Polymeric grafting of acrylic acid onto poly(3-hydroxybutyrate-co-3-hydroxyvalerate): Surface functionalization for tissue engineering applications

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) melt processed disks and solvent cast films were modified by graft co-polyinerization with acrylic acid (AAc) in methanol solution at ambient temperature using gamma irradiation (dose rate of 4.5 kGy/h). To assess the presence of carboxylic acid groups on the surface, reaction with pentafluorophenol was performed prior to X-ray photoelectron spectroscopy analysis. The grafting yield for all samples increased with monomer concentration (2-15%), and for the solvent cast films, it also increased with dose (2-9 kGy). However, the grafting yield of the melt processed disks was largely independent of the radiation dose (2-8 kGy). Toluidine blue was used to stain the modified materials facilitating, visual information about the extent of carboxylic acid functionalization and depth penetration of the grafted copolymer. Covalent linking of glucosamine to the functionalized surface was achieved using carbodimide chemistry verifying that the modified substrates are suitable for biomolecule attachment.

Use of atomic force microscopy for imaging the initial stage of the nucleation of calcium phosphate in Langmuir-Blodgett films of stearic acid

The nucleation of calcium phosphate on the substrate of steatic acid Langmuir-blodgett film at the initial stage was investigated by atomic force microscopy. Nano-dots, nano-wires and nano-islands were observed in sequence for the first time, reflecting the nucleation of calcium phosphate and the molecular arrangement of carboxylic layer. The nucleation rates perpendicular and parallel to the carboxylic terminal group were estimated from the height and diameter of the calcium phosphate crystals, respectively. And this stage was distinct from the late explosive grown stage, in which the change of the morphology was not obvious. The approaches based on this discovery would lead to the development of new strategies in the controlled synthesis of inorganic nano-phases and the assembly of organized composite and ceramic materials.

Poly(lactic-co-glycolic acid): Carbon nanofiber composites for myocardial tissue engineering applications

The objective of the present in vitro research was to investigate cardiac tissue cell functions (specifically cardiomyocytes and neurons) on poly(lactic-co-glycolic acid) (PLGA) (50:50 wt.%)-carbon nanofiber (CNF) composites to ascertain their potential for myocardial tissue engineering applications. CNF were added to biodegradable PLGA to increase the conductivity and cytocompatibility of pure PLGA. For this reason, different PLGA:CNF ratios (100:0, 75:25, 50:50,25:75, and 0:100 wt.%) were used and the conductivity as well as cytocompatibility of cardiomyocytes and neurons were assessed. Scanning electron microscopy, X-ray diffraction and Raman spectroscopy analysis characterized the microstructure, chemistry, and crystallinity of the materials of interest to this study. The results show that PLGA:CNF materials are conductive and that the conductivity increases as greater amounts of CNF are added to PLGA, from OS m(-1) for pure PLGA (100:0 wt.%) to 5.5 x 10(-3) S m(-1) for pure CNF (0:100 wt.%). The results also indicate that cardiomyocyte density increases with greater amounts of CNF in PLGA (up to 25:75 wt.% PLGA:CNF) for up to 5 days. For neurons a similar trend to cardiomyocytes was observed, indicating that these conductive materials promoted the adhesion and proliferation of two cell types important for myocardial tissue engineering applications. This study thus provides, for the first time, an alternative conductive scaffold using nanotechnology which should be further explored for cardiovascular applications. (C) 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Large-scale, uniform DNA network on 11-mercaptoundecanoic acid modified gold (111) surface: Atomic force microscopy study

Large-scale, uniform plasmid deoxyribonucleic acid (DNA) network has been successfully constructed on 11-mercaptoundecanoic acid modified gold (111) surface using a self-assembly technique. The effect of DNA concentration on the characteristics of the DNA network was investigated by atomic force microscopy. It was found that the size of meshes and the height of fibers in the DNA network could be controlled by varying the concentration of DNA with a constant time of assembly of 24 h.

Poly(glutamic acid) nanofibre modified glassy carbon electrode: Characterization by atomic force microscopy, voltammetry and electrochemical impedance

Glassy carbon electrodes (GCE) were modified with poly(glutamic acid) acid films prepared using three different procedures: glutamic acid monomer electropolymerization (MONO), evaporation of poly(glutamic acid) (PAG) and evaporation of a mixture of poly(glutamic acid)/glutaraldehyde (PAG/GLU). All three films showed good adherence to the electrode surface. The performance of the modified GCE was investigated by cyclic voltammetry and differential pulse voltammetry, and the films were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The three poly(glutamic acid) modified GCEs were tested using the electrochemical oxidation of ascorbic acid and a decrease of the overpotential and the improvement of the oxidation peak current was observed. The PAG modified electrode surfaces gave the best results. AFM morphological images showed a polymeric network film formed by well-defined nanofibres that may undergo extensive swelling in solution, allowing an easier electron transfer and higher oxidation peaks. (C) 2007 Elsevier Ltd. All rights reserved.

S��ntesi, caracteritzaci�� qu��mica i estudi de l'activitat biol��gica de nous complexos de Pt(II) amb lligands de tipus diaminocarbox��lic i els respectius ��sters i derivats pept��dics

El cisplat��, PtCl2(NH3)2, ha estat una de les drogues m��s utilitzades en la quimioter��pia del c��ncer des del descobriment de la seva activitat. Per�� degut a la seva alta toxicitat i greus efectes secundaris, s'han sintetitzat nous compostos amb la finalitat de reduir aquests inconvenients. En aquest sentit, el treball desenvolupat en aquesta tesi doctoral ha estat la s��ntesi i caracteritzaci�� de tretze complexos de Pt(II) amb la finalitat d'estudiar llur activitat antitumoral. Aquests complexos presenten unes caracter��stiques estructurals comunes: geometria cis, dos lligands l��bils de tipus clorur i un lligand diaminoquelatant derivat dels ��cids d,l-2,3-diaminopropi��nic (Hdap) i d,l-2,4-diaminobut��ric (Hdab). S'han dissenyat unes estrat��gies sint��tiques a partir de les quals els lligands han estat funcionalitzats amb diferents grups de tipus ��ster, amino��cid i pept��dic: Etdap��2HCl, Etdab��2HCl, [(dap-Metala)��2CF3COOH], [(dab-Metala)��2CF3COOH], [(dap-phe)��2CF3COOH], [(dab-phe)��2CF3COOH], [(dap-Mettrp)��2CF3COOH], [(dab-Mettrp)��2CF3COOH], [(dap-ASTTTNYT-NH2)��2CF3COOH], essent Metala= ��ster met��lic de L-alanina, phe= L-fenilalanina, Mettrp= ��ster met��lic del L-triptof��. Aquests lligands diaminoquelatants s'han utilitzat per sintetitzar els corresponents complexos de Pt(II): PtCl2(Hdap), PtCl2(Hdab), PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe), PtCl2(dab-phe), PtCl2(dap-Mettrp), PtCl2(dab-Mettrp), PtCl2(dap-ASTTTNYT-NH2). A trav��s de diferents t��cniques i assaigs biol��gics (dicroisme circular, electroforesi en gel d'agarosa, microscopia de forces at��miques, citometria de flux, assaigs de proliferaci�� cel��lular) s'ha pogut demostrar l'activitat antitumoral d'aquests compostos. A trav��s de la t��cnica de dicroisme circular (DC) s'ha pogut demostrar que els lligands lliures no interaccionen covalentment amb el DNA de Calf Thymus i no modifiquen l'estructura secund��ria de la doble h��lix. En canvi, els respectius complexos han demostrat tenir capacitat per interaccionar amb el DNA i modificar la seva estructura secund��ria. Els complexos PtCl2(Hdap), PtCl2(Hdab) i PtCl2(dab-phe) mostren un comportament similar al cisplat��, generant adductes cis-bifuncionals que distorcionen la doble h��lix de forma no desnaturalitzant amb obertura de la doble cadena. Els complexos PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-phe), PtCl2(dap-ASTTTNYT-NH2) quan interaccionen amb el DNA generen un canvi en la conformaci�� del DNA de la forma B a la forma C, produint-se un augment de la curvatura de l'h��lix per rotaci�� de les bases nitrogenades. En aquests estudis s'ha comprovat que l'estructura del complex influeix en l'efecte generat sobre l'estructura secund��ria de l'��cid nucleic. En primer lloc, existeix una difer��ncia en el comportament en funci�� del tamany del lligand diaminoquelatant, de manera que els complexos amb el lligand (dab) provoquen un efecte m��s remarcable. Tamb�� s'observa aquest canvi de comportament al passar dels complexos que tenen el grup funcional esterificat als que el tenen protonat. D'aquesta manera, s'observa un major efecte sobre l'estructura secund��ria del DNA en aquells complexos que tenen el lligand diaminoquelatant de tres metilens (dab) i amb el grup carboxilat terminal protonat. Per tal de modelitzar la interacci�� d'aquests complexos amb el DNA, s'ha estudiat la interacci�� d'aquests compostos de Pt(II) amb 5'-GMP a trav��s de RMN-1H, observant la variaci�� dels senyals corresponents al H8 de 5'-GMP. Aix�� s'ha pogut demostrar que aquests compostos interaccionen amb la 5'-GMP a trav��s d'un enlla�� covalent Pt-N7, de la mateixa manera a com interacciona el cisplat��. A trav��s d'electroforesi en gel d'agarosa i microscopia de forces at��miques (AFM) s'ha pogut determinar l'efecte que generen els lligands lliures i els respectius complexos de Pt(II) sobre l'estructura terci��ria del plasmidi pBR322. Els lligands provoquen un augment de l'agregaci�� de les mol��cules de DNA i un lleuger augment de la compactaci�� de l'estructura terci��ria. Aquests resultats s'atribueixen a la capacitat d'aquests compostos a interaccionar per pont d'hidrogen amb el DNA. Els corresponents complexos de Pt(II) provoquen un augment de l'agregaci�� i una important compactaci��, degut per una banda a la capacitat de l'��tom de Pt a interaccionar covalentment amb el DNA, i per altra banda, a la capacitat del lligand a interaccionar per pont d'hidrogen amb l'��cid nucleic. Finalment s'ha estudiat l'activitat citot��xica d'aquests complexos de Pt(II) en diferents l��nies cel��lulars: A431 (l��nia de carcinoma epidermoide), HeLa (l��nia de carcinoma de coll d'��ter) i HL-60 (l��nia promieloc��tica de leuc��mia). Els complexos moderadament solubles en aigua, PtCl2(Hdap), PtCl2(Hdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe) i PtCl2(dab-phe), han demostrat ser actius. L'activitat dep��n de la concentraci�� de complex, del temps d'incubaci�� i de la l��nia cel��lular. Per temps d'incubaci�� alts i concentracions de complex elevades s'observa la m��xima activitat. Els complexos de l'alanina, PtCl2(dap-ala) i PtCl2(dab-ala), s��n els que mostren m��s activitat, mentre que els compostos de la fenilalanina s��n els menys actius, degut probablement a la voluminositat del lligand, la qual pot impedir o dificultar el transport del compost a trav��s de la membrana cel��lular. L'activitat citot��xica dels complexos insolubles en aigua, PtCl2(Etdap) i PtCl2(Etdab), queda bloquejada per l'elevada concentraci�� de DMSO (12%) necess��ria per solubilitzar els compostos. Aquests resultats permeten deduir que la pres��ncia d'un 12% de DMSO anul��la l'activitat d'aquests complexos, ja que el DMSO pot coordinar-se amb el Pt ocupant les posicions l��bils del complex i evitant que es pugui coordinar amb el DNA. Els assaigs de proliferaci�� cel��lular del complex PtCl2(dap-ASTTTNYT-NH2) i del p��ptid lliure ASTTTNYT-NH2 han demostrat que ambd��s compostos s��n actius. Tot i aix��, l'activitat del complex ��s superior a la del p��ptid lliure, ja que el Pt pot interaccionar covalentment amb el DNA i augmentar l'efecte citot��xic. Per tant, el complex presenta un lligand portador biol��gicament actiu que pot transportar el metall a trav��s de la membrana cel��lular i facilitar aix�� la seva interacci�� amb el DNA. A trav��s de la t��cnica de citometria de flux s'ha comprovat que en tots els casos la mort cel��lular produ��da pels complexos ha estat per apoptosi. Per ��ltim, s'ha sintetitzat i caracteritzat un complex trinuclear de Pt(II), {[Pt(Me2Bpy)2][PtCl2(Me2Bpy)]2}, essent Me2Bpy= 4,4'-dimetil-2,2'-dipiridil. La resoluci�� de la seva estructura per difracci�� de Raig-X ha perm��s determinar l'exist��ncia d'una interacci�� intramolecular Pt-Pt de 3.474 ��.

A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate

The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin-methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but they have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in gel-MA based hydrogels, and show that with the incorporation of small quantities of photo-crosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesised ECM throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26 kPa after 8 weeks culture, constructs with HA-MA and CS-MA increased by 96 kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.

Aplica����o da membrana de l��tex natural (NRL) como suporte para cultura das c��lulas osteog��nicas MC3T3-E1 para regenera����o ��ssea guiada (GBR)

P��s-gradua����o em Biotecnologia - IQ

The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo

The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies - thermally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. Over 6 weeks, in vitro degradation produced changes in SCPL scaffold dimension, mass, internal architecture and mechanical properties. TIPS scaffolds produced far less changes in these parameters providing significant advantages over SCPL. In vivo results were based on a microsurgically created arteriovenous (AV) loop sandwiched between two TIPS scaffolds placed in a polycarbonate chamber under rat groin skin. Histologically, a predominant foreign body giant cell response and reduced vascularity was evident in tissue ingrowth between 2 and 8 weeks in TIPS scaffolds. Tissue death occurred at 8 weeks in the smallest pores. Morphometric comparison of TIPS and SCPL scaffolds indicated slightly better tissue ingrowth but greater loss of scaffold structure in SCPL scaffolds. Although advantageous in vitro, large surface area:volume ratios and varying pore sizes in PLGA TIPS scaffolds mean that effective in vivo (AV loop) utilization will only be achieved if the foreign body response can be significantly reduced so as to allow successful vascularisation, and hence sustained tissue growth, in pores less than 300 ��m. �� 2005 Elsevier Ltd. All rights reserved.

Dynamic covalent chemistry on surfaces employing highly reactive cyclopentadienyl moieties

Silicon substrates coated with a bromide-terminated silane are transformed into highly reactive, cyclopentadiene covered analogues. These surfaces undergo rapid cycloaddition reactions with various dienophile-capped polymers. Mild heating of the substrates causes the retro-Diels-Alder reaction to occur, thus reforming the reactive cyclopentadiene surface, generating an efficiently switchable surface.

A simple method for fabricating 3-D multilayered composite scaffolds

One limitation of electrospinning stems from the charge build-up that occurs during processing, preventing further fibre deposition and limiting the scaffold overall thickness and hence their end-use in tissue engineering applications targeting large tissue defect repair. To overcome this, we have developed a technique in which thermally induced phase separation (TIPS) and electrospinning are combined. Thick three-dimensional, multilayered composite scaffolds were produced by simply stacking individual polycaprolactone (PCL) microfibrous electrospun discs into a cylindrical holder that was filled with a 3% poly(lactic-co-glycolic acid) (PLGA) solution in dimethylsulfoxide (a good solvent for PLGA but a poor one for PCL). The construct was quenched in liquid nitrogen and the solvent removed by leaching out in cold water. This technique enables the fabrication of scaffolds composed principally of electrospun membranes that have no limit to their thickness. The mechanical properties of these scaffolds were assessed under both quasi-static and dynamic conditions. The multilayered composite scaffolds had similar compressive properties to 5% PCL scaffolds fabricated solely by the TIPS methodology. However, tensile tests demonstrated that the multilayered construct outperformed a scaffold made purely by TIPS, highlighting the contribution of the electrospun component of the composite scaffold to enhancing the overall mechanical property slate. Cell studies revealed cell infiltration principally from the scaffold edges under static seeding conditions. This fabrication methodology permits the rapid construction of thick, strong scaffolds from a range of biodegradable polymers often used in tissue engineering, and will be particularly useful when large dimension electrospun scaffolds are required.

Thermally induced phase separation in P alpha MSAN/PMMA blends in presence of functionalized multiwall carbon nanotubes: Rheology, morphology and electrical conductivity

The focus of this work is the evaluation and analysis of the state of dispersion of functionalized multiwall carbon nanotubes (CNTs), within different morphologies formed, in a model LCST blend (poly[(alpha-methylstyrene)-co-(acrylonitrile)]/poly(methyl-methacryla te), P alpha MSAN/PMMA). Blend compositions that are expected to yield droplet-matrix (85/15 P alpha MSAN/PMMA and 15/85 P alpha MSAN/PMMA, wt/wt) and co-continuous morphologies (60/40 P alpha MSAN/PMMA, wt/wt) upon phase separation have been combined with two types of CNTs; carboxylic acid functionalized (CNTCOOH) and polyethylene modified (CNTPE) up to 2 wt%. Thermally induced phase separation in the blends has been studied in-situ by rheology and dielectric (conductivity) spectroscopy in terms of morphological evolution and CNT percolation. The state of dispersion of CNTs has been evaluated by transmission electron microscopy. The experimental results indicate that the final blend morphology and the surface functionalization of CNT are the main factors that govern percolation. In presence of either of the CNTs, 60/40 P alpha MSAN/PMMA blends yield a droplet-matrix morphology rather than co-continuous and do not show any percolation. On the other hand, both 85/15 P alpha MSAN/PMMA and 15/85 P alpha MSAN/PMMA blends containing CNTPEs show percolation in the rheological and electrical properties. Interestingly, the conductivity spectroscopy measurements demonstrate that the 15/85 P alpha MSAN/PMMA blends with CNTPEs that show insulating properties at room temperature for the miscible blends reveal highly conducting properties in the phase separated blends (melt state) as a result of phase separation. By quenching this morphology, the conductivity can be retained in the blends even in the solid state. (C) 2011 Elsevier Ltd. All rights reserved.

Systematic selection of solvents for the fabrication of 3D combined macro- and microporous polymeric scaffolds for soft tissue engineering

In this study, we investigate the fabrication of 3D porous poly(lactic-co-glycolic acid) (PLGA) scaffolds using the thermally-induced phase separation technique. The current study focuses on the selection of alternative solvents for this process using a number of criteria, including predicted solubility. toxicity, removability and processability. Solvents were removed via either vacuum freeze-drying or leaching, depending on their physical properties. The residual solvent was tested using gas chromatography-mass spectrometry. A large range of porous, highly interconnected scaffold architectures with tunable pore size and alignment was obtained, including combined macro- and microporous structures and an entirely novel 'porous-fibre' structure. The morphological features of the most promising poly(lactic-co-glycolic acid) scaffolds were analysed via scanning electron microscopy and X-ray micro-computed tomography in both two and three dimensions. The Young's moduli of the scaffolds under conditions of temperature, pH and ionic strength similar to those found in the body were tested and were found to be highly dependent on the architectures.

Porous PLGA microspheres effectively loaded with BSA protein by electrospraying combined with phase separation in liquid nitrogen

Polymer microspheres loaded with bioactive particles, biomolecules, proteins, and/or growth factors play important roles in tissue engineering, drug delivery, and cell therapy. The conventional double emulsion method and a new method of electrospraying into liquid nitrogen were used to prepare bovine serum albumin (BAS)-loaded poly(lactic-co-glycolic acid) (PLGA) porous microspheres. The particle size, the surface morphology and the internal porous structure of the microspheres were observed using scanning electron microscopy (SEM). The loading efficiency, the encapsulation efficiency, and the release profile of the BSA-loaded PLGA microspheres were measured and studied. It was shown that the microspheres from double emulsion had smaller particle sizes (3-50 ���m), a less porous structure, a poor loading efficiency (5.2 %), and a poor encapsulation efficiency (43.5%). However, the microspheres from the electrospraying into liquid nitrogen had larger particle sizes (400-600 ���m), a highly porous structure, a high loading efficiency (12.2%), and a high encapsulation efficiency (93.8%). Thus the combination of electrospraying with freezing in liquid nitrogen and subsequent freeze drying represented a suitable way to produce polymer microspheres for effective loading and sustained release of proteins.