RESUMEN
In this work, three different modified cements, control apatite/beta-tricalcium phosphate cement (CPC), polymeric CPC (p-CPC), and bioactive glass added polymeric cement (p-CPC/BG) were evaluated regarding their physical properties and the responses of primary human osteoblast cells (HObs) and mesenchymal stem cells (MSCs). Although polyacrylic acid (PAA) increased compressive strength and Young's modulus of the cement, it could cause poor apatite phase formation, a prolonged setting time, and a lower degradation rate. Consequently, bioactive glass (BG) was added to PAA/cement to improve its physical properties, such as compressive strength, Young's modulus, setting time, and degradation. For in vitro testing, HObs viability was assessed under two culture systems with cement-preconditioned medium (indirect) and with cement (direct). HObs viability was examined in direct contact with cements treated by different prewashing conditions. HObs presented a more well spread morphology on cement soaked in medium overnight, as compared to other cements with no treatment and washing in PBS. In addition, the proliferation, differentiation, and total collagen production of both HObs and MSCs adhered to the cement were detected. Cells showed excellent proliferation on PAA/cement and PAA/BG/cement. Furthermore, the higher released Si ion and lower acidosis of PAA/BG/cement-conditioned medium resulted in an increase in osteogenic differentiation (HObs and MSCs) and enhanced collagen production (HObs in osteogenic medium and MSCs in control medium). Therefore, our findings suggest that BG incorporated PAA/apatite/ß-TCP cement could be a promising formula for bone repair applications.
Asunto(s)
Apatitas , Células Madre Mesenquimatosas , Humanos , Apatitas/farmacología , Cementos para Huesos/farmacología , Osteogénesis , Fosfatos de Calcio/farmacología , Colágeno , OsteoblastosRESUMEN
Injectable gels that support load are desirable for restoring the mechanical properties of degenerated load-bearing tissue. As these gels become increasingly sophisticated, the need to remotely image them and monitor their swelling increases. However, imaging such gels and monitoring their swelling using noninvasive means is challenging. Here, we use a very low concentration of near-infrared (NIR) core-shell-shell (CSS) reporter nanoparticles to both image and monitor swelling changes of two load-supporting gels. The load-supporting injectable gel consisted of covalently interlinked pH-responsive microgel (MG) particles. The latter gel was not cytotoxic and is termed a doubly cross-linked microgel (DX MG). Inclusion of a complementary fluorescent dye enabled ratiometric monitoring of gel swelling changes in response to pH via nonradiative resonance energy transfer (NRET). In addition, changes in the CSS nanoparticle emission intensity provided a NIR-only method that could also be used to monitor gel swelling. The gel was able to be imaged using NIR light, after being subcutaneously injected into a tissue model. To demonstrate versatility of our approach, CSS and the dye were included within a model implantable gel (poly(acrylamide/acrylic acid)) and fluorescent detection of swelling investigated. Because the concentrations of the reporting species were too low to affect the mechanical properties, our approach to remote gel imaging and swelling monitoring has good potential for application in injectable gels and implants.
Asunto(s)
Materiales Biocompatibles/farmacología , Geles/farmacología , Nanopartículas/química , Núcleo Pulposo/efectos de los fármacos , Materiales Biocompatibles/química , Citotoxinas/química , Citotoxinas/farmacología , Transferencia de Energía , Colorantes Fluorescentes/química , Geles/efectos adversos , Humanos , Concentración de Iones de Hidrógeno , Metacrilatos/química , Metacrilatos/farmacología , Polímeros/química , Polímeros/farmacología , Espectroscopía Infrarroja Corta , Propiedades de SuperficieRESUMEN
Whilst hydrogels and hollow particles both continue to attract much attention in the literature there are few examples of hydrogel composites containing hollow particles. Here, we study composite polyacrylamide (PAAm) hydrogels containing micrometer-sized pH-responsive shell-crosslinked hollow particles (abbreviated as HPXL) based on poly(methylmethacrylate-co-methacrylic acid) functionalised with glycidyl methacrylate (GMA). The HPXL particles were prepared using our scaleable emulsion template method and inclusion of GMA was found to promote spherical hollow particle formation. The pendant vinyl groups from GMA enabled shell-crosslinked hollow particles to be prepared prior to formation of the PAAm/HPXL composite gels. The morphologies of the particles and composite gels were studied by optical microscopy, confocal laser scanning microscopy and scanning electron microscopy. Dynamic rheology measurements for the composite gels showed that the modulus variation with HPXL concentration could be described by a percolation model with a HPXL percolation threshold concentration of 4.4 wt% and a scaling exponent of 2.6. The composite gels were pH-responsive and largely maintained their mechanical properties over the pH range 4.0 to 8.0. Because the composite gels had tuneable mechanical properties (with modulus values up to 530 kPa) and were pH-responsive they are potential candidates for future wound healing or membrane applications.
Asunto(s)
Resinas Acrílicas/química , Hidrogeles/química , Cápsulas/química , Reactivos de Enlaces Cruzados/química , Elasticidad , Concentración de Iones de Hidrógeno , Ácidos Polimetacrílicos/químicaRESUMEN
A new family of pH-responsive microgel-colloidosomes was prepared using microgel particles as the building blocks and macro-crosslinker. Our simple and versatile method used covalent inter-linking of vinyl-functionalised microgel particles adsorbed to oil droplets to form shells of doubly crosslinked microgels (DX MGs) and was demonstrated using two different microgel types.
RESUMEN
The hematon is a three-dimensional aggregate of cells which is able to produce all blood types. To be able to do this, it must be able to create within the cell aggregate a microenvironment which enables haematopoietic stem cell maintenance, renewal and differentiation. A first step was taken towards the creation of artificial hematopoietic stem cell microniches in vitro by the creation with dielectrophoresis of hemispherical cell aggregates of a height of 50-100 mum with a defined internal architecture similar to that of a putative hematon. It is shown that, after their dielectrophoretic manipulation, the cells remain viable and active. Cells within the aggregate are in direct contact with each other, potentially allowing direct cell-cell communication within the cell construct. Some cell immobilisation methods are explored for further stabilising the 3-D organisation of the cell aggregate after its formation. The introduction of traceable individual cells into the artificial microniche is demonstrated.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Animales , Agregación Celular/fisiología , Comunicación Celular , Diferenciación Celular/fisiología , Células Inmovilizadas/citología , Células Inmovilizadas/fisiología , Humanos , Células Jurkat , RatonesRESUMEN
THOC5 is a nuclear/cytoplasmic protein member of the spliceosome complex which potentiates C/EBP expression in adipocyte differentiation. As C/EBP family members are important regulators of myelopoiesis and THOC5 is highly expressed in neutrophil/macrophage progenitor cells we assessed the role of THOC5 in cytokine-stimulated monocytic development. M-CSF stimulated maturation of the NFS60 cell line was associated with enhanced THOC5 expression and phosphorylation. THOC5 was also shown to form a complex with C/EBPbeta. Ectopic expression of THOC5 mimicked M-CSF mediated cell maturation and enhanced protein expression of the myeloid transcription factors C/EBPbeta, C/EBPalpha, Pu-1 and also GAB2 (a PI-3 Kinase and macrophage development regulator). Increased THOC5 expression also mimicked M-CSF stimulated increases in the lipid second messenger PtdInsP(3). Inhibition of THOC5-induced increases in PtdInsP(3) levels abrogated the elevated levels of C/EBPbeta. Thus THOC5 expression can potentiate receptor signalling to transcription factor expression and monocyte differentiation.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/farmacología , Proteínas Nucleares/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular , Humanos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Monocitos/citología , Monocitos/metabolismo , Proteínas Nucleares/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Transducción de Señal , Factores de Transcripción/genética , TransfecciónRESUMEN
The fusion protein TEL/PDGFRB is associated with chronic myelomonocytic leukaemia and has intrinsic tyrosine kinase activity. The effects of TEL/PDGFRB were assessed using the multipotent haemopoietic cell line FDCP-Mix. In the absence of growth factors, TEL/PDGFRB expression increased survival that was associated with elevated levels of phosphatidylinositol 3,4,5 trisphosphate (PIP3). Whilst TEL/PDGFRB had subtle effects on the growth factor requirements it had a profound effect on differentiation. The cells became refractory to cytokine-stimulated development, showing limited maturation but failing to produce fully mature cells. We have previously identified the spliceosome protein THOC5 as a target in macrophage colony-stimulating factor signalling and a protein involved in the regulation of transcription factor expression. TEL/PDGFRB expression increased the expression and phosphorylation of THOC5. Elevated expression of THOC5 increased PIP3 levels and decreased apoptosis. Mass spectrometry was used to identify a site for TEL/PDGFRB-mediated phosphorylation on THOC5, which was shown to be a target for a number of other leukaemogenic tyrosine kinases. Thus, THOC5 is a novel target for modulation of signal transduction with a potential role in leukaemogenesis.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Inositol/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/fisiología , Apoptosis/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Supervivencia Celular/fisiología , Sustancias de Crecimiento/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Proteínas Nucleares/fisiología , Proteínas de Fusión Oncogénica/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRbeta, FIP1/PDGFRalpha, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach.
Asunto(s)
Leucemia/enzimología , Espectrometría de Masas/métodos , Proteínas Oncogénicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma/análisis , Proteómica/métodos , Animales , Línea Celular , Quimiotaxis , Exones , Perfilación de la Expresión Génica , Leucemia/genética , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas/genética , Biosíntesis de Proteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteoma/genética , Proteoma/metabolismoRESUMEN
Hematopoiesis is sustained by the proliferation and development of an extremely low number of hematopoietic stem cells resident in the bone marrow. These stem cells can migrate from their bone marrow microenvironment and can be found at low levels in the peripheral blood. The factors that regulate egress or ingress of the stem cells from the marrow include cytokines and chemokines. This process of stem cell trafficking is fundamental to both stem cell biology and stem cell transplantation. We show that primitive hematopoietic cells with cobblestone area-forming cell activity express receptors for and display enhanced motility in response to a new class of stem cell agonists, namely lysophospholipids. These agents synergistically promote chemokine-stimulated cell chemotaxis, a process that is crucial in stem cell homing. The response to lysophospholipids is mediated by Rac, Rho, and Cdc42 G proteins and the hematopoietic-specific guanyl nucleotide exchange factor Vav 1. Inhibitor studies also show a critical role for phosphatidylinositol 3 kinase (PI3K). Lipid mediators, therefore, regulate the critical process of primitive hematopoietic cell motility via a PI3K- and Vav-dependent mechanism and may govern stem cell movement in vivo. These results are of relevance to understanding stem cell trafficking during bone marrow transplantation.