RESUMO
Endothelial cells emerge from the atrioventricular canal to form coronary blood vessels in juvenile zebrafish hearts. We find that pdgfrb is first expressed in the epicardium around the atrioventricular canal and later becomes localized mainly in the mural cells. pdgfrb mutant fish show severe defects in mural cell recruitment and coronary vessel development. Single-cell RNA sequencing analyses identified pdgfrb+ cells as epicardium-derived cells (EPDCs) and mural cells. Mural cells associated with coronary arteries also express cxcl12b and smooth muscle cell markers. Interestingly, these mural cells remain associated with coronary arteries even in the absence of Pdgfrß, although smooth muscle gene expression is downregulated. We find that pdgfrb expression dynamically changes in EPDCs of regenerating hearts. Differential gene expression analyses of pdgfrb+ EPDCs and mural cells suggest that they express genes that are important for regeneration after heart injuries. mdka was identified as a highly upregulated gene in pdgfrb+ cells during heart regeneration. However, pdgfrb but not mdka mutants show defects in heart regeneration after amputation. Our results demonstrate that heterogeneous pdgfrb+ cells are essential for coronary development and heart regeneration.
Assuntos
Vasos Coronários/crescimento & desenvolvimento , Vasos Coronários/metabolismo , Coração/fisiologia , Organogênese/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Regeneração/fisiologia , Animais , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Miócitos de Músculo Liso/metabolismo , Pericárdio/metabolismo , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologiaRESUMO
In this work, a biosensor based on surface plasmon field-enhanced florescence spectroscopy (SPFS) method was successfully constructed to detect the truncated form of cholera toxin, that is, its beta subunit (CTX-B). CTX-B is a relatively small molecule (12 kDa) and it was chosen as model analyte for the detection of protein toxins originated from waterborne pathogens. Recognition layer was prepared on gold-coated LaSFN9 glasses modified with 11-mercaptoundecanoic acid (11-MUA). Biotin-conjugated anti-CTX-B polyclonal antibody (B-Ab) was immobilized on streptavidin (SA) layer constructed on the 11-MUA-modified surface. CTX-B amount was determined with direct assay using B-Ab in surface plasmon resonance (SPR) mode and with sandwich assay in SPFS mode using Cy5-conjugated anti-CTX-B polyclonal antibody. Minimum detected CTX-B concentrations were 10 and 0.01 µg/ml with SPR and SPFS, respectively, showing the sensitivity of the SPFS system over the conventional one. The detection was done in 2-6 h, which was faster than both culture and polymerase chain reaction (PCR)-based methods. Stability tests were performed with SA-coated sensors (excluding B-Ab). In this form, the layer was stable after 30 days of storage in phosphate-buffered saline (PBS; 0.01 M, pH = 7.4) at +4°C. B-Ab layer was formed immediately on them before each measurement.
Assuntos
Técnicas Biossensoriais , Toxina da Cólera , Biotina/química , Ouro/química , Análise Espectral , Estreptavidina/química , Ressonância de Plasmônio de Superfície/métodosRESUMO
AIMS: Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. METHODS AND RESULTS: A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. CONCLUSION: A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.
Assuntos
Células Endoteliais , Células-Tronco Pluripotentes , Diferenciação Celular , Células-Tronco Embrionárias , Humanos , Análise de Sequência de RNARESUMO
Vascular endothelial growth factor C (Vegfc) activates its receptor, Flt4, to induce lymphatic development. However, the signals that act downstream of Flt4 in this context in vivo remain unclear. To understand Flt4 signaling better, we generated zebrafish bearing a deletion in the Flt4 cytoplasmic domain that eliminates tyrosines Y1226 and 1227. Embryos bearing this deletion failed to initiate sprouting or differentiation of trunk lymphatic vessels and did not form a thoracic duct. Deletion of Y1226/7 prevented ERK phosphorylation in lymphatic progenitors, and ERK inhibition blocked trunk lymphatic sprouting and differentiation. Conversely, endothelial autonomous ERK activation rescued lymphatic sprouting and differentiation in flt4 mutants. Interestingly, embryos bearing the Y1226/7 deletion formed a functional facial lymphatic network enabling them to develop normally to adulthood. By contrast, flt4 null larvae displayed hypoplastic facial lymphatics and severe lymphedema. Thus, facial lymphatic vessels appear to be the first functional lymphatic network in the zebrafish, whereas the thoracic duct is initially dispensable for lymphatic function. Moreover, distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genótipo , Hibridização In Situ , Vasos Linfáticos/embriologia , Mutação/genética , Fosforilação/genética , Fosforilação/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.
Assuntos
Deleção Cromossômica , Inversão Cromossômica , Peixe-Zebra/genética , Animais , Sequência de Bases , Sítios de Ligação , Pontos de Quebra do Cromossomo , Endonucleases/metabolismo , Ordem dos Genes , Células Germinativas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Dedos de ZincoRESUMO
The transcriptional repressor Rest (Nrsf) recruits chromatin-modifying complexes to RE1 'silencer elements', which are associated with hundreds of neural genes. However, the requirement for Rest-mediated transcriptional regulation of embryonic development and cell fate is poorly understood. Conflicting views of the role of Rest in controlling cell fate have emerged from recent studies. To address these controversies, we examined the developmental requirement for Rest in zebrafish using zinc-finger nuclease-mediated gene targeting. We discovered that germ layer specification progresses normally in rest mutants despite derepression of target genes during embryogenesis. This analysis provides the first evidence that maternal rest is essential for repression of target genes during blastula stages. Surprisingly, neurogenesis proceeds largely normally in rest mutants, although abnormalities are observed within the nervous system, including defects in oligodendrocyte precursor cell development and a partial loss of facial branchiomotor neuron migration. Mutants progress normally through embryogenesis but many die as larvae (after 12 days). However, some homozygotes reach adulthood and are viable. We utilized an RE1/NRSE transgenic reporter system to dynamically monitor Rest activity. This analysis revealed that Rest is required to repress gene expression in mesodermal derivatives including muscle and notochord, as well as within the nervous system. Finally, we demonstrated that Rest is required for long-term repression of target genes in non-neural tissues in adult zebrafish. Our results point to a broad role for Rest in fine-tuning neural gene expression, rather than as a widespread regulator of neurogenesis or cell fate.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurogênese , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Movimento Celular , Transcrição Gênica , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Inspired by nature, we designed organohydrogels (OHGs) consisting of a silk fibroin (SF) hydrogel as the continuous phase and the hydrophobic microinclusions based on semicrystalline poly(n-octadecyl acrylate) (PC18A) as the dispersed phase. SF acts as a self-emulsifier to obtain oil-in-water emulsions, and hence, it is a versatile and green alternative to chemical emulsifiers. We first prepared a stable oil-in-water emulsion without an external emulsifier by dispersing the n-octadecyl acrylate (C18A) monomer in an aqueous SF solution. To stabilize the emulsions for longer times, gelation in the continuous SF phase was induced by the addition of ethanol, which is known to trigger the conformational transition in SF from random coil to ß-sheet structures. In the second step, in situ polymerization of C18A droplets in the emulsion system was conducted under UV light in the presence of a photoinitiator to obtain high-strength OHGs with shape-memory function, and good cytocompatibility. The incorporation of hydrophilic N,N-dimethylacrylamide and noncrystallizable hydrophobic lauryl methacrylate units in the hydrogel and organogel phases of OHGs, respectively, further improved their mechanical and shape-memory properties. The shape-memory OHGs presented here exhibit switchable viscoelasticity and mechanics, a high Young's modulus (up to 4.3 ± 0.1 MPa), compressive strength (up to 2.5 ± 0.1 MPa), and toughness (up to 0.68 MPa).
Assuntos
Fibroínas , Fibroínas/química , Seda/química , Emulsões/química , Hidrogéis/química , Água/químicaRESUMO
A detailed understanding of the cell adhesion on polymeric surfaces is required to improve the performance of biomaterials. Quartz crystal microbalance with dissipation (QCM-D) as a surface-sensitive technique has the advantage of label-free and real-time monitoring of the cell-polymer interface, providing distinct signal patterns for cell-polymer interactions. In this study, QCM-D was used to monitor human fetal osteoblastic (hFOB) cell adhesion onto polycaprolactone (PCL) and chitosan (CH) homopolymer films as well as their blend films (75:25 and 25:75). Complementary cell culture assays were performed to verify the findings of QCM-D. The thin polymer films were successfully prepared by spin-coating, and relevant properties, i.e., surface morphology, ζ-potential, wettability, film swelling, and fibrinogen adsorption, were characterized. The adsorbed amount of fibrinogen decreased with an increasing percentage of chitosan in the films, which predominantly showed an inverse correlation with surface hydrophilicity. Similarly, the initial cell sedimentation after 1 h resulted in lesser cell deposition as the chitosan ratio increased in the film. Furthermore, the QCM-D signal patterns, which were measured on the homopolymer and blend films during the first 18 h of cell adhesion, also showed an influence of the different interfacial properties. Cells fully spread on pure PCL films and had elongated morphologies as monitored by fluorescence microscopy and scanning electron microscopy (SEM). Corresponding QCM-D signals showed the highest frequency drop and the highest dissipation. Blend films supported cell adhesion but with lower dissipation values than for the PCL film. This could be the result of a higher rigidity of the cell-blend interface because the cells do not pass to the next stages of spreading after secretion of their extracellular matrix (ECM) proteins. Variations in the QCM-D data, which were obtained at the blend films, could be attributed to differences in the morphology of the films. Pure chitosan films showed limited cell adhesion accompanied by low frequency drop and low dissipation.
RESUMO
BACKGROUND: Magnesium (Mg) enhances the bone regeneration, mineralization and attachment at the tissue/biomaterial interface. OBJECTIVE: In this study, the effect of Mg on mineralization/osseointegration was determined using (Ti,Mg)N thin film coated Ti6Al4V based plates and screws in vivo. METHODS: TiN and (Ti,Mg)N coated Ti6Al4V plates and screws were prepared using arc-PVD technique and used to fix rabbit femur fractures for 6 weeks. Then, mineralization/osseointegration was assessed by surface analysis including cell attachment, mineralization, and hydroxyapatite deposition on concave and convex sides of the plates along with the attachment between the screw and the bone. RESULTS: According to Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) analyses; cell attachment and mineralization were higher on the concave sides of the plates from both groups in comparison to the convex sides. However, mineralization was significantly higher on Mg-containing ones. The mean gray value indicating mineralized area after von Kossa staining was found as 0.48 ± 0.01 and 0.41 ± 0.04 on Mg containing and free ones respectively. Similarly, Fourier Transform Infrared Spectroscopy (FTIR) and X-ray diffraction (XRD) analyses showed that hydroxyapatite growth was abundant on the Mg-containing and concave sides of the plates. Enhanced mineralization and strong attachment to bone were also detected in EDS and SEM analyses of Mg-containing screws. CONCLUSION: These findings indicated that (Ti,Mg)N coatings can be used to increase attachment at the implant tissue interface due to accelerated mineralization, cell attachment, and hydroxyapatite growth.
Assuntos
Magnésio , Titânio , Animais , Coelhos , Magnésio/farmacologia , Titânio/química , Materiais Revestidos Biocompatíveis/química , Osseointegração , Durapatita/química , Microscopia Eletrônica de Varredura , Fêmur/cirurgia , Propriedades de SuperfícieRESUMO
A new dual-channel probe based on rhodamine B derivative (MSB) was successfully designed, synthesized, characterized by Nuclear Magnetic Resonance (NMR) Spectroscopy, Fourier Transform Infrared Spectrophotometer (FTIR), Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), X-ray Photoelectron Spectroscopy (XPS), and Single Crystal X-rayDiffraction, and the sensing abilities toward Fe3+ cation have been demonstrated and the probe was successfully utilized for fluorescence imaging of Fe3+ in living cells. The probe demonstrated quite fast, sensitive, and selective response to Fe3+ by causing an extreme enhancement in UV-vis and fluorescence spectroscopy techniques in the buffered aqueous media which makes MSB a dual-channel probe. While the color of MSB solution was initially light yellow, it turned pink in the presence of Fe3+, which provided highly selective naked-eye determination among several ions as alkaline, alkaline-earth, and transition metal ions. After that, the probe was easily applied to paper strips and real samples such as drinking waters and supplementary iron tablets for sensing Fe3+ in an aqueous solution. The detection limit (LOD) and the response time of the probe were determined as 4.85x10-9 M and 4 min, respectively, which are quite lower compared with other rhodamine based Fe3+ sensors in the literature. According to Job's plot, 1H NMR titration, MALDI-TOF MS, XPS, and DFT study techniques, the complexation ratio between MSB and Fe3+ was found as 1:1. Moreover, the spectral response was reversible with alternately addition of Fe3+ or Na2EDTA to the MSB solution. In addition, fluorescence imaging in NIH/3T3 mouse fibroblast cells and studies in real samples with a quite high recovery rate exhibited that the probe is qualified for detection of Fe3+ ion with multiple practical usages.
Assuntos
Imagem Óptica , Smartphone , Animais , Camundongos , Rodaminas , Espectroscopia Fotoeletrônica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The objective of the study is, for the first time, to construct a new near infrared (NIR) fluorophore, spectrophotometric, colorimetric, ratiometric, and turn-on probe (CSME) based on chromenylium cyanine platform decorated with methionine biomolecule to provide an efficient solution for critical shortcoming to be encountered for analysis of hazardous Hg2+ in environment and living cell. The CSME structure and its interaction with Hg2+ ion were evaluated by NMR, FTIR, MS, UV-Vis and fluorescence methods as well as Density Functional Theory (DFT) calculations. The none fluorescence CSME having spirolactam ring only interacted with Hg2+ in aqueous solution including competing ions. This interaction caused the fluorescence CSME with opened spirolactam form which exhibited spectral and colorimetric changes in the NIR region. The probe based on UV-Vis and fluorescence techniques respond in 90 s, has wide linear ranges (for UV-Vis: 6.29 × 10-8 - 1.86 × 10-4 M; for fluorescence: 9.49 × 10-9 - 1.13 × 10-5 M), and has a lower Limit of Detection (LOD) value (for fluorescence: 4.93 × 10-9 M, 0.99 ng/mL) than the value predicted by the US Environmental Protection Agency (EPA) organization. Hg2+ analysis was performed in drinking and tap water with low Relative Standard Deviation (RSD) values and high recovery. Smartphone and living cell applications were successfully performed for colorimetric sensing Hg2+ in real samples and 3T3 cells, respectively.
Assuntos
Corantes Fluorescentes , Mercúrio , Camundongos , Animais , Corantes Fluorescentes/química , Metionina , Água/química , Racemetionina , Mercúrio/análiseRESUMO
Cell-type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild-type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity.
Assuntos
Relógios Biológicos , Fatores de Crescimento de Fibroblastos/metabolismo , Mesoderma/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas Repressoras/genética , Transdução de Sinais , Transativadores/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Cardiac tissue engineering focusing on biomaterial scaffolds incorporating cells from different sources has been explored to regenerate or repair damaged area as a lifesaving approach.The aim of this study was to evaluate the cardiomyocyte differentiation potential of human adipose mesenchymal stem cells (hAD-MSCs) as an alternative cell source on silk fibroin (SF) scaffolds for cardiac tissue engineering. The change in surface morphology of SF scaffolds depending on SF concentration (1-6%, w/v) and increase in their porosity upon application of unidirectional freezing were visualized by scanning electron microscopy (SEM). Swelling ratio was found to increase 2.4 fold when SF amount was decreased from 4% to 2%. To avoid excessive swelling, 4% SF scaffold with swelling ratio of 10% (w/w) was chosen for further studies.Biodegradation rate of SF scaffolds depended on enzymatic activity was found to be 75% weight loss of SF scaffolds at the day 14. The phenotype of hAD-MSCs and their multi-linage potential into chondrocytes, osteocytes, and adipocytes were shown by flow cytometry and immunohistochemical staining, respectively.The viability of hAD-MSCs on 3D SF scaffolds was determined as 90%, 118%, and 138% after 1, 7, and 14 days, respectively. The use of 3D SF scaffolds was associated with increased production of cardiomyogenic biomarkers: α-actinin, troponin I, connexin 43, and myosin heavy chain. The fabricated 3D SF scaffolds were proved to sustain hAD-MSCs proliferation and cardiomyogenic differentiation therefore, hAD-MSCs on 3D SF scaffolds may useful tool to regenerate or repair damaged area using cardiac tissue engineering techniques.
Assuntos
Fibroínas , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos , Regeneração , Seda/metabolismo , Alicerces Teciduais , Tecido Adiposo/citologia , Materiais Biocompatíveis , Diferenciação Celular , Proliferação de Células , Condrócitos , Humanos , Porosidade , Engenharia Tecidual/métodosRESUMO
The NF-ĸB transcription factor is a critical regulator of immune homeostasis and inflammatory responses and is a critical factor in the pathogenesis of inflammatory disease. The pathways to NF-ĸB activation are paradigms for signal-induced ubiquitination and proteasomal degradation, control of transcription factor function by subcellular localisation, and the control of gene transcription and physiological processes by signal transduction mechanisms. Despite the importance of NF-ĸB in disease, the NF-ĸB pathway remains unexploited for the treatment of inflammatory disease. Our understanding of NF-ĸB comes mostly from studies of transgenic mice and cell lines where components of the pathway have been deleted or over expressed. Recent advances in quantitative proteomics offer new opportunities to understand the NF-ĸB pathway using the absolute abundance of individual pathway components. We have analysed available quantitative proteomic datasets to establish the structure of the NF-ĸB pathway in human immune cells under both steady state and activated conditions. This reveals a conserved NF-κB pathway structure across different immune cell lineages and identifies important differences to the current model of the NF-ĸB pathway. These include the findings that the IKK complex in most cells is likely to consist predominantly of IKKß homodimers, that the relative abundancies of IκB proteins show strong cell type variation, and that the components of the non-canonical NF-ĸB pathway are significantly increased in activated immune cells. These findings challenge aspects of our current view of the NF-κB pathway and identify outstanding questions important for defining the role of key components in regulating inflammation and immunity.
Assuntos
NF-kappa B , Proteômica , Animais , Humanos , Quinase I-kappa B/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de SinaisRESUMO
In this study, three natural biomaterials, Locust bean gum (LBG), Xanthan gum (XG), and Mastic gum (MG), were combined to form cryogel scaffolds. Thermal and chemical characterizations revealed the successful blend formation from LBG-XG (LX) and LBG-XG-MG (LXM) polymers. All blends resulted in macro-porous scaffolds with interconnected pore structures under the size of 400⯵m. The swollen cryogels had similar mechanical properties compared with other polysaccharide-based cryogels. The mean tensile and compressive modulus values of the wet cryogels were in the range of 3.5-11.6â¯kPa and 82-398â¯kPa, respectively. The sustained release of the small molecule Kartogenin from varying concentrations and ratios of cryogels was in between 32 and 66% through 21â¯days of incubation. Physical, mechanical, and chemical properties make LX and LXM polysaccharide-based cryogels promising candidates for cartilage and other soft tissue engineering, and drug delivery applications.
Assuntos
Criogéis/química , Preparações de Ação Retardada/química , Alicerces Teciduais/química , Anilidas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Criogéis/toxicidade , Preparações de Ação Retardada/toxicidade , Liberação Controlada de Fármacos , Galactanos/química , Galactanos/toxicidade , Mananas/química , Mananas/toxicidade , Resina Mástique/química , Resina Mástique/toxicidade , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Ácidos Ftálicos/química , Gomas Vegetais/química , Gomas Vegetais/toxicidade , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/toxicidade , Porosidade , Ratos Sprague-Dawley , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodosRESUMO
Since its discovery over 30 years ago the NF-ĸB family of transcription factors has gained the status of master regulator of the immune response. Much of what we understand of the role of NF-ĸB in immune development, homeostasis and inflammation comes from studies of mice null for specific NF-ĸB subunit encoding genes. The role of inflammation in diseases that affect a majority of individuals with health problems globally further establishes NF-ĸB as an important pathogenic factor. More recently, genomic sequencing has revealed loss of function mutations in the NFKB1 gene as the most common monogenic cause of common variable immunodeficiencies in Europeans. NFKB1 encodes the p105 subunit of NF-ĸB which is processed to generate the NF-ĸB p50 subunit. NFKB1 is the most highly expressed transcription factor in macrophages, key cellular drivers of inflammation and immunity. Although a key role for NFKB1 in the control of the immune system is apparent from Nfkb1-/- mouse studies, we know relatively little of the role of NFKB1 in regulating human macrophage responses. In this study we use the THP1 monocyte cell line and CRISPR/Cas9 gene editing to generate a model of NFKB1-/- human macrophages. Transcriptomic analysis reveals that activated NFKB1-/- macrophages are more pro-inflammatory than wild type controls and express elevated levels of TNF, IL6, and IL1B, but also have reduced expression of co-stimulatory factors important for the activation of T cells and adaptive immune responses such as CD70, CD83 and CD209. NFKB1-/- THP1 macrophages recapitulate key observations in individuals with NFKB1 haploinsufficiency including decreased IL10 expression. These data supporting their utility as an in vitro model for understanding the role of NFKB1 in human monocytes and macrophages and indicate that of loss of function NFKB1 mutations in these cells is an important component in the associated pathology.
Assuntos
Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Inflamação/genética , Macrófagos/metabolismo , Subunidade p50 de NF-kappa B/genética , Transcriptoma , Imunidade Adaptativa , Sistemas CRISPR-Cas , Citocinas/genética , Citocinas/metabolismo , Humanos , Imunidade Celular , Inflamação/imunologia , Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Subunidade p50 de NF-kappa B/deficiência , Fenótipo , RNA-Seq , Células THP-1 , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Bacterial infections and lack of osseointegration may negatively affect the success of titanium (Ti) implants. In the present study, a functional coating composed of chitosan (CS) microspheres and nano hydroxyapatite (nHA) was prepared to obtain antimicrobial Ti implants with enhanced bioactivity. First, the chitosan microspheres were fixed to Ti surfaces activated by alkali and heat treatment, then nHA coatings were precipitated onto these surfaces. Ciprofloxacin was loaded into the microspheres using two different procedures; encapsulation and diffusion. Scanning electron microscopy micrographs of the modified Ti surfaces showed that the coating was successfully deposited onto the Ti surfaces and stable for 30 days in PBS. The drug was completely released from free microspheres loaded by encapsulation in 21 days whereas only 89% release was observed after immobilization. The burst release also decreased from ca. 55% to ca. 35%. The release was further reduced following the nHA precipitation. The modified Ti surfaces showed antimicrobial activity based on the bacterial time-kill assay using S. aureus, but the efficiency was affected by both nHA precipitation and drug loading strategy. Highest antimicrobial activity was seen in the samples without nHA layer, and when the drug was loaded by diffusion. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed that nHA on the surface enhanced HA growth in simulated body fluid for 3 weeks, showing increased osseointegration potential. Therefore, the proposed coating may be used to prevent Ti implant failure originated from bacterial infection and/or low bioactivity.
Assuntos
Antibacterianos/química , Quitosana/química , Materiais Revestidos Biocompatíveis/química , Sistemas de Liberação de Fármacos por Nanopartículas/química , Titânio/química , Antibacterianos/farmacologia , Quitosana/metabolismo , Materiais Revestidos Biocompatíveis/metabolismo , Liberação Controlada de Fármacos , Durapatita/química , Humanos , Microesferas , Osseointegração , Próteses e Implantes , Staphylococcus aureus/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
Magnesium (Mg) based implants such as plates and screws are often preferred to treat bone defects because of the positive effects of magnesium in bone growth and healing. Their low corrosion resistance, however, leads to fast degradation and consequently failure before healing was completed. Previously, we developed Mg doped titanium nitrate (TiN) thin film coatings to address these limitations and demonstrated that <10 at% Mg doping led to enhanced mineralization in vitro. In the present study, in vivo performance of (Ti,Mg)N coated Ti6Al4V based plates and screws were studied in the rabbit model. Bone fractures were formed on femurs of 16 rabbits and then fixed with either (Ti,Mg)N coated (n = 8) or standard TiN coated (n = 8) plates and screws. X-ray imaging and µCT analyses showed enhanced bone regeneration on fracture sites fixed with (Ti,Mg)N coated plates in comparison with the Mg free ones. Bone mineral density, bone volume, and callus volume were also found to be 11.4, 23.4, and 42.8% higher, respectively, in accordance with µCT results. Furthermore, while TiN coatings promoted only primary bone regeneration, (Ti,Mg)N led to secondary bone regeneration in 6 weeks. These results indicated that Mg presence in the coatings accelerated bone regeneration in the fracture site. (Ti,Mg)N coating can be used as a practical method to increase the efficiency of existing bone fixation devices of varying geometry.