Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering

Tissue engineering is a discipline that aims at regenerating damaged biological tissues by using a cell-construct engineered in vitro made of cells grown into a porous 3D scaffold. The role of the scaffold is to guide cell growth and differentiation by acting as a bioresorbable temporary substrate that will be eventually replaced by new tissue produced by cells. As a matter or fact, the obtainment of a successful engineered tissue requires a multidisciplinary approach that must integrate the basic principles of biology, engineering and material science. The present Ph.D. thesis aimed at developing and characterizing innovative polymeric bioresorbable scaffolds made of hydrolysable polyesters. The potentialities of both commercial polyesters (i.e. poly-e-caprolactone, polylactide and some lactide copolymers) and of non-commercial polyesters (i.e. poly-w-pentadecalactone and some of its copolymers) were explored and discussed. Two techniques were employed to fabricate scaffolds: supercritical carbon dioxide (scCO2) foaming and electrospinning (ES). The former is a powerful technology that enables to produce 3D microporous foams by avoiding the use of solvents that can be toxic to mammalian cells. The scCO2 process, which is commonly applied to amorphous polymers, was successfully modified to foam a highly crystalline poly(w-pentadecalactone-co-e-caprolactone) copolymer and the effect of process parameters on scaffold morphology and thermo-mechanical properties was investigated. In the course of the present research activity, sub-micrometric fibrous non-woven meshes were produced using ES technology. Electrospun materials are considered highly promising scaffolds because they resemble the 3D organization of native extra cellular matrix. A careful control of process parameters allowed to fabricate defect-free fibres with diameters ranging from hundreds of nanometers to several microns, having either smooth or porous surface. Moreover, versatility of ES technology enabled to produce electrospun scaffolds from different polyesters as well as “composite” non-woven meshes by concomitantly electrospinning different fibres in terms of both fibre morphology and polymer material. The 3D-architecture of the electrospun scaffolds fabricated in this research was controlled in terms of mutual fibre orientation by properly modifying the instrumental apparatus. This aspect is particularly interesting since the micro/nano-architecture of the scaffold is known to affect cell behaviour. Since last generation scaffolds are expected to induce specific cell response, the present research activity also explored the possibility to produce electrospun scaffolds bioactive towards cells. Bio-functionalized substrates were obtained by loading polymer fibres with growth factors (i.e. biomolecules that elicit specific cell behaviour) and it was demonstrated that, despite the high voltages applied during electrospinning, the growth factor retains its biological activity once released from the fibres upon contact with cell culture medium. A second fuctionalization approach aiming, at a final stage, at controlling cell adhesion on electrospun scaffolds, consisted in covering fibre surface with highly hydrophilic polymer brushes of glycerol monomethacrylate synthesized by Atom Transfer Radical Polymerization. Future investigations are going to exploit the hydroxyl groups of the polymer brushes for functionalizing the fibre surface with desired biomolecules. Electrospun scaffolds were employed in cell culture experiments performed in collaboration with biochemical laboratories aimed at evaluating the biocompatibility of new electrospun polymers and at investigating the effect of fibre orientation on cell behaviour. Moreover, at a preliminary stage, electrospun scaffolds were also cultured with tumour mammalian cells for developing in vitro tumour models aimed at better understanding the role of natural ECM on tumour malignity in vivo.

Adsorption of phenylalanine on layered double hydroxides: effect of temperature and ionic strength

In this work we report the adsorption of phenylalanine (Phe) on Magnesium Aluminum Layered Double Hydroxides (Mg-Al-CO(3)-LDH) at two different temperatures (298 and 310 K) and under two distinct ionic strength conditions (with and without the addition 0.1 M of NaCl). The adsorption isotherms exhibit the same profile in all conditions, and they only differ in the amount of removed Phe. At lower ionic strength, the isotherms are almost identical at both temperatures, except for the last points, where the increase in temperature causes a decrease in the amount of adsorbed Phe. An increase in ionic strength results in a decrease in Phe adsorption. The electrokinetic potential decreases as the amount of adsorbed Phe increases, and only positive values are observed. This indicates that the surface of the adsorbent is not totally neutralized and suggests that more Phe could be removed by adsorption. The presence of Phe on the solid is confirmed by FTIR spectra, which present the specific bands assigned to Phe. The hydrophobicity of the amino acid probably contributes to its extraction, thus enabling the removal of a great amount of Phe. In conclusion, LDH is potentially applicable in the removal of Phe from wastewater.

Molecular engineered porous clays using surfactants

Quaternary ammonium surfactants were used to control the pore structure of bentonite intercalated with a mixed hydro-sol of silicon and titanium. Porous clay heterostructures of alumina and laponite were prepared in the presence of polyethylene oxide (PEO) surfactants. Participation of the surfactants in the synthesis results in significant changes in the structure of porous clay products. Surfactants are involved in different mechanisms, In the case of bentonite, the mean size of the framework pores was directly proportional to the chain length of the quaternary ammonium surfactants. This indicates a molecular templating mechanism, similar to that observed in the synthesis of MCM41. However, in the case of laponite, the size and volume of the mesopores were related to the amount of PEO surfactants used. By using an appropriate surfactant, we can obtain highly porous clays with various pore structures. Introducing surfactants during intercalation is an efficient strategy for the molecular engineering of porous clay adsorbents and catalysts. (C) 2002 Elsevier Science B.V. All rights reserved.

Preparation and Characterization of Polymeric Beads and Membranes for Boron Removal From Water

Boron is an element essential for various biological processes, nevertheless at high concentration it can cause health issues in both plants and animals, thus making boron a pollutant element. Low cost and effective polymeric adsorbents capable of removing boron in aqueous solution at neutral pH were prepared for this purpose. The adsorbent selectivity towards boron was conferred taking advantage of the interaction between boric acid and the alcoholic groups of N-methyl-D-Glucamine, which are able to form specific complexes. Two different kinds of devices were produced and tested: cross-linked chitosan hydrogel beads (CCBMG) and PVA/chitosan membranes, the latter taking advantage of scCO2-assisted phase inversion technique. The capability of the adsorbents to be regenerated and to allow recovery of boric acid from a solution emulating the concentration of boric acid in seawater were evaluated.

Polymeeriadsorbenttien käyttö kromatografisessa erotuksessa

Polymeeriadsorbentteja valmistetaan silloittamalla styreeniä, akrylaattia tai fenoliformaldehydiä. Useimmiten ristisilloittajana toimii divinyylibentseeni. Polymeeriadsorbenteissa ei itsessään ole ioninvaihtoryhmiä, joten ne sopivat ionittomien ja heikosti ionisoitujen aineiden adsorptioon. Usein polymeeriadsorbentteja käytetään vaihtoehtona aktiivihiilelle eri sovelluksissa. Työn kirjallisuusosassa on katsaus polymeeriadsorbenttien sovelluksiin lähinnä elintarviketeollisuudessa. Lisäksi siinä selvitetään polymeeriadsorbenttien rakennetta ja synteesimenetelmiä. Kokeellisessa osassa tutkittiin valittujen styreeni- ja akrylaattipohjaisten polymeeriadsorbenttien soveltuvuutta kromatografisen erotuksen stationaarifaasiksi. Kromatografia-ajoissa käytettiin eluenttina vettä, jonka lämpötila oli pääasiassa joko 75 tai 125 °C. Jälkimmäisessä lämpötilassa vesi on paineistettua neste, jota kutsutaan myös alikriittiseksi vedeksi. Malliaineina oli eri sokereita, aminohappoja sekä bentsoehappoa ja bentsyylialkoholia. Kromatografisen soveltuvuuden lisäksi selvitettiin adsorbenttien termistä kestävyyttä ja rakennetta. Termisesti polymeeriadsorbentit kestivät hyvin lämpötiloja 125 °C:eseen saakka. Polymeeriadsorbenteilla, joilla on suuri ominaispinta-ala, on myös suuri adsorptiokapasiteetti. Styreenipohjaiset adsorbentit erottivat kaikkia tutkittuja malliaineita akrylaattipohjaisia paremmin. Jotkut adsorbentit eivät erottaneet mitään tutkituista yhdisteistä. Lämpötilan nostaminen kavensi piikkejä ja nopeutti malliaineiden retentoitumista, mutta ei parantanut erottumista.

Karboksyylihappojen kromatografinen fraktiointi

Suurin osa alifaattisista karboksyylihapoista tuotetaan nykyään synteettisesti, mutta öljyn hinnan nousu ja ekologisempi ajattelutapa on aiheuttanut kiinnostusta tuottaa näitä karboksyyli- ja hydroksihappoja jatkossa fermentoimalla tai sellun valmistuksen sivuvirtana syntyvästä mustalipeästä. Nykyään mustalipeä poltetaan sellaisenaan soodakattiloissa keittokemikaalien regeneroimiseksi, energiaksi ja sähköksi. Jatkossa mustalipeästä voisi erottaa arvokkaat orgaaniset hapot ennen polttamista. Saadusta happoseoksesta tulisi erottaa yksittäiset alifaattiset karboksyylihapot toisistaan jatkojalostusta varten. Tämän kandidaatintyön tavoitteena oli selvittää, millä kromatografisella erotusmenetelmällä fermentointituotteina ja teollisuuden sivuvirtoina syntyvistä karboksyylihapposeoksista saadaan yksittäiset alifaattiset karboksyylihapot erotettua toisistaan. Mittaukset suoritettiin kolonnilla, jossa hartsipedin halkaisija oli 1,5 cm ja korkeus 15 cm. Kolonnin erototusmateriaaleina kokeiltiin vahvoja ja heikkoja kationinvaihtohartseja, vahvaa anioninvaihtohartsia ja polymeerisiä adsorbentteja. Erotettavaksi happoseokseksi valittiin sitruuna-, viini-, glykoli-, maito- ja etikkahapon seos. Tehokkain erotus saatiin Puroliten valmistamalla Macronet 270:lla, joka on mikrohuokoinen polymeerinen adsorbentti. Macronet 270:lla saatiin erotettua erityisesti viini- ja glykolihappo sitruuna-, maito- ja etikkahaposta. Yksittäisiä happoja ei saatu kuitenkaan kunnolla erotettua. Parhaat koeolosuhteet erotustehokkuuden ja retentioaikojen kannalta saatiin vesieluentin virtausnopeudella 2 mL/min, syöttöpulssin tilavuudella 5 mL ja kolonnin lämpötilassa 75 °C.

BET surface area of carbonaceous adsorbents: verification using geometric considerations and GCMC simulations on virtual porous carbon models

The applicability of BET model for calculation of surface area of activated carbons is checked by using molecular simulations. By calculation of geometric surface areas for the simple model carbon slit-like pore with the increasing width, and by comparison of the obtained values with those for the same systems from the VEGA ZZ package (adsorbate-accessible molecular surface), it is shown that the latter methods provide correct values. For the system where a monolayer inside a pore is created the ASA approach (GCMC, Ar, T = 87 K) underestimates the value of surface area for micropores (especially, where only one layer is observed and/or two layers of adsorbed Ar are formed). Therefore, we propose the modification of this method based on searching the relationship between the pore diameter and the number of layers in a pore. Finally BET; original andmodified ASA; and A, B and C-point surface areas are calculated for a series of virtual porous carbons using simulated Ar adsorption isotherms (GCMC and T = 87 K). The comparison of results shows that the BET method underestimates and not, as it was usually postulated, overestimates the surface areas of microporous carbons.

Newly-developed parameters to quantify texture : application to polymeric porous structures

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Newly-developed parameters to quantify texture : application to polymeric porous structures

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Onset of the Henry constant for supercritical adsorption into carbonaceous porous materials

The Henry constant is commonly used as a measure of how strong an adsorbate is attracted towards a solid surface and is regarded as one of the fundamental parameters in adsorption studies. Having a sound basis in thermodynamics, the Henry Law is often used as a criterion to evaluate the validity of adsorption isotherm equations. However, the application of the Henry Law for microporous materials, especially microporous activated carbon, remains questionable. It is the aim of this paper to examine the Henry Law behavior of supercritical adsorbates in carbonaceous pores of different sizes, and to define the conditions for the Henry Law to be applicable for carbonaceous adsorbents.

Characterisation of pore structure of carbon adsorbents using regularisation procedure

The constrained regularisation procedure was applied to compute the pore size distributions (PSDs, f(x)) for a variety of activated carbons using overall adsorption equation based on the combination of the Kelvin equation and the statistical adsorbed film thickness. The impact of the boundary values of relative nitrogen pressure p/p(0) was analysed on the basis of the corresponding alterations in the PSDs. Changes in microporosity and mesoporosity of activated carbons can be described adequately only when the range of p/p(0) is as wide as possible, as at a high initial p/p(0) value, the f(x) curves can be broadened with shifted maxima especially for micropores and narrow mesopores. Comparative analysis of the PSDs and the adsorption potential, adsorption energy and fractal dimension distributions gives useful information on the complete description of the adsorbent characteristics. (C) 2001 Elsevier Science B.V. All rights reserved.

Development of biocompatible and “smart” porous structures using CO2-assisted processes

Dissertação apresentada para a obtenção do grau de Doutor em Engenharia Química, especialidade Engenharia da Reacção Química, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Development of 2-oxazoline-based hydrogels and porous polyester microparticles in scCO2

Thesis submitted to Faculdade de Ciências e Tecnologia from Universidade Nova de Lisboa in partial fulfillment of the requirements for the obtention of the degree of Master of Science in Biotechnology

Porous structures for the purification of biopharmaceuticals

This work aimed at the development of a (bio)polymeric monolithic support for biopharmaceuticals purification and/or capture. For that, it was assured that functional groups on its surface were ready to be involved in a plethora of chemical reactions for incorporation of the desired and most suitable ligand. Using cryogelation as preparation method a screening on multiple combinations of materials was performed in order to create a potentially efficient support with the minimal footprint, i.e. a monolithic support with reasonable mechanical properties, highly permeable, biocompatible, ready to use, with gravitational performance and minimal unspecific interactions towards the target molecules, but also biodegradable and produced from renewable materials. For the pre-selection all monoliths were characterized physico-chemically and morphologically; one agarose-based and two chitosan-based monoliths were then subjected to further characterizations before and after their modification with magnetic nanoparticles. These three specimens were finally tested towards adenovirus and the recovery reached 84% for the chitosan-GMA plain monolith prepared at -80°C. Monoliths based on chitosan and PVA were prepared in the presence and absence of magnetic particles, and tested for the isolation of GFP directly from crude cellular extracts. The affinity ligand A4C7 previously selected for GFP purification was synthesized on the monolith. The results indicated that the solid-phase synthesis of the ligand directly onto the monolith might require optimization and that the large pores of the monoliths are unsuitable for the purification of small proteins, such as GFP.

Experimental high-throughput adsorption unit for multi-evaluation of adsorbents for gas capture, storage and separation applications

Dissertação para obtenção do grau de Mestre em Engenharia Química e Bioquímica