Human mesenchymal stem cells growth and osteogenic differentiation on piezoelectric poly(vinylidene fluoride) microsphere substrates

The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC) fate when cultured in supports with varying topography. Poly(vinylidene fluoride) (PVDF) culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM). Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride) is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.The authors thank the Portuguese Foundation for Science and Technology (FCT) for financial support under project PTDC/EEI-SII/5582/2014, Strategic Funding UID/FIS/04650/2013 and grants SFRH/BPD/90870/2012 (C.R.) and SFRH/BPD/121526/2016 (D.M.C). The authors acknowledge funding by the Spanish Ministry of Economy and Competitiveness (MINECO) through the project MAT2016-76039-C4-3-R (AEI/FEDER, UE) and from the Basque Government Industry Department under the ELKARTEK program. JLGR, LC, RSS and AS acknowledge funding by the Conselleria de Educación, Investigación, Cultura y Deporte of the Generalitat Valenciana through PROMETEO/2016/063 project. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development. This work was partially financed with FEDER funds (CIBERONC (CB16/12/00284)). The authors acknowledge the assistance and advice of Electron Microscopy Service of the UPVinfo:eu-repo/semantics/publishedVersio

Extracellular matrix electrospun membranes for mimicking natural renal filtration barriers

Kidney diseases are recognized as a major health problem, which affect 10% of the population. Because currently available therapies have many limitations, some tissue engineering strategies have been emerging as promising approaches in this field. In this work, porcine kidneys were decellularized to obtain decellularized kidney extracellular matrix (dKECM). Our results demonstrate a successful protocol of decellularization characterized by the removal of nucleic acid material and preservation of collagen and glycosaminoglycans. Blends of polycaprolactone (PCL) and dKECM were prepared by electrospinning and characterized. The biological performance of the membranes was tested with a human kidney cell line (HK-2) for 7⠯days. It was observed that cellular metabolic activity, proliferation and protein content increased with an increase in dKECM concentrations (30, 50 and 70%). Additionally, the expression of zona occludens-1 was revealed on dKECM-containing membranes but not on pure PCL membranes. To the best of our knowledge this is the first time that natural extracellular matrix is used to mimic the kidney basement membrane as an in vitromodel. This could be a valuable tool for regenerative nephrology and may have an impact on the development of kidney advanced therapies in the future.This work was supported by the European Regional Development Fund (ERDF) on the project FROnTHERA (NORTE-01-0145-FEDER000023); the Portuguese Foundation for Science and Technology (FCT), under the scope of the project SPARTAN (PTDC/CTM-BIO/4388/2014); and the FCT PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/169/2013

Particulate kidney extracellular matrix: bioactivity and proteomic analysis of a novel scaffold from porcine origin

Decellularized matrices are attractive substrates, being able to retain growth factors and proteins present in the native tissue. Several biomaterials can be produced by processing these matrices. However, new substrates capable of being injected that reverse local kidney injuries are currently scarce. Herein, we hypothesized that the decellularized particulate kidney porcine ECM (pKECM) could support renal progenitor cell cultures for posterior implantation. Briefly, kidneys are cut into pieces, decellularized by immersion on detergent solutions, lyophilized and reduced into particles. Then, ECM particles are analyzed for nuclear material remaining by DNA quantification and histological examination, molecular conformation by FITR and structural morphology by SEM. Protein extraction is also optimized for posterior identification and quantification by mass spectrometry. The results obtained confirm the collagenous structure and composition of the ECM, the effective removal of nucleic material and the preservation of ECM proteins with great similarity to human kidneys. Human renal progenitor cells (hRPCs) are seeded in different ratios with pKECM, on 3D suspensions. The conducted assays for cell viability, proliferation and distribution over 7 days of culture suggest that these matrices as biocompatible and bioactive substrates for hRPCs. Also, by analyzing CD133 expression, an optimal ratio for specific phenotypic expression is revealed, demonstrating the potential of these substrates to modulate cellular behavior. The initial hypothesis of developing and characterizing a particulate ECM biomaterial as a consistent substrate for 3D cultures is successfully validated. The findings in this manuscript suggest these particles as valuable tools for regenerative nephrology by minimizing surgeries and locally reversing small injuries which can lead to chronic renal disfunction.This work was supported by the European Regional Development Fund (ERDF) on the project FROnTHERA (NORTE-01-0145-FEDER-000023) and the FCT PhD Grant on the Doctoral Program on Advanced Therapies for Health (PATH) (PD/BD/128102/2016). Mass spectrometry and proteomics were performed at the Proteomics i3S Scientific Platform with the assistance of Hugo Osório. This work was supported by the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145- FEDER-022125)

Quem avalia resiliência e qual resiliência vale? Reflexões sobre a aplicação do Manual de Avaliação da Resiliência em uma fronteira Amazônica contestada

The Brazilian Amazon is a complex social-ecological system, the management of which includes diverse groups of social actors whose values and interests influence decision making and outcomes. Such management requires leaders who appreciate the multiple knowledge systems and historical land occupation trajectories that have shaped this region. Twenty-three emerging leaders from universities, government agencies, the private sector, and social movement organizations participated in a two-year Specialization Course in the state of Mato Grosso, Brazil. Course participants applied the Resilience Assessment (RA) Workbook for Practitioners to analyze three livelihood sub-systems within the municipality of Cotriguaçu. This paper offers reflections on the utility of the RA workbook as a tool for bridging multiple stakeholders’ knowledge, identities, power and interests in collaborative social-ecological management. Our experience points to the risks of conducting expert-led RAs in regions dominated by historical legacies of oppression, weak institutions, and limited governance.A Amazônia brasileira é um sistema socioecológico complexo que inclui diversos grupos de atores sociaiscujos valores, interesses, trajetórias de ocupação do solo e relações de poder influenciam a tomadade decisão sobre a gestão dos recursos naturais. Uma gestão eficaz exige que os líderes estejampreparados com abordagens e ferramentas para facilitar a colaboração entre esses diversos atoressociais. Este artigo apresenta as lições aprendidas a partir de um curso de especialização no estado deMato Grosso, que teve como objetivo capacitar 20 líderes de universidades, agências governamentais,setor privado e movimento social. Os participantes do curso aplicaram a metodologia de Avaliação deResiliência (AR) para analisar os meios de vida de três grupos de atores dentro do município de Cotriguaçu.Nós descrevemos a pedagogia construtivista utilizada no curso, lições aprendidas na aplicaçãoda AR, e reflexões sobre a utilidade da metodologia para gestão colaborativa socioecológica. Nossaexperiência revela os desafios de engajar adequadamente os atores sociais locais em tais análises eos riscos de realização de ARs lideradas por especialistas em regiões dominadas por elites poderosas,desigualdades, e lacunas de governança

Occurrence of infection with Toxoplasma gondii and factors associated with transmission in broiler chickens and laying hens in different raising systems

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. The aim of the present study was to determine the occurrence and identify the risk factors associated with transmission of T. gondii to chickens raised in different systems (free-ranged and confined) to produce eggs or meat. The 810 animals were allocated in two experimental groups according to the production system purpose: 460 broiler chickens (Group 1) and 350 layer chickens (Group 2). In order to analyze the possible factors involved in T. gondii infection in the chickens, an epidemiological questionnaire was developed for all properties.The serological detection of anti-Toxoplasma gondii antibodies was performed by Indirect Immunofluorescence (IFAT) and by Enzime Linked Imunossorbent Assay (ELISA). Since the agreement index (kappa) between these two serological techniques was considered high, 21.2% of the 810 animals were considered reactive. In Group 1, 12.2% (56/460) were positive, while in the Group 2 the positivity rate was 33.1% (116/350). The production system may be influencing the seropositivity of the animals in both groups. However, only in Group 2 it was possible to notice a statistically significant relationship between the breeding system and the frequency of positive sera. This result indicates that, at least for laying hens, the production system is directly involved in T. gondii infection. The contact with cats in Group 1 did not influence the distribution of seroreactive animals, but in Group 2 a significant relationship was observed. The occurrence of anti-T. gondii antibodies was high in both groups (broiler and posture chickens). Free-ranged chickens raised for egg production proved to be the most exposed group to the T. gondii infection. This can be related to the fact that these animals stay for longer periods in the farms, in direct contact with possibly contaminated soil by the presence of domestic cats

Digestible lysine levels in diets supplemented with ractopamine

In order evaluate digestible lysine levels in diets supplemented with 20 ppm of ractopamine on the performance and carcass traits, 64 barrows with high genetic potential at finishing phase were allotted in a completely randomized block design with four digestible lysine levels (0.80, 0.90, 1.00, and 1.10%), eight replicates and two pigs per experimental unit. Initial body weight and pigs' kinship were used as criteria in the blocks formation. Diets were mainly composed of corn and soybean meal supplemented with minerals, vitamins and amino acids to meet pigs' nutritional requirements at the finishing phase, except for digestible lysine. No effect of digestible lysine levels was observed in animal performance. The digestible lysine intake increased linearly by increasing the levels of digestible lysine in the diets. Carcass traits were not influenced by the dietary levels of digestible lysine. The level of 0.80% of digestible lysine in diets supplemented with 20 ppm ractopamine meets the nutritional requirements of castrated male pigs during the finishing phase