Expression, purification and in vitro biological activity from human recombinant BMP-2 produced by a novel approach

Bone morphogenetic proteins have promoted great biomedical interest due to their ability in inducing new bone formation when used as powerful osteoinductive components of several late-stage bone grafting products. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is obtained from mammalian cell expressing systems in low amounts or from bacteria inclusion bodies after timeconsuming refolding methods. Thus, there is a need to establish novel approaches for producing rhBMP-2 in high yields by simple and cheap procedures.Portuguese Foundation for Science and Technolog y, FCT (PhD Grant to PC Bessa , SFRH/BD/17049/2004). This work was also partially supported by the European STREP HIPPOCRATES (NMP3 - CT - 2003 - 505758) and carried out under the scope of European NoE EXPERTISSUES (NMP3 - CT - 2004 - 500283).info:eu-repo/semantics/publishedVersio

Do genes and environment meet to regulate cerebrospinal fluid dynamics? Relevance for schizophrenia

Schizophrenia is a neurodevelopment disorder in which the interplay of genes and environment contributes to disease onset and establishment. The most consistent pathological feature in schizophrenic patients is an enlargement of the brain ventricles. Yet, so far, no study has related this finding with dysfunction of the choroid plexus (CP), the epithelial cell monolayer located within the brain ventricles that is responsible for the production of most of the cerebrospinal fluid (CSF). Enlarged brain ventricles are already present at the time of disease onset (young adulthood) and, of notice, isolated mild ventriculomegaly detected in utero is associated with subsequent mild neurodevelopmental abnormalities similar to those observed in children at high risk of developing schizophrenia. Here we propose that altered CP/CSF dynamics during neurodevelopment may be considered a risk, causative and/or participating factor for development of schizophrenia.Nadine C. Santos and Fernanda Marques are recipients of postdoctoral fellowships by the Switchbox project (European Commission FP7 initiative grant HEALTH-F2-2010-259772) and the Fundação para a Ciência e Tecnologia (FCT, Portugal), respectively

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

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-b 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.The author wishes to acknowledge the Sanger Institute for kindly offering the bacterial clones for cloning of human BMP-9 to -14. This work was supported by Fundacao para a Ciencia e Tecnologia (PhD grant SFRH/BD/17049/2004, project ElastM POCI/CTM/57177/2004 funded by FEDER and the Fundacao para a Ciencia e Tecnologia; and European STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Expression, purification and in vitro biological activity from human recombinant BMP-2 produced by a novel approach

Bone tissue engineering has been an increasing field of research during the last years. The ideal approach for a regenerative application would consist in the use of cells from the patient, scaffolding materials and differentiation growth factors. Bone morphogenetic protein-2 (BMP-2) is one such growth factors with a strong ability to induce new bone and cartilage formation and has been used as a powerful osteoinductive component of several late-stage tissue engineering products for bone grafting. In this work, we aimed at obtaining high yields of human recombinant BMP-2 in a stable, pure and biologically active form by use of a new bacteria expression system that circumvents the disadvantages of conventional recombinant protein preparation methods and to perform a study of the stability conditions and functionality of these peptides in vitro in human mesenchymal stem cells and C2C12 murine cell line.Portuguese Foundation for Science and Technology, FCT (PhD Grant to PC Bessa, to PC Bessa, SFRH/BD/17049/2004 SFRH/BD/17049/2004 ). This work was ). This work was also partially supported by the European STREP HIPPOCRATES (NMP3 also partially supported by the European STREP HIPPOCRATES (NMP3--CTCT--2003 2003--505758) and carried out under the scope of 505758) and carried out under the scope of European NoE EXPERTISSUES (NMP3 European NoE EXPERTISSUES (NMP3--CTCT- -2004 2004 --500283). 500283info:eu-repo/semantics/publishedVersio

Silk fibroin microparticles as carriers for delivery of human recombinant bone morphogenetic protein-2 : in vitro and in vivo bioactivity

The in vitro and in vivo efficiency of fibroin microparticles as a delivery carrier for bone morphogenetic protein-2 (BMP-2) was evaluated. BMP-2 was encapsulated in silk fibroin particles that were produced by a simple and very mild processing method. The dose-response of BMP-2-loaded fibroin particles was examined in C2C12 cells, after 5 days of culture. The BMP-2 retained most of its activity as observed by the increase in alkaline phosphatase activity, which was much higher when BMP-2 was encapsulated into the particles rather than just surface-adsorbed. After 2 weeks of culture, increased mineralization was observed with BMP-2-loaded particles in comparison to soluble added growth factor. No significant cytotoxicity was detected. When implanted in a rat ectopic model, bone formation was observed by in vivo micro-computed tomography after 2 and 4 weeks postimplantation, with particles loaded with 5 or 12.5 microg BMP-2. An increase in bone density was observed over time. Histology revealed further evidence of ectopic bone formation, observed by strong alizarin red staining and osteocalcin immunostaining. Our findings show that fibroin microparticles may present an interesting option for future clinical applications in the bone tissue engineering field, and therefore, further studies have been planned.The authors acknowledge Anna Hofmann and Anna Khadem for additional help with some experiments, and Karin Brenner for animal maintenance. This work was supported by Fundacao para a Ciencia e Tecnologia (Ph.D. grant SFRH/BD/17049/2004), project ElastM (POCI/CTM/57177/2004 funded by FEDER and the Fundacao para a Ciencia e Tecnologia), Marie Curie Alea Jacta EST short-term grant (MEST-CT-2004-8104), and European STREP Project HIP-POCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

In Vitro Toxicity of Industrially Relevant Engineered Nanoparticles in Human Alveolar Epithelial Cells: Air-Liquid Interface versus Submerged Cultures

This article belongs to the Special Issue Engineered Nanomaterials Exposure and Risk Assessment: Occupational Health and SafetyDiverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air-liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions.This research was funded by CERASAFE (www.cerasafe.eu; accessed on 26 October 2021), with the support of ERA-NET SIINN (project id:16) and the Portuguese Foundation for Science and Technology (FCT; SIINN/0004/2014). This work was also supported by the NanoBioBarriers project (PTDC/MED-TOX/31162/2017), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR) and FCT; Spanish Ministry of Science and Innovation (projects PCIN-2015-173-C02-01 and CEX2018-000794- S-Severo Ochoa), and by the Romanian National Authority for Scientific Research and Innovation (CCCDI-UEFISCDI, project number 29/2016 within PNCDI III). M.J. Bessa (SFRH/BD/120646/2016) and F. Brandão (SFRH/BD/101060/2014) are recipients of FCT PhD scholarships under the framework of Human Capital Operating Program (POCH) and European Union funding. The Doctoral Program in Biomedical Sciences, of the ICBAS—University of Porto, offered additional funds. S. Fraga thanks FCT for funding through program DL 57/2016–Norma transitória (Ref. DL-57/INSA-06/2018). Thanks are also due to FCT/MCTES for the financial support to EPIUnit (UIDB/04750/2020).info:eu-repo/semantics/publishedVersio

Osteoinduction in human fat derived stem cells by recombinant human bone morphogenetic protein-2 produced in Escherichia coli

Bioactive recombinant human bone morphogenetic protein-2 (rhBMP-2) was obtained using Escherichia coli pET-25b expression system: 55 mg purified rhBMP-2 were achieved per g cell dry wt, with up to 95% purity. In murine C2C12 cell line, rhBMP-2 induced an increase in the transcription of Smads and of osteogenic markers Runx2/Cbfa1 and Osterix, measured by semi-quantitative RT-PCR. Bioassays performed in human fat-derived stem cells showed an increased activity of the early osteogenic marker, alkaline phosphatase, and the absence of cytotoxicity

2017 update of the WSES guidelines for emergency repair of complicated abdominal wall hernias

Emergency repair of complicated abdominal wall hernias may be associated with worsen outcome and a significant rate of postoperative complications. There is no consensus on management of complicated abdominal hernias. The main matter of debate is about the use of mesh in case of intestinal resection and the type of mesh to be used. Wound infection is the most common complication encountered and represents an immense burden especially in the presence of a mesh. The recurrence rate is an important topic that influences the final outcome. A World Society of Emergency Surgery (WSES) Consensus Conference was held in Bergamo in July 2013 with the aim to define recommendations for emergency repair of abdominal wall hernias in adults. This document represents the executive summary of the consensus conference approved by a WSES expert panel. In 2016, the guidelines have been revised and updated according to the most recent available literature.Peer reviewe