca de Aguiar pluton, but the role of geologic faults is probably the most important decisive factor to increase the indoor radon concentration in dwellings. Hence, the development of awareness campaigns in order to inform population about the incurred radiological risks to radon exposure are highly recommended for this specific area.

Safety and long-term effects of renal denervation: Rationale and design of the Dutch registry.

BACKGROUND: Percutaneous renal denervation (RDN) has recently been introduced as a treatment for therapy-resistant hypertension. Also, it has been suggested that RDN may be beneficial for other conditions characterised by increased sympathetic nerve activity. There are still many uncertainties with regard to efficacy, safety, predictors for success and long-term effects. To answer these important questions, we initiated a Dutch RDN registry aiming to collect data from all RDN procedures performed in the Netherlands. METHODS: The Dutch RDN registry is an ongoing investigator-initiated, prospective, multicentre cohort study. Twenty-six Dutch hospitals agreed to participate in this registry. All patients who undergo RDN, regardless of the clinical indication or device that is used, will be included. Data are currently being collected on eligibility and screening, treatment and follow-up. RESULTS: Procedures have been performed since August 2010. At present, data from 306 patients have been entered into the database. The main indication for RDN was hypertension (n = 302, 99%). Patients had a mean office blood pressure of 177/100 (±29/16) mmHg with a median use of three (range 0-8) blood pressure lowering drugs. Mean 24-hour blood pressure before RDN was 157/93 (±18/13) mmHg. RDN was performed with different devices, with the Simplicity™ catheter currently used most frequently. CONCLUSION: Here we report on the rationale and design of the Dutch RDN registry. Enrolment in this investigator-initiated study is ongoing. We present baseline characteristics of the first 306 participants.

Ticagrelor Does Not Inhibit Adenosine Transport at Relevant Concentrations: A Randomized Cross-Over Study in Healthy Subjects In Vivo.

BACKGROUND AND PURPOSE: In patients with myocardial infarction, ticagrelor reduces cardiovascular and sepsis-related mortality, and can cause dyspnea. It is suggested that this is caused by adenosine receptor stimulation, because in preclinical studies, ticagrelor blocks the nucleoside transporter and increases cellular ATP release. We now investigated the effects of ticagrelor on the adenosine system in humans in vivo. EXPERIMENTAL APPROACH: In a double-blinded, placebo-controlled cross-over trial in 14 healthy subjects, we have tested whether ticagrelor (180 mg) affects adenosine- and dipyridamole-induced forearm vasodilation, as surrogates of nucleoside uptake inhibition and adenosine formation, respectively. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was measured. Primary endpoint was adenosine-induced vasodilation. KEY RESULTS: Ticagrelor did not affect adenosine- or dipyridamole-induced forearm vasodilation. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was not affected by ticagrelor. In vitro, ticagrelor dose-dependently inhibited nucleoside uptake, but only at supra-physiological concentrations. CONCLUSION AND IMPLICATIONS: In conclusion, at relevant plasma concentration, ticagrelor does not affect adenosine transport, nor adenosine formation in healthy subjects. Therefore, it is unlikely that this mechanism is a relevant pleiotropic effect of ticagrelor. TRIAL REGISTRATION: ClinicalTrials.gov NCT01996735.

Biological evaluation of intervertebral disc cells in different formulations of gellan gum-based hydrogels.

Gellan gum (GG)-based hydrogels are advantageous in tissue engineering not only due to their ability to retain large quantities of water and provide a similar environment to that of natural extracellular matrix (ECM), but also because they can gelify in situ in seconds. Their mechanical properties can be fine-tuned to mimic natural tissues such as the nucleus pulposus (NP). This study produced different formulations of GG hydrogels by mixing varying amounts of methacrylated (GG-MA) and high-acyl gellan gums (HA-GG) for applications as acellular and cellular NP substitutes. The hydrogels were physicochemically characterized by dynamic mechanical analysis. Degradation and swelling abilities were assessed by soaking in a phosphate buffered saline solution for up to 170 h. Results showed that as HA-GG content increased, the modulus of the hydrogels decreased. Moreover, increases in HA-GG content induced greater weight loss in the GG-MA/HA-GG formulation compared to GG-MA hydrogel. Potential cytotoxicity of the hydrogel was assessed by culturing rabbit NP cells up to 7 days. An MTS assay was performed by seeding rabbit NP cells onto the surface of 3D hydrogel disc formulations. Viability of rabbit NP cells encapsulated within the different hydrogel formulations was also evaluated by Calcein-AM and ATP assays. Results showed that tunable GG-MA/HA-GG hydrogels were non-cytotoxic and supported viability of rabbit NP cells.

An investigation of the potential application of chitosan/aloe-based membranes for regenerative medicine.

A significant number of therapeutics derived from natural polymers and plants have been developed to replace or to be used in conjunction with existing dressing products. The use of the therapeutic properties of aloe vera could be very useful in the creation of active wound dressing materials. The present work was undertaken to examine issues concerning structural features, topography, enzymatic degradation behavior, antibacterial activity and cellular response of chitosan/aloe vera-based membranes. The chitosan/aloe vera-based membranes that were developed displayed satisfactory degradation, roughness, wettability and mechanical properties. A higher antibacterial potency was displayed by the blended membranes. Moreover, in vitro assays demonstrated that these blended membranes have good cell compatibility with primary human dermal fibroblasts. The chitosan/aloe vera-based membranes might be promising wound dressing materials.

Bioactive starch-based scaffolds and human adipose stem cells are a good combination for bone tissue engineering.

Silicon is known to have an influence on calcium phosphate deposition and on the differentiation of bone precursor cells. This study explores the effect of the incorporation of silanol (Si-OH) groups into polymeric scaffolds on the osteogenic differentiation of human adipose stem cells (hASC) cultured under dynamic and static conditions. A blend of corn starch with polycaprolactone (30/70 wt.%, SPCL) was used to produce three-dimensional fibre meshes scaffolds by the wet-spinning technique, and a calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups. In vitro assessment, using hASC, of functionalized and non-functionalized scaffolds was evaluated in either α-MEM or osteogenic medium under static and dynamic conditions (provided by a flow perfusion bioreactor). The functionalized materials, SPCL-Si, exhibit the capacity to sustain cell proliferation and induce their differentiation into the osteogenic lineage. The formation of mineralization nodules was observed in cells cultured on the SPCL-Si materials. Culturing under dynamic conditions using a flow perfusion bioreactor was shown to enhance the hASC proliferation and differentiation and a better distribution of cells within the material. The present work demonstrates the potential of these functionalized materials for future applications in bone tissue engineering. Additionally, these results highlight the simplicity, economic and reliable production process of those materials.

Osteogenic differentiation of two distinct subpopulations of human adipose-derived stem cells: an in vitro and in vivo study.

The first stem cells considered for the reconstruction of bone were bone marrow mesenchymal stem cells (BMSCs). Subsequently, cells with similar marker expression panel and differentiation potential were found in new sources of cells, such as adipose tissue. This source of stem cells has a promising future in tissue-engineering applications, considering the abundance of this tissue in the human body, the easy harvesting and the high number of stem cells that are available from such a small amount of tissue. The isolation of the adipose stem cells is generally performed by means of enzymatic digestion of the tissues, followed by a natural selection of the stem cells based on their capacity to adhere to the culture flasks, leading to a quite heterogeneous population. This constitutes a major drawback for the use of these cells, since the heterogeneity of the cell culture obtained can compromise their proliferation and differentiation potential. In the present study we have analysed the in vitro and in vivo behaviour of two selected subpopulations with high osteogenic potential. For this purpose, ASCs(CD29+) and ASCs (STRO-1+)subpopulations were isolated and in vitro cultured onto a biodegradable polymeric scaffold, using osteogenic medium, before implantation in a nude mice model. The biodegradable polymeric scaffold used is a fibre-mesh structure based on a blend of starch and polycaprolatone (SPCL) that has been successfully used in several bone tissue-engineering studies. The implanted ASCs-scaffold constructs promoted the formation of new bone tissue in nude mice. However, the results obtained show differences in the behaviour of the two ASCs subpopulations under study, particularly regarding their potential to differentiate into the osteogenic lineage, and allowed the indentification of ASCs (STRO-1+) as the best subpopulation for bone tissue-engineering applications.

High dose ascorbic acid does not reverse central sympathetic overactivity in chronic heart failure.

WHAT IS KNOWN AND OBJECTIVE: The increased central sympathetic activity typically associated with chronic heart failure (CHF) is probably mediated by formation of reactive oxygen species (ROS) in the brain. Our objective was to undertake a trial to test our hypothesis that administration of the well-known antioxidant and ROS scavenger ascorbic acid, would reverse or reduce the sympathetic overactivity in CHF patients. METHODS: In a prospective, randomized, placebo-controlled, double-blind, cross-over trial, 11 CHF patients were treated with ascorbic acid 2 g/day or placebo for 3 days. At the end of each treatment period, sympathetic nervous system activity was measured by microneurography for direct muscle sympathetic nerve activity (MSNA) recording, analysis of heart rate variability (HRV) and measurement of plasma norepinephrine concentrations. RESULTS: During ascorbic acid administration, plasma vitamin C levels were higher than during placebo (74·9 ± 6·0 µmol/L vs. 54·8 ± 4·6 µmol/L, P = 0·03). Ascorbic acid had no effect on sympathetic activity: MSNA (ascorbic acid: 66·8 ± 3·3 vs. placebo 66·9 ± 3·2 bursts/100 beats, P = 0·98). In addition, HRV and plasma norepinephrine levels did not differ. WHAT IS NEW AND CONCLUSION: Short-term administration of the antioxidant ascorbic acid in CHF patients does not reverse the increased sympathetic activity as measured by microneurography, HRV and plasma norepinephrine levels. The use of higher oral dosages seems not feasible due to accompanying side effects.

Fosfatase ácida resistente ao tartarato como biomarcador do metabolismo ósseo no cão / Tartrate-resistant acid phosphatase as a biomarker of bone turnover in dog

Values of serum tartrate-resistant acid phosphatase ( TRAP) activity were obtained in adult dogs and its biological variability was assessed. Nine healthy skeletally mature Portuguese Podengo dogs were used for the determination of TRAP, total and bone alkaline phosphatase serum activities, and also to study their relationship with serum minerals, namely calcium (Ca), phosphorous (P), and magnesium (Mg). The serum TRAP activity was 2.19±0.56IU/mL, with intra-individual variation of 18.3 percent and inter-individual variation of 25.6 percent. Significant correlations were observed between serum TRAP activity and Ca (r=-0.3431; P<0.05), Ca and Mg (r=-0.787; P<0.01), and TRAP and Mg (r=0.397; P<0.05). The results indicate that serum TRAP activity in dog could be of great value in research and in clinical practice, providing complementary non-invasive information on bone metabolism.

Determinaram-se os valores da atividade da fosfatase ácida resistente ao tartarato (FART) e avaliou-se a sua variabilidade biológica. Neste estudo, foram utilizados nove cães adultos e saudáveis de raça Podengo Português para as determinações das atividades da FART, da fosfatase alcalina total, da isoenzima óssea da fosfatase alcalina e da concentração dos minerais séricos - cálcio, fósforo e magnésio. A atividade sérica obtida da FART foi de 2,19±0,56 UI/mL, com uma variação intra-individual de 18,3 por cento e interindividual de 25,6 por cento. Foram observadas correlações significativas ao longo do tempo entre FART e cálcio (r=-0,3431; P<0,05), entre FART e magnésio (r=0,3974; P<0,05) e entre cálcio e magnésio (r=-0,787; P<0,01). Os resultados indicam que este marcador de reabsorção óssea pode ser de grande valor na prática clínica e na investigação e, ainda, ser utilizado como um método auxiliar não invasivo para avaliação do metabolismo ósseo.

A new route to produce starch-based fiber mesh scaffolds by wet spinning and subsequent surface modification as a way to improve cell attachment and proliferation.

This study proposes a new route for producing fiber mesh scaffolds from a starch-polycaprolactone (SPCL) blend. It was demonstrated that the scaffolds with 77% porosity could be obtained by a simple wet-spinning technique based on solution/precipitation of a polymeric blend. To enhance the cell attachment and proliferation, Ar plasma treatment was applied to the scaffolds. It was observed that the surface morphology and chemical composition were significantly changed because of the etching and functionalization of the fiber surfaces. XPS analyses showed an increase of the oxygen content of the fiber surfaces after plasma treatment (untreated scaffolds O/C:0.32 and plasma-treated scaffolds O/C:0.41). Both untreated and treated scaffolds were examined using a SaOs-2 human osteoblast-like cell line during 2 weeks of culture. The cell seeded on wet-spun SPCL fiber mesh scaffolds showed high viability and alkaline phosphatase enzyme activity, with those values being even higher for the cells seeded on the plasma-treated scaffolds.

Proliferation and differentiation of goat bone marrow stromal cells in 3D scaffolds with tunable hydrophilicity.

We have synthesized methacrylate-endcapped caprolactone networks with tailored water sorption ability, poly(CLMA-co-HEA), in the form of three-dimensional (3D) scaffolds with the same architecture but exhibiting different hydrophilicity character (x(HEA)=0, 0.3, 0.5), and we investigated the interaction of goat bone marrow stromal cells (GBMSCs) with such structures. For this purpose, GBMSCs were seeded and cultured for 3, 7, 14, 21, and 28 days onto the developed scaffolds. Cells have proliferated throughout the whole scaffold volume. Cell adhesion and morphology were analyzed by SEM, whereas cell viability and proliferation was assessed by MTS test and DNA quantification concluding that numbers of cells increased as a function of the culturing time (until day 14) and also with the hydrophobic content in the samples (from 50 to 100% of CLMA). No significant difference between samples with 100% and 70% of CLMA were detected in some cases. Osteoblastic differentiation was followed by assessing the alkaline phosphatase activity of cells, as well as type I collagen and osteocalcin expressions levels until day 21. The three markers were positive at days 14 and 21 when cells were cultured in 100% CLMA substrates which suggests osteoblastic differentiation of mesenchymal stem cells within these scaffolds. On the other hand, when the CLMA content decreases (until 50%), type I collagen and osteocalcin were positive but ALP was negative indicating that the differentiation process is affected by hydrophilic content. We suggest that such system may be useful to extract information on the effect of materials' wettability on the corresponding biological performance in a 3D environment. Such general insights may be relevant in the context of biomaterials selection for tissue engineering strategies.

Expression, purification and osteogenic bioactivity of recombinant human BMP-4, -9, -10, -11 and -14.

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-beta superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.

Novel method for the isolation of adipose stem cells (ASCs).

Adipose stem cells (ASCs) represent a cell population with great potential for tissue engineering applications. Several articles have been published showing the proliferation and differentiation potential, the markers and the wide range of potential applications of these cells. In the majority of these studies the ASCs are isolated using a basic enzymatic procedure, which results in a quite heterogeneous cell population that compromises their proliferation and differentiation. This paper reports the development and optimization of a new isolation/purification method that allows populations of ASCs to be obtained, which significantly reduces (and eventually eliminates) the 'contamination' of other cell types. This method is based on the use of immunomagnetic beads coated with specific antibodies. The first part of the study described here analysed the expression of marker genes for stem cells and the colony-forming unit (CFU) capacity of the cells isolated, while the second part is dedicated to the osteogenic differentiation potential of the isolated cells. The results showed that, using the isolation method based on immunomagnetic beads, it was possible to obtain ASCs and also underline the existence of several subpopulations of stem cells in the adipose tissue.

Starch-poly(epsilon-caprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering applications: structure, mechanical properties and degradation behaviour.

In scaffold-based tissue engineering strategies, the successful regeneration of tissues from matrix-producing connective tissue cells or anchorage-dependent cells (e.g. osteoblasts) relies on the use of a suitable scaffold. This study describes the development and characterization of SPCL (starch with epsilon-polycaprolactone, 30:70%) and SPLA [starch with poly(lactic acid), 30:70%] fibre-meshes, aimed at application in bone tissue-engineering strategies. Scaffolds based on SPCL and SPLA were prepared from fibres obtained by melt-spinning by a fibre-bonding process. The porosity of the scaffolds was characterized by microcomputerized tomography (microCT) and scanning electron microscopy (SEM). Scaffold degradation behaviour was assessed in solutions containing hydrolytic enzymes (alpha-amylase and lipase) in physiological concentrations, in order to simulate in vivo conditions. Mechanical properties were also evaluated in compression tests. The results show that these scaffolds exhibit adequate porosity and mechanical properties to support cell adhesion and proliferation and also tissue ingrowth upon implantation of the construct. The results of the degradation studies showed that these starch-based scaffolds are susceptible to enzymatic degradation, as detected by increased weight loss (within 2 weeks, weight loss in the SPCL samples reached 20%). With increasing degradation time, the diameter of the SPCL and SPLA fibres decreases significantly, increasing the porosity and consequently the available space for cells and tissue ingrowth during implantation time. These results, in combination with previous cell culture studies showing the ability of these scaffolds to induce cell adhesion and proliferation, clearly demonstrate the potential of these scaffolds to be used in tissue engineering strategies to regenerate bone tissue defects.