Apatite-Polymer Composites for the Controlled Dual Delivery of BMP-2 and BMP-6 for Bone Tissue Engineering

The release of growth factors from tissue engineering scaffolds provides signals that influence the migration, differentiation, and proliferation of cells. The incorporation of a drug delivery platform that is capable of tunable release will give tissue engineers greater versatility in the direction of tissue regeneration. We have prepared a novel composite of two biomaterials with proven track records - apatite and poly(lactic-co-glycolic acid) (PLGA) – as a drug delivery platform with promising controlled release properties. These composites have been tested in the delivery of a model protein, bovine serum albumin (BSA), as well as therapeutic proteins, recombinant human bone morphogenetic protein-2 (rhBMP-2) and rhBMP-6. The controlled release strategy is based on the use of a polymer with acidic degradation products to control the dissolution of the basic apatitic component, resulting in protein release. Therefore, any parameter that affects either polymer degradation or apatite dissolution can be used to control protein release. We have modified the protein release profile systematically by varying the polymer molecular weight, polymer hydrophobicity, apatite loading, apatite particle size, and other material and processing parameters. Biologically active rhBMP-2 was released from these composite microparticles over 100 days, in contrast to conventional collagen sponge carriers, which were depleted in approximately 2 weeks. The released rhBMP-2 was able to induce elevated alkaline phosphatase and osteocalcin expression in pluripotent murine embryonic fibroblasts. To augment tissue engineering scaffolds with tunable and sustained protein release capabilities, these composite microparticles can be dispersed in the scaffolds in different combinations to obtain a superposition of the release profiles. We have loaded rhBMP-2 into composite microparticles with a fast release profile, and rhBMP-6 into slow-releasing composite microparticles. An equi-mixture of these two sets of composite particles was then injected into a collagen sponge, allowing for dual release of the proteins from the collagenous scaffold. The ability of these BMP-loaded scaffolds to induce osteoblastic differentiation in vitro and ectopic bone formation in a rat model is being investigated. We anticipate that these apatite-polymer composite microparticles can be extended to the delivery of other signalling molecules, and can be incorporated into other types of tissue engineering scaffolds.

Modificació de fibres de jute per l'elaboració de plàstics reforçats reciclables

El treball que s'ha dut a terme es centra en la recerca d'agents modificants per a fibres cel·lulòsiques capaços de reduir la polaritat de les funcions alcohol de la seva estructura per formació de funcions ester. Les fibres de jute se sotmeten a reacció en un sistema tancat provist d'atmòsfera de nitrogen a fi d'evitar reaccions laterals que no són del nostre interés.L'obtenció dels resultats perseguits està lligat a les condicions experimentals aplicades durant les reaccions. La influència de les diferents variables escollides facilitarà en major o menor grau la reacció entre les molècules d'agent d'acoblament i cel·lulosa vinculades. Una gran part de l'atenció es centrarà en l'estudi de l'etapa de modificació, sobretot en l'efectivitat dels reactius addicionats per la reacció amb els grups hidroxil. Un cop comparats els experiments realitzats tant amb clorur d'oleïl com amb anhídrid metacrílic, es conclou que la majoria de condicions provades permeten assolir valors de modificació prou significatius. L'excepció ve donada quan la temperatura utilitzada és de 20ºC, llavors les mateixes condicions que a una temperatura de 60ºC condueixen cap a resultats poc satisfactoris. La reactivitat per part dels dos agents d'acoblament utilitzats no ha estat la mateixa. Els resultats per condicions experimentals del mateix tipus han conduït cap a valors força diferents. Pel que fa a la determinació dels paràmetres òptims es conclou que les variables amb les que el grau de modificació millora considerablement són: una temperatura de 60ºC, 10% de catalitzador respecte la quantitat de clorur d'oleïl o anhídrid addicionada, relació OH reactiu estequiomètrica 1:1 i 40mL de solvent. Un cop modificada la fibra, se sotmet a reacció amb el monòmer estirè. Es comprova que el grau de polimerització segueix el valor de modificació prèviament obtingut, a menor quantitat de funcions alcohol lliures major interacció amb el monòmer estirè. Les propietats inicials de la fibra no es corresponen amb les obtingudes després del tractament, l'increment de la resistència a l'atac de microorganismes i a l'absorció d'humitat s'explica per una reducció de la presència de funcions alcohol polars i per la capa d'estirè polimeritzat per unió amb els dobles enllaços introduïts amb els agents d'acoblament. Pel que fa referència a les dues tècniques de caracterització més utilitzades, l'anàlisis elemental permet quantificar d'una manera precisa la reacció de la fibra amb els agents d'acoblament i la posterior reacció de la fibra modificada amb el monòmer estirè. La caracterització per espectroscopia d'infraroig permet comprovar qualitativament la reactivitat del clorur d'oleïl i qualitativament-quantitativament la de l'anhídrid metacrílic amb les funcions alcohol de la cel·lulosa present en les fibres de jute. Els pics més característics apareguts seran utilitzats per avaluar la reactivitat de la funció carbonílica del reactiu modificant amb l'estructura cel·lulòsica i del doble enllaç de la cel·lulosa modificada amb la matriu polimèrica.

Analysis and Simulation of Transverse Random Fracture of Long Fibre Reinforced Composites

La present tesi proposa una metodología per a la simulació probabilística de la fallada de la matriu en materials compòsits reforçats amb fibres de carboni, basant-se en l'anàlisi de la distribució aleatòria de les fibres. En els primers capítols es revisa l'estat de l'art sobre modelització matemàtica de materials aleatoris, càlcul de propietats efectives i criteris de fallada transversal en materials compòsits. El primer pas en la metodologia proposada és la definició de la determinació del tamany mínim d'un Element de Volum Representatiu Estadístic (SRVE) . Aquesta determinació es du a terme analitzant el volum de fibra, les propietats elàstiques efectives, la condició de Hill, els estadístics de les components de tensió i defromació, la funció de densitat de probabilitat i les funcions estadístiques de distància entre fibres de models d'elements de la microestructura, de diferent tamany. Un cop s'ha determinat aquest tamany mínim, es comparen un model periòdic i un model aleatori, per constatar la magnitud de les diferències que s'hi observen. Es defineix, també, una metodologia per a l'anàlisi estadístic de la distribució de la fibra en el compòsit, a partir d'imatges digitals de la secció transversal. Aquest anàlisi s'aplica a quatre materials diferents. Finalment, es proposa un mètode computacional de dues escales per a simular la fallada transversal de làmines unidireccionals, que permet obtenir funcions de densitat de probabilitat per a les variables mecàniques. Es descriuen algunes aplicacions i possibilitats d'aquest mètode i es comparen els resultats obtinguts de la simulació amb valors experimentals.

Simulation of delamination in composites under quasi-static and fatigue loading using cohesive zone models

Es desenvolupa una eina de disseny per l'anàlisi de la tolerància al dany en composites. L'eina pot predir el inici i la propagació de fisures interlaminars. També pot ser utilitzada per avaluar i planificar la necessitat de reparar o reemplaçar components durant la seva vida útil. El model desenvolupat pot ser utilitzat tan per simular càrregues estàtiques com de fatiga. El model proposat és un model de dany termodinàmicament consistent que permet simular la delaminació en composites sota càrregues variables. El model es formula dins el context de la Mecànica del Dany, fent ús dels models de zona cohesiva. Es presenta un metodologia per determinar els paràmetres del model constitutiu que permet utilitzar malles d'elements finits més bastes de les que es poden usar típicament. Finalment, el model és també capaç de simular la delaminació produïda per càrregues de fatiga.

Simulation of interlaminar and intralaminar damage in polymer-based composites for aeronautical applications under impact loading

La aplicación de materiales compuestos de matriz polimérica reforzados mediante fibras largas (FRP, Fiber Reinforced Plastic), está en gradual crecimiento debido a las buenas propiedades específicas y a la flexibilidad en el diseño. Uno de los mayores consumidores es la industria aeroespacial, dado que la aplicación de estos materiales tiene claros beneficios económicos y medioambientales. Cuando los materiales compuestos se aplican en componentes estructurales, se inicia un programa de diseño donde se combinan ensayos reales y técnicas de análisis. El desarrollo de herramientas de análisis fiables que permiten comprender el comportamiento mecánico de la estructura, así como reemplazar muchos, pero no todos, los ensayos reales, es de claro interés. Susceptibilidad al daño debido a cargas de impacto fuera del plano es uno de los aspectos de más importancia que se tienen en cuenta durante el proceso de diseño de estructuras de material compuesto. La falta de conocimiento de los efectos del impacto en estas estructuras es un factor que limita el uso de estos materiales. Por lo tanto, el desarrollo de modelos de ensayo virtual mecánico para analizar la resistencia a impacto de una estructura es de gran interés, pero aún más, la predicción de la resistencia residual después del impacto. En este sentido, el presente trabajo abarca un amplio rango de análisis de eventos de impacto a baja velocidad en placas laminadas de material compuesto, monolíticas, planas, rectangulares, y con secuencias de apilamiento convencionales. Teniendo en cuenta que el principal objetivo del presente trabajo es la predicción de la resistencia residual a compresión, diferentes tareas se llevan a cabo para favorecer el adecuado análisis del problema. Los temas que se desarrollan son: la descripción analítica del impacto, el diseño y la realización de un plan de ensayos experimentales, la formulación e implementación de modelos constitutivos para la descripción del comportamiento del material, y el desarrollo de ensayos virtuales basados en modelos de elementos finitos en los que se usan los modelos constitutivos implementados.

Conductive elastomeric composites

Conductive elastic materials are formed by distributing conductive particles within an elastic polymer. We consider a novel composite based on dendritic nickel particles that exhibit remarkably strong negative piezoresistivity with an increase in conductivity of up to 10 orders of magnitude with strains of the order of 0.2. A vital factor for the conductivity of conductive elastomers is the concentration of conductive fillers and many aspects can be understood in terms of percolation theory. In this system the concentration of particles within the composite does not change with strain, yet due to the shape of the particles, the concentration of electrical contacts between the particles does change. We have developed a new model based on the concentration of contact sites, rather than particles which enables us to successfully model this remarkable strain-dependence of conductivity.

Influence of fibrolytic enzymes on the hydrolysis and fermentation of pure cellulose and xylan by mixed ruminal microorganisms in vitro

A series of in vitro studies was, conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 muL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20degreesC. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 muL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 muL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its consequences for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its conscequence for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Cellulose microfibril angle in the cell wall of wood fibres

The term microfibril angle (MFA) in wood science refers to the angle between the direction of the helical windings of cellulose microfibrils in the secondary cell wall of fibres and tracheids and the long axis of cell. Technologically, it is usually applied to the orientation of cellulose microfibrils in the S2 layer that makes up the greatest proportion of the wall thickness, since it is this which most affects the physical properties of wood. This review describes the organisation of the cellulose component of the secondary wall of fibres and tracheids and the various methods that have been used for the measurement of MFA. It considers the variation of MFA within the tree and the biological reason for the large differences found between juvenile (or core) wood and mature (or outer) wood. The ability of the tree to vary MFA in response to environmental stress, particularly in reaction wood, is also described. Differences in MFA have a profound effect on the properties of wood, in particular its stiffness. The large MFA in juvenile wood confers low stiffness and gives the sapling the flexibility it needs to survive high winds without breaking. It also means, however, that timber containing a high proportion of juvenile wood is unsuitable for use as high-grade structural timber. This fact has taken on increasing importance in view of the trend in forestry towards short rotation cropping of fast grown species. These trees at harvest may contain 50% or more of timber with low stiffness and therefore, low economic value. Although they are presently grown mainly for pulp, pressure for increased timber production means that ways will be sought to improve the quality of their timber by reducing juvenile wood MFA. The mechanism by which the orientation of microfibril deposition is controlled is still a matter of debate. However, the application of molecular techniques is likely to enable modification of this process. The extent to which these techniques should be used to improve timber quality by reducing MFA in juvenile wood is, however, uncertain, since care must be taken to avoid compromising the safety of the tree.

Infrared actuation in aligned polymer-nanotube composites

Rubber composites containing multiwalled carbon nanotubes have been irradiated with near-infrared light to study their reversible photomechanical actuation response. We demonstrate that the actuation is reproducible across differing polymer systems. The response is directly related to the degree of uniaxial alignment of the nanotubes in the matrix, contracting the samples along the alignment axis. The actuation stroke depends on the specific polymer being tested; however, the general response is universal for all composites tested. We conduct a detailed study of tube alignment induced by stress and propose a model for the reversible actuation behavior based on the orientational averaging of the local response. The single phenomenological parameter of this model describes the response of an individual tube to adsorption of low-energy photons; its experimentally determined value may suggest some ideas about such a response.

Cellulose microfibril angle in the cell wall of wood fibres

The term microfibril angle (MFA) in wood science refers to the angle between the direction of the helical windings of cellulose microfibrils in the secondary cell wall of fibres and tracheids and the long axis of cell. Technologically, it is usually applied to the orientation of cellulose microfibrils in the S2 layer that makes up the greatest proportion of the wall thickness, since it is this which most affects the physical properties of wood. This review describes the organisation of the cellulose component of the secondary wall of fibres and tracheids and the various methods that have been used for the measurement of MFA. It considers the variation of MFA within the tree and the biological reason for the large differences found between juvenile (or core) wood and mature (or outer) wood. The ability of the tree to vary MFA in response to environmental stress, particularly in reaction wood, is also described. Differences in MFA have a profound effect on the properties of wood, in particular its stiffness. The large MFA in juvenile wood confers low stiffness and gives the sapling the flexibility it needs to survive high winds without breaking. It also means, however, that timber containing a high proportion of juvenile wood is unsuitable for use as high-grade structural timber. This fact has taken on increasing importance in view of the trend in forestry towards short rotation cropping of fast grown species. These trees at harvest may contain 50% or more of timber with low stiffness and therefore, low economic value. Although they are presently grown mainly for pulp, pressure for increased timber production means that ways will be sought to improve the quality of their timber by reducing juvenile wood MFA. The mechanism by which the orientation of microfibril deposition is controlled is still a matter of debate. However, the application of molecular techniques is likely to enable modification of this process. The extent to which these techniques should be used to improve timber quality by reducing MFA in juvenile wood is, however, uncertain, since care must be taken to avoid compromising the safety of the tree.