Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987. Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/ Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP

Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP

Attachment and Proliferation of Osteoblasts on Lithium-Hydroxyapatite Composites

The biocompatibility and bioactivity properties of hydroxyapatites (HAs) modified through lithium addition were investigated. Hydroxyapatites obtained from bovine bone were mixed with lithium carbonate (Li), in the proportions of 0.25, 0.50, 1.00, and 2.00% wt, and sintered at 900°, 1000°, 1100°, 1200°, and 1300°C, creating LiHA samples. The osteoblast culture behavior was assessed in the presence of these LiHA compositions. The cellular interactions were analyzed by evaluating the viability and cellular proliferation, ALP production and collagen secretion. The cytotoxic potential was investigated through measurement of apoptosis and necrosis induction. The process of cellular attachment in the presence of the product of dissolution of LiHA, was evaluated trough fluorescence analysis. The physical characteristics of these materials and their cellular interactions were examined with SEM and EDS. The results of this study indicate that the LiHA ceramics are biocompatible and have variable bioactivities, which can be tailored by different combinations of the concentration of lithium carbonate and the sintering temperature. Our findings suggest that LiHA 0.25% wt, sintered at 1300°C, combines the necessary physical and structural qualities with favorable biocompatibility characteristics, achieving a bioactivity that seems to be adequate for use as a bone implant material

Endothelial Differentiation of Human Stem Cells Seeded onto Electrospun Polyhydroxybutyrate/Polyhydroxybutyrate-Co-Hydroxyvalerate Fiber Mesh

Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with endothelial-differentiated cells to improve vascularization in engineered bone tissues

Meeting Report: Aging Research and Drug Discovery

Aging is the single largest risk factor for most chronic diseases, and thus possesses large socioeconomic interest to continuously aging societies. Consequently, the field of aging research is expanding alongside a growing focus from the industry and investors in aging research. This year's 8th Annual Aging Research and Drug Discovery ARDD) meeting was organized as a hybrid meeting from August 30th to September 3rd 2021 with more than 130 attendees participating on-site at the Ceremonial Hall at University of Copenhagen, Denmark, and 1800 engaging online. The conference comprised of presentations from 75 speakers focusing on new research in topics including mechanisms of aging and how these can be modulated as well as the use of AI and new standards of practices within aging research. This year, a longevity workshop was included to build stronger connections with the clinical community

Avaliação da adesão, proliferação e diferenciação endotelial de células-tronco humanas cultivadas em nanofibras de polihidroxibutirato

Exportado OPUSMade available in DSpace on 2019-08-12T07:15:43Z (GMT). No. of bitstreams: 1 disserta__o_alessandra__versao_final.pdf: 1339109 bytes, checksum: 46bbc5a069aa006b2262a8a80d48de6c (MD5) Previous issue date: 5A engenharia de tecidos é um campo da medicina regenerativa que se baseia no cultivo de células em matrizes estruturais associadas à incorporação de moléculas sinalizadoras, visando a regeneração tecidual. Apesar do enorme potencial, essa estratégia enfrenta um grande desafio para a manutenção da viabilidade celular após a implantação desses constructos. A falta de uma vascularização no interior do implante compromete a nutrição e a remoção de metabólitos pelas células, o que pode levar a falência do implante. Nesse sentido, o nosso grupo de pesquisa pretende criar uma nova abordagem visando solucionar o problema da falta de vascularização no produto da engenharia de tecido, em especial para reparo de tecido ósseo. Dessa forma, a primeira etapa desse projeto visa estabelecer o cultivo de células-tronco humanas derivadas do tecido adiposo (hASC), avaliar a biocompatibilidade de membranas derivadas do polihidroxibutirato (PHB) e do seu co-polímero poli-3-hidroxibutirato-co-3-hidroxivalerato (PHB-HV) e promover a diferenciação dessas células-tronco em células endoteliais. Através de análises in vitro foi possível demonstrar que a membrana de nanofibras baseada em PHB/PHBHV apresenta biocompatibilidade com as células-tronco isoladas do tecido adiposo humano, favorecendo a adesão, a viabilidade e a proliferação dessas células. Ao serem implantas in vivo, a membrana de PHB/PHB-HV não induziu uma resposta inflamatória intensa e não afetou a função dos tecidos adjacentes ao implante, possibilitando sua utilização in vivo. Além disso, quando induzidas a diferenciação endotelial, as hASC passaram a expressar marcadores específicos de células endoteliais, tornando possível a utilização dessas células diferenciadas, juntamente com a membrana de PHB/PHB-HV, para auxiliar a vascularização de matrizes tridimensionais.Tissue engineering is a field of regenerative medicine based on the association of cell culture in structural matrices and the incorporation of signaling molecules, targeting tissue regeneration. Despite its enormous potential, this strategy faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the nutrition and removal of metabolites by cells, which can lead to implant failure. In this context, our research group is trying to create a new strategy for enhancing vascularization in tissue engineering, especially for bone tissue. The aim of this project is to establish a culture of human adipose tissue derived stem cells (hASC) in order to evaluate the biocompatibility of nanofiber meshes derived from polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3- hydroxyvalerate (PHB-HV), and to promote the differentiation of stem cells into endothelial cells. Through in vitro assays, it was demonstrated that PHB/PHB-HV nanofiber meshes is biocompatible with stem cells isolated from human adipose tissue, and that it promotes cell adhesion, cell viability and proliferation. When implanted in vivo, the PHB/PHB-HV nanofiber meshes did not induce a high inflammatory response and did not affect the function of tissues in the region of the implant. Moreover, when cultured with endothelial differentiation medium, the hASC expressed endothelial markers. Taken together, the data presented above allow to suggest that it would be possible to use hASC derived differentiated cells cultured in PHB/PHB-HV nanofiber meshes to enhance the vascularization of tissue-engineered grafts

Modulation of Angiogenic Activity by Light-Activatable miRNA-Loaded Nanocarriers

"This document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication in [JournalTitle], copyright © American Chemical Society after peer review. To access the final edited and published work see [insert ACS Articles on Request author-directed link to Published Work, see https://pubs.acs.org/doi/ipdf/10.1021/acsnano.7b0753

Proliferation of human mesenchymal stem cells derived from adipose tissue on polyurethanes with tunable biodegradability

Polyurethanes (PUs) have been considered good candidates to be used in biomedical temporary devices that require mechanical properties comparable to soft tissues. However, toxicity of some PUs is still a concern, since these polyurethanes can contain potential toxic components and residual organic solvents derived from their synthesis. In this work, in vitro tests to measure viability and proliferation of human mesenchymal stem cells (hMSCs) in contact with PUs with tunable biodegradability were performed by employing MTT, alkaline phosphatase and collagen secretion assays. PUs were produced in an aqueous environment by employing isophorone diisocyanate/hydrazine (hard segment) and poly(caprolactone diol)/2,2-bis (hydroxymethyl) propionic acid (soft segment) as the main reagents. Three series of PUs having different soft segment contents were synthesized. These PUs had their chemical structure, morphology and hydrolytic degradation investigated. The rate of hydrolysis of the obtained PUs was tailored by modifying the soft segment content of the polymers. In vitro results showed that PUs can provide a satisfactory environment for the adhesion and proliferation of hMSCs