A 3-D in vitro co-culture model of mammary gland involution.

Involution is a process whereby the mammary gland undergoes extensive tissue remodelling involving exquisitely coordinated cell death, extracellular matrix degradation and adipose tissue regeneration following the weaning of offspring. These processes are mediated in part through Jak/Stat signalling pathways, which can be deregulated in breast cancer. Synthetic in vitro analogues of the breast could become important tools for studying tumorigenic processes, or as personalized drug discovery platforms and predictors of therapeutic response. Ideally, such models should support 3D neo-tissue formation, so as to recapitulate physiological organ function, and be compatible with high-throughput screening methodologies. We have combined cell lines of epithelial, stromal and immunological origin within engineered porous collagen/hyaluronic acid matrices, demonstrating 3D-specific molecular signatures. Furthermore seeded cells form mammary-like branched tissues, with lobuloalveolar structures that undergo inducible involution phenotypes reminiscent of the native gland under hormonal/cytokine regulation. We confirm that autophagy is mediated within differentiated mammary epithelial cells in a Stat-dependent manner at early time points following the removal of a prolactin stimulus (H/WD). In addition, epithelial cells express markers of an M2 macrophage lineage under H/WD, a process that is attenuated with the introduction of the monocyte/macrophage cell line RAW 264.7. Thus, such 3D models are suitable platforms for studying cell-cell interactions and cell death mechanisms in relation to cancer.This is the accepted manuscript. The final version is available from RSC at http://pubs.rsc.org/en/content/articlehtml/2014/ib/c3ib40257f

Impact of UV- and carbodiimide-based crosslinking on the integrin-binding properties of collagen-based materials.

Collagen constructs are widely used for tissue engineering. These are frequently chemically crosslinked, using EDC, to improve their stability and tailor their physical properties. Although generally biocompatible, chemical crosslinking can modify crucial amino acid side chains, such as glutamic acid, that are involved in integrin-mediated cell adhesion. Instead UV crosslinking modifies aromatic side chains. Here we elucidate the impact that EDC, in combination with UV, exerts on the activity of integrin-binding motifs. By employing a model cell line that exclusively utilises integrin α2β1, we found that whilst EDC crosslinking modulated cell binding, from cation-dependent to cation-independent, UV-mediated crosslinking preserved native-like cell binding, proliferation and surface colonisation. Similar results were observed using a purified recombinant I-domain from integrin α1. Conversely, binding of the I-domain from integrin α2 was sensitive to UV, particularly at low EDC concentrations. Therefore, from this in vitro study, it appears that UV can be used to augment EDC whist retaining a specific subset of integrin-binding motifs in the native collagen molecule. These findings, delineating the EDC- and UV-susceptibility of cell-binding motifs, permit controlled cell adhesion to collagen-based materials through specific integrin ligation in vitro. However, in vivo, further consideration of the potential response to UV wavelength and dose is required in the light of literature reports that UV initiated collagen scission may lead to an adverse inflammatory response. STATEMENT OF SIGNIFICANCE: Recently, there has been rapid growth in the use of extracellular matrix-derived molecules, and in particular collagen, to fabricate biomaterials that replicate the cellular micro-environment. Often chemical or physical crosslinkers are required to enhance the biophysical properties of these materials. Despite extensive use of these crosslinkers, the cell-biological consequences have not been ascertained. To address this, we have investigated the integrin-binding properties of collagen after chemically crosslinking with EDC and physically crosslinking with UV-irradiation. We have established that whilst EDC crosslinking abates all of the integrin binding sites in collagen, UV selectively inhibits interaction with integrin-α2 but not -α1. By providing a mechanistic model for this behaviour, we have, for the first time, defined a series of crosslinking parameters to systematically control the interaction of collagen-based materials with defined cellular receptors.The authors would like to thank the EPSRC [Fellowship EP/N019938/1] the ERC [Advanced Grant 320598 3D-E] and the British Heart Foundation [Special Project SP/15/7/31561] for providing financial support for this project. DVB was funded by the People's Programme of the EU 7th Framework Programme [RAE no: PIIF-GA-2013-624904]

Composites dentales fotocurados con diferentes diluyentes y rellenos inorgánicos

Se realizó un estudio cinético de la fotopolimerización de composites preparados con óxidos de silicio de diferente tamaño de partícula y morfología (cuarzo y aerosil) utilizando como diluyentes y-metacriloxipropil trimetoxisilano (y-MPS) y dimetacrilato de tetraetilenglicol (DMTEEG). Como monomero base se empleó Bis-GMA y como sistema iniciador el par canforquinona-metacrilato de 2-N,N-dimetilaminoetilo en concentraciones de 0,5 % en peso con respecto a la matriz orgánica. Se pudo observar que la velocidad de polimerización (Rp) de los composites de cuarzo con diferentes diluyentes crece con el aumento de la intensidad de luz incidente (Io) y que la dependencia de Rp con la raíz cuadrada de Io tiene un carácter lineal. En presencia de DMTEEG los valores de Rp son aproximadamente dos veces mayores que en el caso del y-MPS. A los composites híbridos (cuarzo y aerosil) con y-MPS como diluyente se les realizaron estudios cinéticos tanto de las muestras recién preparadas como después de 3 semanas de almacenamiento con el objetivo de estimar la estabilidad de este sistema en el tiempo. Se pudo determinar que para ambos sistemas los valores de Rp y de las conversiones son muy próximos aunque un poco superiores para los composites que estuvieron 3 semanas almacenados lo que puede ser la consecuencia de una mayor compatibilidad entre el relleno y la matriz orgánica que se alcanza. Al comparar entre sí los composites con macro-relleno (cuarzo) y los híbridos se puede afirmar que las características del relleno prácticamente no influyen sobre los valores de Rp

Optimisation of UV irradiation as a binding site conserving method for crosslinking collagen-based scaffolds.

Short wavelength (λ = 254 nm) UV irradiation was evaluated over a range of intensities (0.06 to 0.96 J/cm(2)) as a means of cross-linking collagen- and gelatin-based scaffolds, to tailor their material characteristics whilst retaining biological functionality. Zero-link carbodiimide treatments are commonly applied to collagen-based materials, forming cross-links from carboxylate anions (for example the acidic E of GFOGER) that are an essential part of integrin binding sites on collagen. Cross-linking these amino acids therefore disrupts the bioactivity of collagen. In contrast, UV irradiation forms bonds from less important aromatic tyrosine and phenylalanine residues. We therefore hypothesised that UV cross-linking would not compromise collagen cell reactivity. Here, highly porous (~99 %) isotropic, collagen-based scaffolds were produced via ice-templating. A series of scaffolds (pore diameters ranging from 130-260 μm) with ascending stability in water was made from gelatin, two different sources of collagen I, or blends of these materials. Glucose, known to aid UV crosslinking of collagen, was added to some lower-stability formulations. These scaffolds were exposed to different doses of UV irradiation, and the scaffold morphology, dissolution stability in water, resistance to compression and cell reactivity was assessed. Stabilisation in aqueous media varied with both the nature of the collagen-based material employed and the UV intensity. Scaffolds made from the most stable materials showed the greatest stability after irradiation, although the levels of cross-linking in all cases were relatively low. Scaffolds made from pure collagen from the two different sources showed different optimum levels of irradiation, suggesting altered balance between stabilisation from cross-linking and destabilisation from denaturation. The introduction of glucose into the scaffold enhanced the efficacy of UV cross-linking. Finally, as hypothesized, cell attachment, spreading and proliferation on collagen materials were unaffected by UV cross-linking. UV irradiation may therefore be used to provide relatively low level cross-linking of collagen without loss of biological functionality.The authors would like to thank the British Heart Foundation (Grants NH/11/1/28922 and RG/15/4/31268), The Welcome Trust (Grant 094470/Z/10/Z), the ERC Advanced Grant 320598 3D-E and EPSRC Doctoral Training Account for providing financial support for this project. D. V. Bax is funded by the Peoples Programme of the EU 7th Framework Programme (RAE no: PIIF-GA-2013-624904) and also supported by an EPSRC IKC Proof of Concept Award.This is the final version of the article. It was first available from Springer via http://dx.doi.org/10.1007/s10856-015-5627-

The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen.

Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons.The work was supported in Department of Biochemistry by New Horizons and Programme grants from British Heart Foundation (NH/11/1/28922 and RG/09/003/27122) and a Biomedical Resource grant from the Wellcome Trust (094470/Z/10/Z). In Department of Materials Science, funding was from the Peoples Programme of the EU 7th Framework Programme (RAE no: PIIFGA-2013-624904, to DVB), a Proof of Concept grant from the EPSRC Medical Technologies IKC, and an ERC Advanced Grant 320598 3D-E (to REC).This is the final version of the article. It first appeared from Elsevier via https://doi.org/10.1016/j.biomaterials.2016.01.04

Composites de hidroxiapatita con matrices biodegradables

Mucho se ha publicado sobre el empleo de la hídroxíapatita (HAP) como un sustituto del hueso en diferentes áreas de la medicina Desde hace algunos años, se viene criticando el uso de este sistema debido a la capacidad de migración de las partí­culas de HAP del sitio del implante. Su posible estabilización se viene investigando y se han desarrollado métodos para lograrlo. Se prepararon composites de hídroxíapatita con quitosana (CHI) y ácido acrílico (AA) con el objetivo de obtener sistemas biodegradables con características osteoconductoras. Se estudió la influencia de dife­rentes factores: método de preparación, contenido de HAP y la concentración de CID sobre la cinética de fotopolimerización de estos composites bajo irradación con luz visible. Como sistema de iniciación se empleó el par canforquinona-alcohol N,N­dirnetilaminobencílico, ambos al 1 % en peso con respecto al AA. Se encontró que el método de preparación influye sobre las propiedades fisico-quínúcas y cinéticas de los composites. Por ejemplo, para los sistemas preparados sobre la base de geles de AA y CHl previamente hinchada con agua, se observan menores velocidades de polimeriza­ción (R,) que para los composites obtenidos a partir de los llamados geles inversos, donde el AA se pone previamente en contacto con la CHI (antes de la formación de su gel con agua)

Síntesis y caracterización del copolímero poli(metacrilato de metilo-estireno) obtenido mediante polimerización en suspensión

Se reporta el estudio del efecto de la composición del copolímero metacrilato de metilo (MMA) y estireno (STY) sobre la distribución de masas moleculares y la capacidad de embebimiento de los polímeros utilizados para cementos óseos. La síntesis de los copolímeros se llevó a cabo empleando la polimerización en suspensión. La fracción molar de MMA en la mezcla reaccionante (f.) se modificó entre 0,96 y 0,60. La composición real del copolímero fue analizada por RMN-H' y se determinó que variaba entre 0,98 y 0,76. Con el aumento de la concentración de STY en la mezcla reaccionante se obtuvieron disminuciones importantes en el peso molecular del copolímero. Los resultados indican que el copolímero de composición 0,80 MMA y 0,20 STY en la mezcla de alimentación resulta más apropiado para la preparación de cementos óseos. Se estudió el efecto de la velocidad de agitación (N) en el intervalo de 400 a 700 r/min sobre el tamaño de partícula. Se encontró que el aumento de N en el intervalo de 400 a 600 r/min produce una disminución significativa del tamaño de partícula. Sin embargo, la variación del tamaño de partícula con el aumento de N de 600 a 700 r/min fue poco significativa. Se comprobó que la relación entre el diámetro final promedio de las partículas y la velocidad de agitación puede ser descrita mediante la ecuación simplificada de Hopff. Los polímeros obtenidos con MMA/ST 0,8/0,2 y velocidades de agitación entre 600 y 700 r/min reúnen los requisitos de masas moleculares promedio

Evaluación de resinas compuestas fotopolimerizables preparadas con dos tipos de aerosil y dos sistemas monoméricos

Se prepararon formulaciones de composites dentales fotopolimerizables, las cuales fueron evaluadas mediante la determinación de la profundidad de curado, resistencia a la compresión, resistencia a la compresión diametral, absorción y solubilidad en agua. Las formulaciones se prepararon con los sistemas Bis-GMA/DMTEEG/SIPERNAT D 10 (SD); Bis-GMA/ DMTEEG/FK310 (F) y Bis-GMA/MPS/FK310 (M). Con los dos primeros sistemas se trata de estudiar el comportamiento del SIPERNAT D 10 (relleno hidrófobo) y el FK310 (relleno hidrófilo). El tercer sistema cambia el monómero diluente, tratando el relleno hidrófilo directamente con el silano con vistas a mejorar la adhesión matriz relleno y de esta forma, las propiedades ya mencionadas. Los composites preparados con los sistemas SD y F presentan las mejores propiedades, cumpliendo la mayoría de ellas con los valores reportados en la norma. Los composites preparados con el sistema M presentan valores pequeños de profundidad de curado y propiedades mecánicas y elevados de absorción y solubilidad en agua. La adición directa del silano en este caso no ha sido efectiva

Cementos óseos acrílicos modificados con hidroxiapatita. Parte I. Cinética de polimerización

En Cirugía Ortopédica uno de los principales problemas es el método de fijación de las prótesis al cuerpo humano. Desde finales de los años cincuenta se ideó la técnica de fijar prótesis metálicas con cementos de poli(metacrilato de metilo) al canal femoral en reconstrucciones de cadera. Ello dio lugar a que por primera vez se obtuvieran resultados satisfactorios en este tipo de cirugía y hoy día se estima que existan más de 12 millones de personas con implantes artificiales. En estos 40 años la técnica de cementación ha demostrado una efectividad de un 80 % a los 12 a 15 años de implantación, lo cual, no es suficiente para el tratamiento de personas jóvenes. Debido a ello diferentes modificaciones se han realizado a la composición de los cementos con vistas a mejorar las deficiencias que conllevan al fracaso de las prótesis a largo plazo. Una de las variantes que puede resultar en una mejora de la fijación secundaria de los cementos es el empleo de rellenos bioactivos de hidroxiapatita en la matriz acrílica. En el presente trabajo se determinó el efecto de tres tipos de hidroxiapatitas con diferente tratamiento térmico en la cinética de polimerización (grado de conversión y temperatura máxima de polimerización) de un cemento óseo. Para ello se realizaron experimentos de fraguado del cemento y se siguió la cinética de reacción mediante Calorimetría Diferencial de Barrido

Natural Biomaterials for Cardiac Tissue Engineering: A Highly Biocompatible Solution.

Cardiovascular diseases (CVD) constitute a major fraction of the current major global diseases and lead to about 30% of the deaths, i.e., 17.9 million deaths per year. CVD include coronary artery disease (CAD), myocardial infarction (MI), arrhythmias, heart failure, heart valve diseases, congenital heart disease, and cardiomyopathy. Cardiac Tissue Engineering (CTE) aims to address these conditions, the overall goal being the efficient regeneration of diseased cardiac tissue using an ideal combination of biomaterials and cells. Various cells have thus far been utilized in pre-clinical studies for CTE. These include adult stem cell populations (mesenchymal stem cells) and pluripotent stem cells (including autologous human induced pluripotent stem cells or allogenic human embryonic stem cells) with the latter undergoing differentiation to form functional cardiac cells. The ideal biomaterial for cardiac tissue engineering needs to have suitable material properties with the ability to support efficient attachment, growth, and differentiation of the cardiac cells, leading to the formation of functional cardiac tissue. In this review, we have focused on the use of biomaterials of natural origin for CTE. Natural biomaterials are generally known to be highly biocompatible and in addition are sustainable in nature. We have focused on those that have been widely explored in CTE and describe the original work and the current state of art. These include fibrinogen (in the context of Engineered Heart Tissue, EHT), collagen, alginate, silk, and Polyhydroxyalkanoates (PHAs). Amongst these, fibrinogen, collagen, alginate, and silk are isolated from natural sources whereas PHAs are produced via bacterial fermentation. Overall, these biomaterials have proven to be highly promising, displaying robust biocompatibility and, when combined with cells, an ability to enhance post-MI cardiac function in pre-clinical models. As such, CTE has great potential for future clinical solutions and hence can lead to a considerable reduction in mortality rates due to CVD