RESUMO
Omega-3 polyunsaturated fatty acids (PUFAs) play a vital role in human health, well-being, and the management of inflammatory diseases. Insufficient intake of omega-3 is linked to disease development. Specialized pro-resolving mediators (SPMs) are derived from omega-3 PUFAs and expedite the resolution of inflammation. They fall into categories known as resolvins, maresins, protectins, and lipoxins. The actions of SPMs in the resolution of inflammation involve restricting neutrophil infiltration, facilitating the removal of apoptotic cells and cellular debris, promoting efferocytosis and phagocytosis, counteracting the production of pro-inflammatory molecules like chemokines and cytokines, and encouraging a pro-resolving macrophage phenotype. This is an experimental pilot study in which ten healthy subjects were enrolled and received a single dose of 6 g of an oral SPM-enriched marine oil emulsion. Peripheral blood was collected at baseline, 3, 6, 9, 12, and 24 h post-administration. Temporal increases in plasma and serum SPM levels were found by using LC-MS/MS lipid profiling. Additionally, we characterized the temporal increases in omega-3 levels and established fundamental pharmacokinetics in both aforementioned matrices. These findings provide substantial evidence of the time-dependent elevation of SPMs, reinforcing the notion that oral supplementation with SPM-enriched products represents a valuable source of essential bioactive SPMs.
Assuntos
Ácidos Docosa-Hexaenoicos , Ácidos Graxos Ômega-3 , Humanos , Voluntários Saudáveis , Cromatografia Líquida , Projetos Piloto , Espectrometria de Massas em Tandem , Inflamação , Fator de Ativação de Plaquetas , Mediadores da InflamaçãoRESUMO
Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is highly heterogeneous, ranging from asymptomatic to severe and fatal cases. COVID-19 has been characterized by an increase of serum pro-inflammatory cytokine levels which seems to be associated with fatal cases. By contrast, the role of pro-resolving lipid mediators (SPMs), involved in the attenuation of inflammatory responses, has been scarcely investigated, so further studies are needed to understand SPMs metabolism in COVID-19 and other infectious diseases. Our aim was to analyse the lipid mediator metabolome, quantifying pro- and anti-inflammatory serum bioactive lipids by LC-MS/MS in 7 non-infected subjects and 24 COVID-19 patients divided into mild, moderate, and severe groups according to the pulmonary involvement, to better understand the disease outcome and the severity of the pulmonary manifestations. Statistical analysis was performed with the R programming language (R Foundation for Statistical Computing, Vienna, Austria). All COVID-19 patients had increased levels of Prostaglandin E2. Severe patients showed a significant increase versus controls, mild- and moderate-affected patients, expressed as median (interquartile range), in resolvin E1 [112.6 (502.7) vs 0.0 (0.0) pg/ml in the other groups], as well as in maresin 2 [14.5 (7.0) vs 8.1 (4.2), 5.5 (4.3), and 3.0 (4.0) pg/ml, respectively]. Moreover, 14-hydroxy docosahexaenoic acid (14-HDHA) levels were also increased in severe vs control and mild-affected patients [24.7 (38.2) vs 2.4 (2.2) and 3.7 (6.4) ng/mL, respectively]. Resolvin D5 was also significantly elevated in both moderate [15.0 (22.4) pg/ml] and severe patients [24.0 (24.1) pg/ml] versus controls [0.0 (0.0) pg/ml]. These results were confirmed by sparse partial least squares discriminant analysis which highlighted the contribution of these mediators to the separation between each of the groups. In conclusion, the potent inflammatory response to SARS-CoV-2 infection involves not only pro- but also anti-inflammatory lipid mediators that can be quantified in easily accessible serum samples, suggesting the need to perform future research on their generation pathways that will help us to discover new therapeutic targets.
Assuntos
COVID-19 , Humanos , Projetos Piloto , Cromatografia Líquida , SARS-CoV-2/metabolismo , Espectrometria de Massas em Tandem , Pulmão/metabolismo , Eicosanoides/metabolismo , Anti-Inflamatórios , Gravidade do PacienteRESUMO
The advantages of additive manufactured scaffolds, as custom-shaped structures with a completely interconnected and accessible pore network from the micro- to the macroscale, are nowadays well established in tissue engineering. Pore volume and architecture can be designed in a controlled fashion, resulting in a modulation of scaffold's mechanical properties and in an optimal nutrient perfusion determinant for cell survival. However, the success of an engineered tissue architecture is often linked to its surface properties as well. The aim of this study was to create a family of polymeric pastes comprised of poly(ethylene oxide therephthalate)/poly(butylene terephthalate) (PEOT/PBT) microspheres and of a second biocompatible polymeric phase acting as a binder. By combining microspheres with additive manufacturing technologies, we produced 3D scaffolds possessing a tailorable surface roughness, which resulted in improved cell adhesion and increased metabolic activity. Furthermore, these scaffolds may offer the potential to act as drug delivery systems to steer tissue regeneration.
RESUMO
Substrates for neuron culture and implantation are required to be both biocompatible and display surface compositions that support cell attachment, growth, differentiation, and neural activity. Laminin, a naturally occurring extracellular matrix protein is the most widely used substrate for neuron culture and fulfills some of these requirements, however, it is expensive, unstable (compared to synthetic materials), and prone to batch-to-batch variation. This study uses a high-throughput polymer screening approach to identify synthetic polymers that supports the in vitro culture of primary mouse cerebellar neurons. This allows the identification of materials that enable primary cell attachment with high viability even under "serum-free" conditions, with materials that support both primary cells and neural progenitor cell attachment with high levels of neuronal biomarker expression, while promoting progenitor cell maturation to neurons.
Assuntos
Células-Tronco Neurais , Neurônios , Animais , Diferenciação Celular , Células Cultivadas , Laminina , Camundongos , PolímerosRESUMO
The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not been possible to efficiently generate Purkinje neuron (PN) progenitors from human or mouse pluripotent stem cells, let alone to develop a methodology for in vivo transplantation in the adult cerebellum. Here, we present a protocol to obtain an expandable population of cerebellar neuron progenitors from mouse embryonic stem cells. Our protocol is characterized by applying factors that promote proliferation of cerebellar progenitors. Cerebellar progenitors isolated in culture from cell aggregates contained a stable subpopulation of PN progenitors that could be expanded for up to 6 passages. When transplanted into the adult cerebellum of either wild-type mice or a strain lacking Purkinje cells (L7cre-ERCC1 knockout), GFP-labeled progenitors differentiated in vivo to establish a population of calbindin-positive cells in the molecular layer with dendritic trees typical of mature PNs. We conclude that this protocol may be useful for the generation and maturation of PNs, highlighting the potential for development of a regenerative medicine approach to the treatment of cerebellar neurodegenerative diseases.
Assuntos
Diferenciação Celular , Cerebelo/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Potenciais de Ação , Fatores Etários , Animais , Biomarcadores , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultura , Feminino , Imunofluorescência , Expressão Gênica , Genes Reporter , Imunofenotipagem , Masculino , Camundongos , Transplante de Células-TroncoRESUMO
This data article contains seven figures and two tables supporting the research article entitled: spatiotemporal proliferation of human stromal cells adjusts to nutrient availability and leads to stanniocalcin-1 expression in vitro and in vivo[1]. The data explain the culture of stromal cells in vitro in three culture systems: discs, scaffolds and scaffolds in a perfusion bioreactor system. Also, quantification of extracellular matrix components (ECM) in vitro and staining of ECM components in vivo can be found here. Finally the quantification of blood vessels dimensions from CD31 signals and representative histograms of stanniocalcin-1 fluorescent signals in negative controls and experimental conditions in vivo are presented.
RESUMO
Cells and tissues are intrinsically adapted to molecular gradients and use them to maintain or change their activity. The effect of such gradients is particularly important for cell populations that have an intrinsic capacity to differentiate into multiple cell lineages, such as bone marrow derived mesenchymal stromal cells (MSCs). Our results showed that nutrient gradients prompt the spatiotemporal organization of MSCs in 3D culture. Cells adapted to their 3D environment without significant cell death or cell differentiation. Kinetics data and whole-genome gene expression analysis suggest that a low proliferation activity phenotype predominates in stromal cells cultured in 3D, likely due to increasing nutrient limitation. These differences implied that despite similar surface areas available for cell attachment, higher cell concentrations in 3D reduced MSCs proliferation, while activating hypoxia related-pathways. To further understand the in vivo effects of both proliferation and cell concentrations, we increased cell concentrations in small (1.8 µl) implantable wells. We found that MSCs accumulation and conditioning by nutrient competition in small volumes leads to an ideal threshold of cell-concentration for the induction of blood vessel formation, possibly signaled by the hypoxia-related stanniocalcin-1 gene.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Meios de Cultura/farmacocinética , Glicoproteínas/biossíntese , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Neovascularização Fisiológica/fisiologia , Adaptação Fisiológica/fisiologia , Disponibilidade Biológica , Proliferação de Células/fisiologia , Células Cultivadas , Desenho de Equipamento , Humanos , Análise Espaço-Temporal , Alicerces TeciduaisRESUMO
Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain µl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.
Assuntos
Materiais Biocompatíveis/farmacologia , Condrócitos/química , Matriz Extracelular/química , Células-Tronco Mesenquimais/química , Engenharia Tecidual/métodos , Animais , Bovinos , Condrócitos/citologia , Técnicas de Cocultura , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos NusRESUMO
Cells react to various forms of physical phenomena that promote and maintain the formation of tissues. The best example of this are cells of musculoskeletal origin, such as mesenchymal stem cells (MSCs), which consistently proliferate or differentiate under cues from hydrostatic pressure, diffusive mass transport, shear stress, surface chemistry, mechanotransduction, and molecular kinetics. To date, no other cell type shows greater receptiveness to macroscopic and microscopic cues, highlighting the acute sensitivity of MSCs and the importance of physical principles in tissue homeostasis. In this review, we describe the literature that has shown how physical phenomena govern MSCs biology and provide insight into the mechanisms and strategies that can spur new biotechnological applications with tissue biology.
Assuntos
Fenômenos Mecânicos , Células-Tronco Mesenquimais/fisiologia , Estresse Fisiológico , Animais , Proliferação de Células , HumanosRESUMO
The nutritional requirements of stem cells have not been determined; in particular, the amino acid metabolism of stem cells is largely unknown. In this study, we investigated the amino acid metabolism of human mesenchymal stem cells (hMSCs), with focus on two questions: Which amino acids are consumed and/or secreted by hMSCs and at what rates? To answer these questions, hMSCs were cultured on tissue culture plastic and in a bioreactor, and their amino acid profile was analyzed. The results showed that the kinetics of hMSCs growth and amino acid metabolism were significantly higher for hMSCs in tissue culture plastic than in the bioreactor. Despite differences in culture conditions, 8 essential and 6 nonessential amino acids were consumed by hMSCs in both tissue culture plastic and bioreactor cultures. Glutamine was the most consumed amino acid with significantly higher rates than for any other amino acid. The metabolism of nonessential amino acids by hMSCs deviated significantly from that of other cell lines. The secretion of alanine, glycine, glutamate, and ornithine by hMSCs showed that there is a strong overflow metabolism that can be due to the high concentrations of amino acids provided in the medium. In addition, the data showed that there is a metabolic pattern for proliferating hMSCs, which can contribute to the design of medium without animal serum for stem cells. Further, this study shows how to implement amino acid rates and metabolic principles in three-dimensional stem cell biology.
Assuntos
Aminoácidos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Reatores Biológicos , Células Cultivadas , Feminino , Humanos , Cinética , MasculinoRESUMO
A practical method of photocrosslinking high molecular weight poly(trimethylene carbonate)(PTMC) is presented. Flexible, elastomeric and biodegradable networks could be readily prepared by UV irradiating PTMC films containing pentaerythritol triacrylate (PETA) and a photoinitiator. The network characteristics, mechanical properties, wettability, and in vitro enzymatic erosion of the photocrosslinked PTMC films were investigated. Densely crosslinked networks with gel contents up to 98% could be obtained in this manner. Upon photocrosslinking, flexible and tough networks with excellent elastomeric properties were obtained. To illustrate the ease with which the properties of the networks can be tailored, blends of PTMC with mPEG-PTMC or with PTMC-PCL-PTMC were also photocrosslinked. The wettability and the enzymatic erosion rate of the networks could be tuned by blending with block copolymers. Tissue engineering scaffolds were also fabricated using these flexible photocrosslinkable materials. After crosslinking, the fabricated PTMC-based scaffolds showed inter-connected pores and extensive microporosity. Human mesenchymal stem cell (hMSC) culturing studies showed that the photocrosslinked scaffolds prepared from PTMC and PTMC/PTMC-PCL-PTMC blends are well-suited for tissue engineering applications.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Dioxanos/farmacologia , Elastômeros/farmacologia , Polímeros/farmacologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Raios Ultravioleta , Animais , Sobrevivência Celular/efeitos dos fármacos , Glucose/metabolismo , Humanos , Ácido Láctico/metabolismo , Fenômenos Mecânicos/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Varredura , Peso Molecular , Sus scrofa , Molhabilidade/efeitos dos fármacosRESUMO
The development of 3D scaffolds consisting of stacked multi-layered porous sheets featuring microchannels is proposed and investigated in this work. In this concept, the inner-porosity of the sheets allows diffusion of nutrients and signalling products between the layers whereas the microchannels facilitate nutrient supply on all layers as they provide space for the culture medium to be perfused throughout the scaffold. Besides the above, these scaffolds have excellent distribution of the cells as seeding and attaching of the cells occurs on individual layers that are subsequently stacked. In addition, these scaffolds enable gaining local data from within the scaffolds as unstacking of the stacked layers allows for determination of various parameters per layer. Here, we show the proof of this concept by culturing C2C12 pre-myoblasts and A4-4 cells on stacked Poly(l-lactic acid) (PLLA) sheets featuring microchannels. The results obtained for culturing under static conditions clearly indicate that despite inhibited cell proliferation due to nutrient limitations, diffusion between the layers takes place and cells on various layers stay viable and also affect each other. Under dynamic conditions, medium flow through the channels improves nutrient availability to the cells on the various layers, drastically increasing cell proliferation on all layers.
Assuntos
Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Células CHO , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Ácido Láctico/química , Ácido Láctico/farmacologia , Camundongos , Microscopia , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Poliésteres , Polímeros/química , Polímeros/farmacologiaRESUMO
The biological effect of the perfluorocarbon-based artificial oxygen carrier (Oxygent) was investigated in tissue-engineered trachea (TET) construction. Media supplemented with and without 10% Oxygent were compared in all assessments. Partial tissue oxygen tension (PtO(2)) was measured with polarographic microprobes; epithelial metabolism was monitored by microdialysis inside the TET epithelium perfused with the medium underneath. Chondrocyte-DegraPol constructs were cultured for 1 month with the medium before glycosaminoglycan assessment and histology. Tissue reaction of TET epithelial scaffolds immersed with the medium was evaluated on the chick embryo chorioallantoic membrane. Oxygent perfusion medium increased the TET epithelial PtO(2) (51.2 +/- 0.3 mm Hg vs. 33.4 +/- 0.3 mm Hg at 200 microm thickness; 12.5 +/- 0.1 mm Hg vs. 3.1 +/- 0.1 mm Hg at 400 microm thickness, p < 0.01) and decreased the lactate concentration (0.63 +/- 0.08 vs. 0.80 +/- 0.06 mmol/L, p < 0.05), lactate/pyruvate (1.87 +/- 0.26 vs. 3.36 +/- 10.13, p < 0.05), and lactate/glucose ratios (0.10 +/- 0.00 vs. 0.29 +/- 0.14, p < 0.05). Chondrocyte-DegraPol in Oxygent group presented lower glycosaminoglycan value (0.03 +/- 0.00 vs. 0.13 +/- 0.00, p < 0.05); histology slides showed poor acid mucopolysaccharides formation. Orthogonal polarization spectral imaging showed no difference in functional capillary density between the scaffolds cultured on chorioallantoic membranes. The foreign body reaction was similar in both groups. We conclude that Oxygent increases TET epithelial PtO(2), improves epithelial metabolism, does not impair angiogenesis, and tends to slow cartilage tissue formation.
Assuntos
Fluorocarbonos/farmacologia , Oxigênio/metabolismo , Engenharia Tecidual , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Animais , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Membrana Corioalantoide/citologia , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/transplante , Derme/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Reação a Corpo Estranho/patologia , Glicosaminoglicanos/metabolismo , Humanos , Hidrocarbonetos Bromados , Microdiálise , Pressão Parcial , Poliésteres/farmacologia , Poliuretanos/farmacologia , Ratos , Sus scrofa , Alicerces TeciduaisRESUMO
Better quantitative understanding of human mesenchymal stem cells (hMSCs) metabolism is needed to identify, understand, and subsequently optimize the processes in expansion of hMSCs in vitro. For this purpose, we analyzed growth of hMSCs in vitro with a mathematical model based on the mass balances for viable cell numbers, glucose, lactate, glutamine, and glutamate. The mathematical modeling had two aims: (1) to estimate kinetic parameters of important metabolites for hMSC monolayer cultures, and (2) to quantitatively assess assumptions on growth of hMSCs. Two cell seeding densities were used to investigate growth and metabolism kinetics of MSCs from three human donors. We analyzed growth up to confluency and used metabolic assumptions described in literature. Results showed a longer initial phase, a slower growth rate, and a higher glucose, lactate, glutamine, and glutamate metabolic rates at the lower cell seeding density. Higher metabolic rates could be induced by a lower contact inhibition effect when seeding at 100 cells/cm2 than when seeding at 1000 cells/cm2. In addition, parameter estimation describing kinetics of hMSCs in culture, depending on the seeding density, showed doubling times in the order of 17-32h, specific glucose consumption in the order of 1.25 x 10(-1) to 3.77 x 10(-1) pmol/cell/h, specific lactate production in the order of 2.48 x 10(-1) to 7.67 x 10(-1)pmol/cell/h, specific glutamine production in the order of 7.04 x 10(-3) to 2.27 pmol/cell/h, and specific glutamate production in the order of 4.87 x 10(-1) to 23.4 pmol/cell/h. Lactate-to-glucose yield ratios confirmed that hMSCs use glucose via anaerobic glycolysis. In addition, glutamine and glutamate metabolic shifts were identified that could be important for understanding growth of hMSCs in vitro. This study showed that the mathematical modeling approach supports quantitative analysis of important mechanisms in proliferation of hMSCs in vitro.