Rozprawy doktorskie na temat „Fabrication of polymeric scaffolds”

Designing tissue engineering scaffolds with the required mechanical properties and favourable microstructure to promote cell attachment, growth and new tissue formation is one of the key challenges in the tissue engineering field. An important class of scaffolds for bone tissue engineering is based on bioceramics and bioactive glasses. The primary disadvantage of these materials is their low fracture resistance under load and their high brittleness. These drawbacks are exacerbated by the fact that optimal scaffolds must be highly porous (>90% porosity). As a main focus of this thesis, a novel approach was investigated to enhance the structural integrity, fracture strength and toughness of partially sintered 45S5 Bioglass® based glass-ceramic scaffolds by polymer infiltration and to develop an understanding of the interaction of these two phases in the final composite structure. Commercially available synthetic poly(D,L-Lactic acid) (PDLLA) was incorporated as a coating onto the partially sintered Bioglass® based scaffolds by dipping technique. Two natural polymers synthesised from bacteria, which exhibit different properties to those of PDLLA, were also investigated: i.e. poly(3-hydroxybutryate) (P(3HB)) and poly(3- hydroxyoctanoate) (P(3HO)). The work of fracture of partially sintered 45S5 Bioglass® scaffolds was significantly improved by forming interpenetrating polymerbioceramic microstructures which mimic the composite structure of bone. It was demonstrated that coating with polymers such as PDLLA, P(3HB) and P(3HO) does not impede the bioactivity of the scaffolds but the extent of bioactivity, given by the kinetic of HA formation, was seen to depend on polymer type and on scaffold sintering conditions. Polymer coated 45S5 Bioglass® pellets sintered at the same condition as the scaffolds and immersed in SBF were investigated to better evaluate the bioactivity mechanism and interfacial properties of the materials. It was demonstrated that polymer coated 45S5 Bioglass® based glass-ceramic scaffolds can have higher bioactivity and improved fracture toughness when the basic scaffold structure is sintered at relative lower sintering temperatures leaving residual open porosity which can be efficiently infiltrated by the polymer. A bilayered scaffold structure was also designed and fabricated to develop for the first time a porous bioactive glass-ceramic scaffold coated with PDLLA nanofibers. Electrospinning was used to deposit a PDLLA fibrous layer on top of the bioactive glass scaffold. These scaffolds were developed for osteochondral tissue engineering applications. SBF studies showed that the extent of mineralisation of the PDLLA fibres depended on the fibrous mesh thickness. PDLLA fibres deposited for 2 hours did not mineralise when immersed for 7, 14 and 28 days in SBF making the structure suitable for osteochondral defect applications. Initial in vitro cell response studies showed that the bilayered scaffolds were non toxic and chondrocyte cells were able to proliferate on the PDLLA fibre layers, demonstrating the potential of the novel scaffolds for osteochondral tissue engineering.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Elliot L. Chaikof; Committee Member: Ajit Yoganathan; Committee Member: Larry McIntire; Committee Member: Marc Levenston; Committee Member: Mark Allen. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Wang, Yadong; Committee Member: Bao, Gang; Committee Member: Bellamkonda, Ravi; Committee Member: Boyan, Barbara; Committee Member: Chaikof, Elliot; Committee Member: Meredith, J. Carson.

High levels of ionizing radiation are known to cause degradation and/or cross-linking in polymers. Lower levels of ionizing radiation, such as x-rays, are commonly used in the treatment of cancers. Material characterization has not been fully explored for polymeric materials exposed to therapeutic radiation levels. This study investigated the effects of therapeutic radiation on three porous scaffolds: polycaprolactone (PCL), polyurethane (PU) and gelatin. Porous scaffolds were fabricated using solvent casting and/or salt leaching techniques. Scaffolds were placed in phosphate buffered saline (PBS) and exposed to a typical cancer radiotherapy schedule. A total dose of 50 Gy was broken into 25 dosages over a three-month period. PBS was collected over time and tested for polymer degradation through high performance liquid chromatography (HPLC) and bicinchoninic acid (BCA) protein assay. Scaffolds were characterized by changes in microstructure using Scanning Electron Microscopy (SEM), and crystallization using Differential Scanning Calorimetry (DSC). Additionally, gelatin ε-amine content was analyzed using Trinitrobenzene Sulfonic Acid Assay (TNBSA). Gelatin scaffolds immersed in PBS for three months without radiation served as a control. Each scaffold responded differently to radiation. PCL showed no change in molecular weight or microstructure. However, the degree of crystallinity decreased 32% from the non-irradiated control. PU displayed both changes in microstructure and a decrease in crystallinity (85.15%). Gelatin scaffolds responded the most dramatically to radiotherapy. Samples were observed to swell, yet maintain shape after exposure. As gelatin was considered a tissue equivalent, further studies on tissues are needed to better understand the effects of radiotherapy.
Master of Science

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.

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.

Advancements in the biomedical field are driven by the design of novel materials with controlled physical and bio-interactive properties. To develop such materials, researchers rely on the use of highly efficient reactions for the assembly of advanced polymeric scaffolds that meet the demands of a functional biomaterial. In this thesis two main strategies for such materials have been explored; these include the use of off-stoichiometric thiol-ene networks and dendritic polymer scaffolds. In the first case, the highly efficient UV-induced thiol-ene coupling (TEC) reaction was used to create crosslinked polymeric networks with a predetermined and tunable excess of thiol or ene functionality. These materials rely on the use of readily available commercial monomers. By adopting standard molding techniques and simple TEC surface modifications, patterned surfaces with tunable hydrophobicity could be obtained. Moreover, these materials are shown to have great potential for rapid prototyping of microfluidic devices. In the second case, dendritic polymer scaffolds were evaluated for their ability to increase surface interactions and produce functional 3D networks. More specifically, a self-assembled dendritic monolayer approach was explored for producing highly functional dendronized surfaces with specific interactions towards pathogenic E. coli bacteria. Furthermore, a library of heterofunctional dendritic scaffolds, with a controllable and exact number of dual-purpose azide and ene functional groups, has been synthesized. These scaffolds were explored for the production of cell interactive hydrogels and primers for bone adhesive implants. Dendritic hydrogels decorated with a selection of bio-relevant moieties and with Young’s moduli in the same range as several body tissues could be produced by facile UV-induced TEC crosslinking. These gels showed low cytotoxic response and relatively rapid rates of degradation when cultured with normal human dermal fibroblast cells. When used as primers for bone adhesive patches, heterofunctional dendrimers with high azide-group content led to a significant increase in the adhesion between a UV-cured hydrophobic matrix and the wet bone surface (compared to patches without primers).

QC 20140116

La chirurgia protesica è oggi una metodica ad altissima percentuale di successo. La più temibile causa di fallimento è sicuramente l’infezione, non sempre di agevole diagnosi, in particolare nei casi tardivi cronici: pertanto è fondamentale la tempestività con cui si attua la terapia antibiotica. Partendo da questa problematica, il progetto ha sviluppato un gel termosensibile e uno scaffold compositi costituiti da chitosano e granuli di osso bovino che oltre ad essere biocompatibili, biodegradabili sono in grado di veicolare a livello dell’osso gentamicina come tale e/o incapsulata in micro-nanoparticelle.

Tissue engineering is a multidisciplinary field that is rapidly emerging as a promising new approach in the restoration and reconstruction of tissues. In this approach, three dimensional (3D) scaffolds are of great importance. Scaffolds function both as supports for cell growth and depot for sustained release of required active agents (e.g. enzymes, genes, antibiotics, growth factors). Scaffolds should possess certain properties in accordance with usage conditions. Wet-spinning is a simple technique that has been widely used for the fabrication of porous scaffolds for tissue engineering applications. Natural polymers can effectively be used in scaffold fabrication due to their biocharacteristics. Among natural polymers, chitosan and alginate are two of the most studied ones in tissue engineering and drug delivery fields because of being biologically renewable, biodegradable, biocompatible, non-antigenic, non-toxic and biofunctional. In this study, two kinds of porous scaffolds were produced as chitosan and alginate coated chitosan fibrous scaffolds by wet-spinning technique In order to investigate the delivery characteristics of the scaffolds, loading of gentamicin as a model antibiotic and bovine serum albumin (BSA) as a model protein was carried out in different loading models. Resultant scaffolds were characterized in terms of their structural formation, biodegradation, biomineralization, water uptake and retention ability and mechanical properties. Additionally, release kinetics of gentamicin and BSA were examined. Efficiency of gentamicin on Escherichia coli (E.coli) was examined. Characterization of scaffolds revealed their adequacy to be used in bone tissue engineering applications and capability to be employed as bioactive agent delivery systems.

Functional polymers are emerging as strong candidates for a variety of biomedical applications, but progress in this field is slow due to the difficulties associated with the synthesis of libraries of polymers. Polymeric scaffolds facilitate the rapid synthesis of such functional polymers by employing click chemistries as a tool for post-polymerisation modification. Acrylic and acetylene based polyhydrazides have been explored as potential scaffolds for the in situ screening of functionalised polymers for biomedical applications. Poly(acryloyl hydrazide) was prepared from commercially available starting materials using RAFT polymerisation in a three step synthesis, and its postpolymerisation modification using a variety of hydrophilic and hydrophobic aldehydes was investigated. Biocompatible solvents and reaction conditions were determined such that the postpolymerisation modification could be achieved with good yields or better. The applicability of the scaffold was shown during the in situ screening of functional polymers for siRNA delivery, which required no isolation or purification of candidate polymers. Poly(4-ethynylbenzohydrazide) was synthesised using rhodium catalysed polymerisation conditions, towards achieving a helical polymer scaffold. Despite the lack of solubility in aqueous solvents, the stability and post-polymerisation modification was analysed in a variety of conditions, opening the possibility of synthesising biodegradable mimics to naturally occurring helical moieties.

Electrospinning is a versatile method of producing nanofibrous polymeric material with potential applications as tissue engineering scaffolds. The main aim of this project was to produce and characterise electrospun polymeric scaffolds based on chitosan and bacterial polyhydroxybutyrate. The effect of the parameters used in the electrospinning process were studied and optimised by electrospinning polyvinyl alcohol from 8 wt% and 10 wt% solutions under a variable applied voltage from 10-25 kV. Attempts were made to electrospun chitosan however it was found that the creation of a polymer blend was necessary to facilitate fibre formation. PVA-chitosan blends were successfully electrospun at blend ratios of up to 80:20. A chitosan-hydroxybenzotriazole-PVA aqueous solution was successfully prepared enabling the production of chitosan/PVA nanofibres without the need for the use of an organic solvent. Polyhydroxybutyrate produced by bacterial synthesis from R. Eutropha using three different carbon sources; olive oil, rapeseed oil and glucose were electrospun and characterized. The choice of carbon source did not have a significant effect on the morphology or crystallinity of the produced fibres. PHB fibre diameters were reduced by 30% through the addition of the salt Benzyl tributylammonium chloride to the electrospinning solution.

[EN] Articular cartilage is a tissue that consists of chondrocytes surrounded by a dense extracellular matrix (ECM). The ECM is mainly composed of type II collagen and proteoglycans. The main function of articular cartilage is to provide a lubricated surface for articulation. Articular cartilage damage is common and may lead to osteoarthritis. Articular cartilage does not have blood vessels, nerves or lymphatic vessels and therefore has limited capacity for intrinsic healing and repair. Tissue engineering (TE) is a powerful approach for healing degenerated cartilage. TE uses three-dimensional (3D) scaffolds as cellular culture supports. The scaffold provides a structure that facilitates chondrocyte adhesion and expansion while maintaining a chondrocytic phenotype and limiting dedifferentiation, which is a problem in two-dimensional (2D) systems. Cell attachment to the scaffolds depends on the physical and chemical characteristics of their surface (morphology, rigidity, equilibrium water content, surface tension, hydrophilicity, presence of electric charges). The primary aim of this thesis was to study the influence of different kinds of biomaterials on the response of chondrocytes to in vitro culture. 3D scaffold constructs must have an interconnected porous structure in order to allow cell development through the network, to maintain their differentiated function, as well as to allow the entry and exit of nutrients and metabolic waste removal. Therefore, the effect of the hydrophilicity and pore architecture of the scaffolds was studied. A series of polymer and copolymer networks with varying hydrophilicity was synthesised and biologically tested in monolayer culture. Cell viability, proliferation and aggrecan expression were quantified. When human chondrocytes were cultured on polymer substrates in which the hydrophilic groups were homogeneously distributed, adhesion, proliferation and viability decreased with the content of hydrophilic groups. Nevertheless, copolymers in which hydrophilic and hydrophobic domains alternate showed better results than the corresponding homopolymers. Biostable and biodegradable scaffolds with different hydrophilicity and porosity were synthesised using a template of sintered microspheres of controlled size. This technique allows the interconnectivity between pores and their size to be controlled. Periodic and regular pore architectures and reproducible structures were obtained. The mechanical behaviour of the porous samples was significantly different from that of the bulk material of the same composition. Cells fully colonised the scaffolds when the pores' size and their interconnection were sufficiently large. Another objective was to assess the chondrogenic redifferentiation in a biodegradable 3D scaffold of polycaprolactone (PCL) of human autologous chondrocytes previously expanded in monolayer. This study demonstrated that chondrocytes cultured in PCL scaffolds without fetal bovine serum (FBS) efficiently redifferentiated, expressing a chondrocytic phenotype characterised by their ability to synthesise cartilage-specific ECM proteins. The influence that pore connectivity and hydrophilicity of caprolactone-based scaffolds has on the chondrocyte adhesion to the pore walls, proliferation and composition of the ECM produced was studied. The number of cells inside polycaprolactone scaffolds increased as porosity was increased. A minimum of around 70% porosity was necessary for this scaffold architecture to allow seeding and viability of the cells within. The results suggested that some of the cells inside the scaffold adhered to the pore walls and kept the dedifferentiated phenotype, while others redifferentiated. In conclusion, the findings of this thesis provide valuable insight into the field of cartilage regeneration using TE techniques. The studies carried out shed light on the right composition, porosity and hydrophilicity of the scaffolds to be used for optimal cartilage production.
[ES] El cartílago articular es un tejido compuesto por condrocitos rodeados por una densa matriz extracelular (MEC). La MEC se compone principalmente de colágeno tipo II y de proteoglicanos. La función principal del cartílago articular es proporcionar una superficie lubricada para las articulaciones. Las lesiones en el cartílago articular son comunes y pueden derivar a osteoartritis. El cartílago articular no tiene vasos sanguíneos, nervios o vasos linfáticos y, por tanto, tiene una capacidad limitada de auto-reparación. La ingeniería tisular (IT) es un área prometedora en la regeneración de cartílago. En la IT se utilizan "andamiajes" (scaffolds) tridimensionales (3D) como soportes para el cultivo celular y tisular. Los scaffolds proporcionan una estructura que facilita la adhesión y la expansión de los condrocitos, manteniendo un fenotipo condrocítico limitando su desdiferenciación; que es el mayor problema en los sistemas bidimensionales (2D). La adhesión celular a los scaffolds depende de las características físicas y químicas de su superficie (morfología, rigidez, contenido de agua en equilibrio, tensión superficial, hidrofilicidad, presencia de cargas eléctricas). El objetivo general de esta tesis fue estudiar la influencia de diferentes tipos de biomateriales en la respuesta de los condrocitos en cultivo in vitro. Los scaffolds deben tener una estructura porosa interconectada para permitir el desarrollo celular a través de toda la estructura 3D, potenciando que los condrocitos mantengan su fenotipo, así como permitiendo entrada de nutrientes y eliminación de desechos metabólicos. Se estudió el efecto de la hidrofilicidad y de la arquitectura de poro. Se cuantificó la viabilidad celular, la proliferación y la expresión de agrecano. Cuando los condrocitos humanos se cultivaron en sustratos poliméricos donde los grupos hidrófilos se distribuyeron de manera homogénea, la adhesión, la proliferación y la viabilidad disminuyó con el contenido de grupos hidrófilo. Sin embargo, los copolímeros en los que los dominios hidrófilos e hidrófobos se alternaban mostraron mejores resultados que los homopolímeros correspondientes. Se sintetizaron series de scaffolds bioestables y series biodegradables con diferente hidrofilicidad y porosidad utilizando plantillas de microesferas sinterizadas. Se obtuvieron arquitecturas de poros regulares y reproducibles. Las células colonizaron el scaffold en su totalidad cuando los poros y la interconexión entre ellos era lo suficientemente grande. Se evaluó la rediferenciación condrogénica de condrocitos autólogos humanos, previamente expandidos en monocapa, sembrados en un scaffold biodegradable de policaprolactona (PCL). Se demostró que los condrocitos cultivados en scaffolds de PCL con medio sin suero bovino fetal (FBS), se rediferenciaban de manera eficiente; expresando un fenotipo condrocítico, caracterizado por su capacidad de sintetizar proteínas de la MEC específicas de cartílago hialino. Se estudió la influencia de la hidrofilicidad y la conectividad de los poros de los scaffolds de caprolactona sobre la adhesión de los condrocitos a las paredes de los poros, su capacidad proliferativa y la composición de MEC sintetizada. Se observó que un mínimo de 70% de porosidad era necesario para permitir la siembra de los condrocitos en el scaffold y su posterior viabilidad. El número de células aumentaba a medida que aumentaba la porosidad del scaffold. Los resultados sugieren que parte de las células que se adherían a las paredes internas de los poros mantenían el fenotipo desdiferenciado de condrocitos cultivados en monocapa, mientras que otros se rediferenciaban. En conclusión, los resultados de esta tesis aportan un avance en el campo de la regeneración de cartílago articular utilizando técnicas de IT. Los estudios realizados proporcionan directrices sobre la composición, la porosidad y la hidrofilicidad más adecuada para l
[CAT] El cartílag articular és un teixit format per condròcits envoltats per una densa matriu extracel·lular (MEC). La MEC es compon principalment de col·lagen tipus II i de proteoglicans. La funció principal del cartílag articular és proporcionar una superfície lubricada a les articulacions. Les lesions en el cartílag articular són comuns i poden derivar en osteoartritis. El cartílag articular no té vasos sanguinis, nervis ni vasos limfàtics i, per tant, té una capacitat limitada d'auto-reparació. L'enginyeria tissular (IT) és una àrea prometedora en la regeneració del cartílag. A la IT s'utilitzen "bastiments" (scaffolds) tridimensionals (3D) com a suports per al cultiu cel·lular i tissular. Els scaffolds proporcionen una estructura que facilita l'adhesió i l'expansió dels condròcits, mantenint un fenotip condrocític limitant la seua desdiferenciació; que és el major problema en els sistemes bidimensionals (2D). L'adhesió cel·lular als scaffolds depèn de les característiques físiques i químiques de la superfície (morfologia, rigidesa, contingut d'aigua en equilibri, tensió superficial, hidrofilicitat i presència de càrregues elèctriques). L'objectiu general d'aquesta tesi va ser estudiar la influència de diferents tipus de biomaterials en la resposta dels condròcits en cultiu in vitro. Els scaffolds han de tindre una estructura porosa interconnectada per a permetre el desenvolupament cel·lular a través de tota l'estructura 3D, potenciant que els condròcits mantinguen el seu fenotip així com permetent l'entrada de nutrients i l'eliminació de productes metabòlics. S'ha estudiat l'efecte de la hidrofilicitat i de l'arquitectura de porus dels scaffolds. Es va quantificar la viabilitat cel·lular, la proliferació i l'expressió de agrecà. Quan els condròcits humans es van cultivar en substrats polimèrics en els quals els grups hidròfils es van distribuir de manera homogènia, l'adhesió, la proliferació i la viabilitat van disminuir amb el contingut de grups hidròfils. No obstant això, els copolímers en els quals els dominis hidròfils i hidròfobs s'alternaven van mostrar millors resultats que els homopolímers corresponents. Es van sintetitzar sèries de scaffolds bioestables i sèries biodegradables amb diferent hidrofilicitat i porositat utilitzant plantilles de microesferes sinteritzades. Es van obtindre arquitectures de porus regulars i reproduïbles. Les cèl·lules van colonitzar el scaffold en la seua totalitat quan els porus i la interconnexió entre ells era suficientment gran. Es van avaluar la rediferenciació condrogènica de condròcits autòlegs humans, prèviament expandits en monocapa, en un scaffold biodegradable de policaprolactona (PCL). Es va demostrar que els condròcits cultivats en scaffolds de PCL sense sèrum boví fetal (FBS) es rediferenciaven de manera eficient, expressant un fenotip condrocític caracteritzat per la seua capacitat de sintetitzar proteïnes de la MEC específiques de cartílag hialí. També es va estudiar la influència de la hidrofilicitat i la connectivitat dels porus dels scaffolds de caprolactona sobre l'adhesió dels condròcits a les parets dels porus, la seua capacitat proliferativa i la composició de MEC sintetitzada. Es va observar que un mínim del 70% de porositat sembla ser necessari per permetre la sembra dels condròcits i la seua posterior viabilitat en el scaffold. El nombre de cèl·lules augmentava a mesura que augmentava la porositat del scaffold. Els resultats suggereixen que part de les cèl·lules que s'adherien a les parets internes dels porus mantenien el fenotip desdiferenciat de condròcits cultivats en monocapa, mentre que altres es rediferenciaven. En conclusió, els resultats d'aquesta tesi proporcionen informació valuosa en el camp de la regeneració de cartílag utilitzant tècniques d'IT. Els estudis realitzats proporcionen directrius sobre la composició, la porositat i la hidrofilicitat m
Pérez Olmedilla, M. (2015). Tissue engineering techniques to regenerate articular cartilage using polymeric scaffolds [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58987
TESIS

Thesis (Ph.D)--Chemical Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Henderson, Clilfford; Committee Member: Ludovice, Peter; Committee Member: Meredith, Carson; Committee Member: Prausnitz, Mark; Committee Member: Rosen, David; Committee Member: Wang, Yadong. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Tissue engineering is a promising alternative strategy to produce artificial bone substitutes
however, the control of the cell organization and cell behavior to create fully functional 3-D constructs has not yet been achieved. To overcome these, activities have been concentrated on the development of multi-functional tissue engineering scaffolds capable of delivering the required bioactive agents to initiate and control cellular activities. The aim of this study was to prepare tissue engineered constructs composed of polymeric scaffolds seeded with mesenchymal stem cells (MSCs) carrying a nanoparticulate growth factor delivery system that would sequentially deliver the growth factors in order to mimic the natural bone healing process. To achieve this, BMP-2 and BMP-7, the osteogenic growth factors, were encapsulated in different polymeric nanocapsules (poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)) with different properties (degradation rates, crystallinity) and, therefore, different release rates to achieve the early release of BMP-2 followed by the release of BMP-7, as it is in nature. Initially, these nanoparticulate delivery systems were characterized and then the effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from these delivery systems was studied in vitro using rat bone marrow MSCs. The effect of using these two growth factors in a sequential manner by mimicking their natural bioavailability timing was shown with maximized osteogenic activity results. BMP-2 loaded PLGA nanocapsules were subcutaneously implanted into Wistar rats and according to initial results, their biocompatibility as well as the positive effect of BMP-2 release on the formation of osteoclast-like cells was shown. To complete the construction of the bioactive scaffold, this nanoparticulate sequential delivery system was incorporated into two different types of polymeric systems
natural (chitosan) and synthetic (poly(&
#949
-caprolactone) (PCL)). 3-D fibrous scaffolds were produced using these materials by wet spinning and 3-D plotting. Incorporation of nanocapsules into 3-D chitosan scaffolds was studied by two different methods: incorporation within and onto chitosan fibers. Incorporation into 3-D PCL scaffolds was achieved by coating the nanocapsules onto the fibers of the scaffolds in an alginate layer. With both scaffold systems, incorporation of nanocapsule populations capable of delivering BMP-2 and BMP-7 in single, simultaneous and sequential fashion was achieved. As with free nanocapsules, the positive effect of sequential delivery on the osteogenic differentiation of MSCs was shown with both scaffold systems, creating multi-functional scaffolds capable of inducing bone healing.

Thesis (Ph.D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008.
Committee Chair: Weck, Marcus; Committee Member: Bunz, Uwe H. F.; Committee Member: Dickson, Robert M; Committee Member: Fahrni, Christoph J; Committee Member: Jones, Christopher W; Committee Member: Murthy, Niren.

This PhD Thesis is focused on the development of fibrous polymeric scaffolds for tissue engineering applications and on the improvement of scaffold biomimetic properties. Scaffolds were fabricated by electrospinning, which allows to obtain scaffolds made of polymeric micro or nanofibers. Biomimetism was enhanced by following two approaches: (1) the use of natural biopolymers, and (2) the modification of the fibers surface chemistry. Gelatin was chosen for its bioactive properties and cellular affinity, however it lacks in mechanical properties. This problem was overcome by adding poly(lactic acid) to the scaffold through co-electrospinning and mechanical properties of the composite constructs were assessed. Gelatin effectively improves cell growth and viability and worth noting, composite scaffolds of gelatin and poly(lactic acid) were more effective than a plain gelatin scaffold. Scaffolds made of pure collagen fibers were fabricated. Modification of collagen triple helix structure in electrospun collagen fibers was studied. Mechanical properties were evaluated before and after crosslinking. The crosslinking procedure was developed and optimized by using - for the first time on electrospun collagen fibers - the crosslinking reactant 1,4-butanediol diglycidyl ether, with good results in terms of fibers stabilization. Cell culture experiments showed good results in term of cell adhesion and morphology. The fiber surface chemistry of electrospun poly(lactic acid) scaffold was modified by plasma treatment. Plasma did not affect thermal and mechanical properties of the scaffold, while it greatly increased its hydrophilicity by the introduction of carboxyl groups at the fiber surface. This fiber functionalization enhanced the fibroblast cell viability and spreading. Surface modifications by chemical reactions were conducted on electrospun scaffolds made of a polysophorolipid. The aim was to introduce a biomolecule at the fiber surface. By developing a series of chemical reactions, one oligopeptide every three repeating units of polysophorolipid was grafted at the surface of electrospun fibers.

This PhD Thesis is focused on the development of fibrous polymeric scaffolds for tissue engineering applications and on the improvement of scaffold biomimetic properties. Scaffolds were fabricated by electrospinning, which allows to obtain scaffolds made of polymeric micro or nanofibers. Biomimetism was enhanced by following two approaches: (1) the use of natural biopolymers, and (2) the modification of the fibers surface chemistry. Gelatin was chosen for its bioactive properties and cellular affinity, however it lacks in mechanical properties. This problem was overcome by adding poly(lactic acid) to the scaffold through co-electrospinning and mechanical properties of the composite constructs were assessed. Gelatin effectively improves cell growth and viability and worth noting, composite scaffolds of gelatin and poly(lactic acid) were more effective than a plain gelatin scaffold. Scaffolds made of pure collagen fibers were fabricated. Modification of collagen triple helix structure in electrospun collagen fibers was studied. Mechanical properties were evaluated before and after crosslinking. The crosslinking procedure was developed and optimized by using - for the first time on electrospun collagen fibers - the crosslinking reactant 1,4-butanediol diglycidyl ether, with good results in terms of fibers stabilization. Cell culture experiments showed good results in term of cell adhesion and morphology. The fiber surface chemistry of electrospun poly(lactic acid) scaffold was modified by plasma treatment. Plasma did not affect thermal and mechanical properties of the scaffold, while it greatly increased its hydrophilicity by the introduction of carboxyl groups at the fiber surface. This fiber functionalization enhanced the fibroblast cell viability and spreading. Surface modifications by chemical reactions were conducted on electrospun scaffolds made of a polysophorolipid. The aim was to introduce a biomolecule at the fiber surface. By developing a series of chemical reactions, one oligopeptide every three repeating units of polysophorolipid was grafted at the surface of electrospun fibers.

Thesis: S.B., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2015.
Cataloged from PDF version of thesis.
Includes bibliographical references (page 33).
In vitro liver models are a critical tool in pharmaceutical research, yet standard hepatocyte cultures fail to capture the complexity of in vivo tissue behavior. One of the most critical features of the in vivo liver is the extensive microvasculature which allows for the delivery of nutrients and metabolites without exposing hepatocytes to de-differentiating fluidic shear stresses. A new liver tissue scaffold design able to capture this histological organization may therefore improve the functional longevity of seeded hepatocytes. The additive manufacturing technique of projection micro-stereolithography (PuSL) proved capable of building non-cytotoxic and highly complex 3D structures with microvasculature on the order of 20 um inner diameter. While extensive biological testing remains to be carried out, the built structures reveal much promise in PuSL as a method of tissue scaffold fabrication in terms of in vivo mimicking architecture.
by Albert Keisuke Matsushita.
S.B.

Aligned fibrillar scaffolds (AFSs) have been widely studied for their application in regenerative medicine, providing possible transplantable tissue replacements for nerve, spinal cord, tendon, ligament, muscle, etc. However, researches in AFSs are technically challenging mainly due to the complex fabrication and characterization processes, especially when the AFSs are made to be fully three-dimensional (3D). As the structure is linked to the quality and function of the engineered tissue product, there is an urgent need for novel techniques to characterize AFSs non-invasively and non-destructively and to link their characteristics to their functions and outcome. In this thesis AFS fabrication and characterization were explored. By combining second harmonic generation (SHG) imaging, multiphoton microscopy (MPM), and various image processing tools, the whole process of 3D tissue characterization could be achieved in a non-invasive, precise, and quantitative way. A proof-of-concept AFS with blended fibers made of polycaprolactone and porcine gelatin was used to demonstrate the feasibility of implementing such a strategy. The data indicated that, in terms of scaffold characterization, the proposed MPM method was capable of measuring the porosity of homogenous scaffolds precisely from deconvolved 3D images. Furthermore, the method could also be used to illustrate the orientation of the aligned nanofibers. Next, when SH-SY5Y neurons were cultured on the AFS, the MPM imaging was capable of evaluating the cell viability ratio, cell-localization in AFS, and neurite outgrowth. This provided guidance for selecting the alignment method for AFS functional recovery. Lastly, when employing this non-invasive imaging-based characterization method, it was possible to illustrate the relationship between the alignment of collagen arrays in decellularized corneal stroma and the transparency. In summary, the proposed strategy can provide some essential scaffold/tissue properties (such as alignment of fiber, porosity of scaffold, and cell viability ratio) quantitatively and non-invasively, which will help both scaffold processing design and characterization.

There is a need for development of versatile soft tissue scaffolds which can interface with tissue junctions and a range of tissue architectures. Two approaches were developed. A bone capable ceramic scaffold was infused with gel and polymer components to suit cartilage resurfacing. Several biphasic prototypes were successfully developed and evaluated mechanically and biologically. Whilst they supported chondrocyte culture within the infused gel layer they lacked sufficient mechanical stability. Two therapeutic agents were investigated for potential roles in modulating cartilage degradation. Gene expression (RT-PCR) measurements demonstrated that whilst zoledronic acid reduced cartilage degradation, S100A9 increased ECM breakdown. In a subsequent more substantive approach, a versatile polymer composite scaffold (variotis™) was designed and synthesised using a novel, high-throughput batch coagulation method. Methods were developed for formation of scaffolds with a range of shapes and properties. This novel scaffold could be readily post formed to impart graded interconnected porosity and mechanical response. Characterisation was undertaken for physical structure and mechanical behavior. The soft, elastic scaffolds were highly porous with excellent void interconnectivity and tensile properties within the range of skin. A range of novel bioactive glass compositions were developed for incorporation with the scaffolds. Glasses were analysed compositionally, mechanically, for bioactivity and radiopacity. During in vitro studies, composites with bioactive glass and bulk metallic glass constituents were shown to support fibroblast cell culture and exhibited less inflammation than stainless steel and cell controls, negligible inflammation. In vivo subcutaneous implantation of 124 polymer and polymer-composite variotis™ scaffolds into rats demonstrated complete tissue filling by 14 days. Histological staining confirmed high proportions of mature collagen content and vascularization within the polymer-Bioglass® composite scaffolds. Low inflammatory levels were confirmed by circulating and wound fluid cell counts.

Functional bone tissue engineering has been necessitated by the need to treat critical size defects in bones due to birth abnormalities, trauma, and pathological conditions. Appropriate conditions for in vitro osteogenesis need to be identified to establish protocols for engineering bone tissues. The success of in vitro osteogenesis lies on the type of cell source, stimuli, and scaffold material used for engineering bone constructs. Recent investigations have established the pluripotency of mesenchymal stem cells (MSCs) and their ability to differentiate down a multitude of pathways including osteogenenic. In vivo studies have shown that MSCs are primarily responsible for bone growth and regeneration and therefore have become a major candidate for bone tissue engineering. Osteogenic differentiation of MSCs via chemical stimuli has been extensively investigated using both monolayer and three-dimensional (3D) culture conditions. These investigations provided useful information on media conditions, cell seeding densities, and differentiation capabilities of MSCs. However, chemical stimulation alone might not be sufficient to accelerate osteogenesis and impart necessary mechanical strength to the final tissue construct. Mechanical strength of the final tissue construct is vital to maintain its structural integrity when exposed to physiological stresses in vivo. Stimulation of MSCs using mechanical strain might provide another method to induce MSC osteogenesis while also obtaining desired mechanical strength of the final tissue constructs. Although in vivo studies and experimental models have indicated that cyclic tensile strain could induce MSC osteogenesis, its effect on MSC osteogenesis in 3D cultures in vitro has not been investigated. The need to maintain cell viability and be able to provide chemical or mechanical cues to cells in 3D cultures requires improvements in scaffold architecture and design. While collagen provides a natural matrix for cell adhesion and growth, its contraction during culture can greatly limit culture duration and mechanical stability of the matrix. Although fibrous scaffolds can be used as an alternative to collagen scaffolds, insufficient media diffusion to the center of these 3D scaffolds could detrimentally affect uniform cell growth throughout the scaffold; hence, scaffolds with better diffusional properties need to be developed. This study investigated the use of 3D collagen matrices as a scaffold material to determine the effects of strain and chemical stimuli on osteogenic differentiation of human MSCs (hMSCs). Major attention was given to the analyses of: cell viability, matrix contraction, nuclei morphology, expression of osteogenic markers and proinflammatory cytokines, as well as changes in mechanical properties of the final tissue construct. As an approach to develop 3D fibrous scaffolds with enhanced diffusional properties, fabrication of melt spun microporous fibers using a blend of poly (lactic acid) (PLA) and sulfopolyester that could be used in 3D nonwoven scaffolds was also investigated. The findings of this study clearly illustrated the ability of cyclic tensile strain to induce osteogenic differentiation of hMSCs when cultured in a 3D environment. Expression of proinflammatory cytokines by strained hMSCs suggested that cyclic strain might have induced modulation of bone resorption in hMSCs. The results also illustrated the effects of strain on the mechanical properties of the final tissue construct. Microporous fibers created from melt spun composite fibers using binary blends of poly (lactic acid) and sulfopolyester could enhance diffusional properties of 3D nonwoven scaffolds fabricated using these fibers. As this body of work demonstrates, use of cyclic tensile strain combined with chemical stimulation to induce osteogenic differentiation of hMSCs could greatly assist the engineering of functional bone tissues in vitro. Microporous fibers created using polymer blends could provide an effective method to improve diffusional properties of 3D polymeric scaffolds.

In situ forming implants (ISIs) are popular due to their ease of use and local drug delivery potential, but they suffer from high initial drug burst, and release behavior is tied closely to solvent exchange and polymer properties. Additionally, such systems are traditionally viewed purely as drug delivery devices rather than potential scaffold materials due to their poor mechanical properties and minimal porosity. The aim of this research was to develop an injectable ISI with drug release, mechanical, and microstructural properties controlled by micro- and nanoparticle additives. First, an injectable ISI was developed with appropriate drug release kinetics for orthopedic applications. Poly(β-amino ester) (PBAE) microparticles were loaded with simvastatin or clodronate, and their loading efficiency and drug retention after washing was quantified. Drug-loaded PBAE microparticles and hydroxyapatite (HA) microparticles were added to a poly(lactic-co-glycolic acid) (PLGA)–based ISI. By loading simvastatin into PBAE microparticles, release was extended from 10 days to 30 days, and burst was reduced from 81% to 39%. Clodronate burst was reduced after addition of HA, but was unaffected by PBAE loading. Scaffold mass and porosity fluctuated as the scaffolds swelled and then degraded over 40 days. Next, the mechanical properties of these composite ISIs were quantified. Both micro- and nanoparticulate HA as well as PBAE microparticle content were varied. Increasing HA content generally improved compressive strength and modulus, with a plateau occurring at 30% nano-HA. Injectability remained clinically acceptable for up to 10% w/w PBAE microparticles. Ex vivo injections into trabecular bone improved both strength and modulus. Lastly, HA-free ISIs were investigated for drug delivery into the gingiva to treat periodontitis. Doxycycline and simvastatin were co-delivered, with delivery of doxycycline over 1 week accompanied by simvastatin release over 30 days. PBAE-containing ISIs exhibited higher initial and progressive porosity and accessible volume than PBAE-free ISIs over the course of degradation. Additionally, PBAE-containing ISIs provided superior tissue retention within a simulated periodontal pocket. The ISIs investigated here have a wide range of potential applications due to their flexible material and drug release properties, which can be controlled by both the chemistry and concentration of various particulate additives.

Biomechatronics is an emerging field in robotics where living organisms are interfaced with classical artificial electromechanical systems. Applications include the development of medical microrobotic devices and targeted drug delivery, artificial prosthetics and biomimetic robotic machines. Actuation is a core element of the mechatronic system, which allows the movement and locomotion of the machine. In the field of bio-hybrid actuation, higly efficient muscular cells are employed for this task, allowing higher performace and also capable of self healing and autonomous adaptation to the external stimuli. For this purpose, the development of suitable scaffolds to support the cells is necessary, requiring the tailoring of mechanical properties and the overall 3D structuring of the material. In this thesis, a polymeric material scaffold with a three-dimensional structure in the micrometer range based on polyurethane is investigated. Design and synthesis of the material with tailored mechanical properties is carried out, focusing on the biological interaction with the muscular cells. The stiffness of the polymer material structure is related to the output movement of the actuator and the overall performance of the device is optimized at the scaffold material level.

This PhD thesis studies fabrication of superhydrophobic polymeric surfaces, their wetting properties, and their antibacterial activities as potential application to medical sciences. A femtosecond laser technique was used to fabricate mico/nano- structures on the surface of PTFE and PU. The effect of laser parameters (fluence, scanning speed, and overlap) on the wettability of the resulted micro/nano-patterns was studied. Two techniques were used to laser-scan the surface, namely uniaxial and biaxial scan. Uniaxial scan creates channeled morphology with direction-dependent wettability. To produce uniform wettability independent of direction, biaxial scanning was examined, which creates well-defined pillars with very high contact angle (CA) and very low contact angle hysteresis (CAH). To facilitate and speed up the surface micro/nano-structuring, laser-ablation was coupled with thermal imprinting. The metallic femtosecond laser-ablated templates were employed to imprint micron/submicron periodic structures onto the surface of several polymers. The CA of imprinted polymers increased to above 160°, while their CAH varied significantly depending on the surface thermophysical and chemical properties. A unique technique was developed to create superomniphobic patterns on HDPE through hot embossing. The filefish skin dual scale superoleophobic patterns were used as a biological model to develop angled microfiber arrays on HDPE. The obtained bioinspired surface is highly capable of repelling both water and liquids with low surface tensions that meets the superomniphobic criteria. The effect of superhydrophobicity on protein adsorption and bacterial adhesion of laser-ablated PTFE substrates were investigated. Samples were incubated in Gram negative (E.coli) and Gram positive (S.aureus) bacteria cultures, BSA solution, IgG solution, and blood plasma for 4 hours. All superhydrophobic surfaces were found to be more resistant to protein /bacteria adhesion compared to the corresponding smooth samples. However, some of the most superhydrophobic PTFE surfaces were found to exhibit the highest adherence with protein/bacteria; while some other did not allow any adsorption/adherence of protein/bacteria respectively towards the end of the incubation. Besides the CA, CAH, average height of pillars, and spacing distance between iii the pillars, this study showed that there are other roughness factors, which play crucial role in the durability of the superhydrophobic surfaces such as the distribution of pillar heights.
Applied Science, Faculty of
Chemical and Biological Engineering, Department of
Graduate

Le polymère poreux (PM) associe les avantages double des matériaux poreux et des polymères, ayant la structure unique de pore, la porosité supérieure et la densité inférieure, ce qui possède une valeur d’application importante dans les domaines de l'adsorption, le soutien de catalyseur, le séparateur de batterie, la filtration, etc. Actuellement, il existe plusieurs façons de préparer le PM, comme la méthode de gabarit, la méthode de séparation de phase, la méthode d'imagerie respiratoire, etc. Chacune des méthodes ci-dessus existe ses propres avantages, mais la préparation à grande échelle de PM à structure de pore contrôlable et aux fonctions spécifiques est toujours un objectif à long terme sur le domaine et l'un des principaux objectifs de ce mémoire. La co-extrusion de microcouche est une méthode pour produire de façon efficace et successive des polymères avec des structures de couches alternées, ayant les avantages de haute efficacité et faible coût. Par conséquent, sur les exigences structurelles de PM de l’application spécifique, ce mémoire a conçu le PM avec une structure spécifique et une co-extrusion de microcouche de manière créative combinée avec la méthode traditionnelle de préparation de PM (méthode de gabarit, méthode de séparation de phase), en combinant les avantages des deux méthodes, les PM avec une structure de pore idéale peuvent être préparés en grande quantité et l’on peut également explorer son application dans les séparateurs de batteries au lithium-ion et l'adsorption d'hydrocarbures aromatiques polycycliques.Le plus important, dans la deuxième partie de cet essai, se trouve que la simulation micro-numérique est utilisée pour étudier le transport et le dépôt de particules dans des milieux poreux pour explorer le mécanisme des matériaux poreux dans les domaines de l'adsorption et du séparateur de batterie. Le code de 3D-PTPO (un modèle tridimensionnel de suivi des particules combinant Python® et OpenFOAM®) est utilisé pour étudier le transport et le dépôt de particules colloïdales dans des milieux poreux, l’on adopte trois modèles (colonne, venturi et tube conique) pour représenter différentes formes de matériaux poreux. Les particules sont considérées comme des points matériaux pendant le transport, le volume des particules sera reconstitué et déposé comme partie de la surface du matériau poreux pendant le dépôt, la caractéristique principale de ce code est de considérer l'influence du volume des particules déposées sur la structure des pores, les lignes d'écoulement et le processus du dépôt des autres particules. Les simulations numériques sont d'abord conduites dans des capillaires simples, le travail de chercheurs de Lopez et d’autres est réexaminé en établissant un modèle géométrique tridimensionnel plus réaliste et il explore les mécanismes cachés derrière les règles de transmission et de dépôt. Par la suite, des simulations numériques sont effectuées dans des capillaires convergents-divergents pour étudier la structure des pores et l'effet de nombre Peclet sur le dépôt de particules. Enfin, l’on étudie l’effet double de l'hétérogénéité de surface et de l'hydrodynamique sur le comportement de dépôt de particules
Due to the combination of the advantages of porous media and polymer materials, polymeric porous media possess the properties of controllable porous structure, easily modifiable surface properties, good chemical stability, etc., which make them applicable in a wide range of industrial fields, including adsorption, battery separator, catalyst carrier, filter, energy storage, etc. Although there exist various preparation methods, such as template technique, emulsion method, phase separation method, foaming process, electrospinning, top-down lithographic techniques, breath figure method, etc., the large-scale preparation of polymeric porous media with controllable pore structures and specified functions is still a long-term goal in this field, which is one of the core objectives of this thesis. Therefore, in the first part of the thesis, polymeric porous media are firstly designed based on the specific application requirements. Then the designed polymeric porous media are prepared by the combination of multilayer coextrusion and traditional preparation methods (template technique, phase separation method). This combined preparation method has integrated the advantages of the multilayer coextrusion (continuous process, economic pathway for large-scale fabrication, flexibility of the polymer species, and tunable layer structures) and the template/phase separation method (simple preparation process and tunable pore structure). Afterwards, the applications of the polymeric porous media in polycyclic aromatic hydrocarbons adsorption and lithium-ion battery separator have been investigated.More importantly, in the second part of the thesis, numerical simulations of particle transport and deposition in porous media are carried out to explore the mechanisms that form the theoretical basis for the above applications (adsorption, separation, etc.). Transport and deposition of colloidal particles in porous media are of vital important in other applications such as aquifer remediation, fouling of surfaces, and therapeutic drug delivery. Therefore, it is quite worthy to have a thorough understanding of these processes as well as the dominant mechanisms involved. In this part, the microscale simulations of colloidal particle transport and deposition in porous media are achieved by a novel colloidal particle tracking model, called 3D-PTPO (Three-Dimensional Particle Tracking model by Python® and OpenFOAM®) code. The particles are considered as a mass point during transport in the flow and their volume is reconstructed when they are deposited. The main feature of the code is to take into account the modification of the pore structure and thus the flow streamlines due to deposit. Numerical simulations were firstly carried out in a capillary tube considered as an element of an idealized porous medium composed of capillaries of circular cross sections to revisit the work of Lopez and co-authors by considering a more realistic 3D geometry and also to get the most relevant quantities by capturing the physics underlying the process. Then microscale simulation is approached by representing the elementary pore structure as a capillary tube with converging/diverging geometries (tapered pipe and venturi tube) to explore the influence of the pore geometry and the particle Péclet number (Pe) on particle deposition. Finally, the coupled effects of surface chemical heterogeneity and hydrodynamics on particle deposition in porous media were investigated in a three-dimensional capillary with periodically repeating chemically heterogeneous surfaces

Conventional bone grafts are fraught with limitations and three dimensional (3D) electrospun fibrous nanocomposites of gelatin and hydroxyapatite (HA) similar to the extracellular matrix (ECM) of bone are viable bone graft substitutes but there is limited research in this area. In this project, fibrous scaffolds of gelatin-HA nanocomposites were fabricated using electrospinning and crosslinked using glutaraldehyde vapour. The microstructural, physical and mechanical properties of the scaffolds were measured and the effects of applied voltage, HA concentration and crosslinking duration on scaffold properties were determined and used to optimise the electrospinning process. Human foetal osteoblast cells were grown on the scaffolds and cell seeding efficiency, cell proliferation, cell viability, alkaline phosphatase activity, collagen matrix synthesis and mineralisation were quantified. Tissue engineered 3D bone grafts were developed by stacking together optimised seeded scaffolds using the three-stack and the four-stack models and also cultured under dynamic conditions in a perfusion bioreactor to improve nutrient and oxygen transport to cells. Mathematical models were developed for nutrient and oxygen transport and cellular response in the scaffolds and layer-by-layer oxygen and cell concentrations were predicted in the 3D bone graft models. Models were developed for cell growth and oxygen consumption rates and their constants were determined and used as input parameters for the mathematical models along with the determined physical and biological scaffold properties in the computer simulations of bone tissue engineering. The scaffolds exhibited a good degree of fibre alignment and both fibre and pore diameters exhibited inverse relationships with applied voltage and HA concentration. The scaffolds possessed reasonable levels of porosity and permeability which were a function of the fibre diameter. Young’s modulus and ultimate tensile strength were functions of fibre diameter, porosity and direction of loading and exhibited proportional relationships with applied voltage, HA concentration and crosslinking duration. Initial cell seeding efficiency was over 90% in all scaffolds with cell proliferation, alkaline phosphatase activity, collagen synthesis and mineralisation all exhibiting inverse relationships with applied voltage and proportional relationships with HA concentration as a result of the concomitant variations in fibre diameter, pore diameter and porosity of the scaffolds. The 25 wt% HA scaffold electrospun at 20 kV had optimum osteogenic, physical and mechanical properties and contained mineralised bone tissue after 18 days of cell culture. Functional 3D bone grafts were obtained with favourable cell proliferation, which improved under dynamic culture, albeit with limited cell migration and a 3D multi-thin-layered stacked bone graft model was proposed based on these findings. Finally, the mathematical models were successfully validated against the experimental cell concentration and migration depth data.

Advanced biomaterials and sophisticated processing technologies aim to fabricate tissue-engineering scaffolds that can predictably interact within a biological environment at a cellular level. Sterilization of such scaffolds is at the core of patient safety and is an important regulatory issue that needs to be addressed prior to clinical translation. In addition, it is crucial that meticulously engineered micro- and nano- structures are preserved after sterilization. Conventional sterilization methods involving heat, steam and radiation are not compatible with engineered polymeric systems because of scaffold degradation and loss of architecture. Using electrospun scaffolds made from polycaprolactone (PCL), a low melting polymer, and employing spores of Bacillus atrophaeus as biological indicators, we compared ethylene oxide, autoclaving and 80% ethanol to a known chemical sterilant, peracetic acid (PAA), for their ability to sterilize as well as their effects on scaffold properties. PAA diluted in 20% ethanol to 1000 ppm or above, sterilized electrospun scaffolds in 15 min at room temperature while maintaining nano-architecture and mechanical properties. Scaffolds treated with PAA at 5000 ppm were rendered hydrophilic, with contact angles reduced to zero degrees. Therefore, PAA can provide economical, rapid and effective sterilization of heat-sensitive polymeric electrospun scaffolds used in tissue-engineering.

No
This title provides a comprehensive and authoritative review on recent advancements in the application and use of composite scaffolds in tissue engineering. Chapters focus on specific tissue/organ (mostly on the structure and anatomy), the materials used for treatment, natural composite scaffolds, synthetic composite scaffolds, fabrication techniques, innovative materials and approaches for scaffolds preparation, host response to the scaffolds, challenges and future perspectives, and more. Bringing all the information together in one major reference, the authors systematically review and summarise recent research findings, thus providing an in-depth understanding of scaffold use in different body systems.

Thesis: S.M., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2014.
35
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 65-67).
Recent advances in material processing are presenting groundbreaking opportunities for biomedical engineers. Projection-micro-stereolithography, or PuSL, is an additive manufacturing technique in which complex parts are built out of UV-curable resins using ultraviolet light. The primary strength of PuSL is its capacity to translate CAD files into three-dimensional parts with unusually small feature sizes (~0.5 microns). It is an ideal candidate, therefore, for making tissue scaffolds with sophisticated microscopic architecture. Nearly all multicellular biological tissues display a hierarchy of scale. In human tissues, this means that the mechanics and function of an organ are defined by structural organization on multiple levels. Macroscopically, a branching blood supply creates a patent network for nutrient delivery and gas exchange. Microscopically, these vessels spread into capillary beds shaped in an organ-specific orientation and organization, helping to define the functional unit of a given tissue. On a nano-scale, the walls of these capillaries have a tissue-specific structure that selectively mediates the diffusion of nutrients and proteins. To craft a histologically accurate tissue, each of these length scales must be considered and mimicked in a space-filling fashion. In this project, I sought to generate a cellular, degradable tissue scaffolds that mimicked native extracellular matrix across length scales. The research described here lays the groundwork for the generation of degradable, vascularized cell scaffolds that might be used to build architecturally complex multi-cellular tissues suitable for both pharmacological modeling and regenerative medicine.
by Micha Sam Brickman Raredon.
S.M.

L'objectif de ce travail est de développer une méthode d'ingénierie de scaffolds multidimensionnels pour la culture cellulaire et l’ingénierie tissulaire. Nous avons d'abord appliqué une technique d'impression 3D pour produire un scaffold en PEGDA et ensuite rempli l'espace libre du scaffold avec du gel de gélatine. Après la congélation et le séchage, un scaffold hybride en PEGDA avec des structures fine de gélatine a été obtenu, qui a été ensuite valisé par la culture et la différenciation des cellules progénitrices neuronales. Pour intégrer plus facilement dans un dispositif microfluidique, nous avons également conçu un scaffold 2D sous forme d’une couche mince de nid d'abeilles de PEGDA rempli des structures poreuses auto-assemblée de PCL. Ce scaffold 2D a été utilisé pour la culture cellulaire et la transfection des gènes, montrant des avantages par rapport aux méthodes classiques en termes d'absorption des nutriments et des facteurs solubles. Enfin, nous avons fabriqué un scaffold mous constitué d’une couche mince de nid d'abeilles en élastomère de PDMS et d’une monocouche de nanofibres de gélatine pour faciliter la différenciation cardiaque à partir des cellules souches pluripotentes humaine. Comme prévu, nous avons réalisé une génération cardiaque avec une contraction plus forte et une homogénéité de battement plus élevée par rapport aux approches classiques. Tous ensemble, nous avons démontré l'utilité des scaffolds hybrides pour l'ingénierie micro-tissulaire qui pourraient avoir un impact sur les études futures dans les domaines de l'ingénierie tissulaire, du criblage des médicaments et de la médecine régénératrice
The objective of this work is to develop a method of engineering multi-dimensional scaffolds for cell culture and tissue formation. We firstly applied a 3D printing technique to produce the designed frame in PEGDA and then filled the free-space of the frame with a gelatin gel. After freezing and drying, a hybrid 3D scaffold made of gelatin porous structures and PEDGA backbone was obtained, which supported culture and differentiation of neural progenitor cells. To more easily integrate into a microfluidic device, we also designed a 2D scaffold in form of a thin layer of honeycomb frame of PEGDA and self-assembled porous structure of PCL. Such a patch form scaffold could be used for cell culture and gene transfection, showing advantages over the conventional methods in terms of nutrients and soluble factors uptake. Finally, we fabricated a soft patch made of an elastic frame in PDMS and a monolayer of gelatin nanofibers to facilitate cardiac differentiation from human induced pluripotent stem cells. As expected, we achieved a cardiac generation with higher contraction strength and a higher beating homogeneity comparing to the conventional approaches. All together, we demonstrated the utility of hybrid scaffolds for micro-tissue engineering which could impact the future studies in the fields of tissue engineering, drug screening and regenerative medicine

Mestrado em Materiais e Dispositivos Biomédicos
Bone is an extremely important connective tissue in the human body, as it provides support and protection of internal organs, being also metabolically relevant as the main mineral reservoir and assuring haematopoiesis through the bone marrow. Due to the current ageing of the population, an increase in bone tissue related diseases is noticeable. Thus, more efficient therapies for treating bone diseases is crucial. Tissue Engineering appears as a promising technology for treating several of those problems, such as bone loss and joint problems. In the present work, composite biomaterials composed of a polymeric hydrogel matrix reinforced with bioactive glass particles were prepared. Individually, these materials have a high water content, which enhances their diffusive transport properties, and display osteogenic properties, respectively. The selected polymer was RGD functionalized pectin, due to its interesting properties, such as biocompatibility, cell-adhesive characteristics and adequacy for cell entrapment, and the bioactive glass selected was a novel alkali-free formulation of 70% diopside and 30% tricalcium phosphate (Di-70), composed of SiO2, CaO, MgO and P2O5. Several different composite formulations were tested, in which pectin concentration, bioactive glass content and glass particle size were varied. The biocomposite’s viscoelastic properties were assessed, as well as their biological behaviour through cytotoxicity assays, and osteogenic character by incubating mesenchymal stem cell (MSC)-laden composites into both basal and osteogenic media for up to 21 days. The results obtained demonstrated that a composite biomaterial with tuneable mechanical properties was successfully prepared, with in situ crosslinking ability within therapeutically relevant timeframes, and not requiring additional crosslinking strategies besides its own composition. Furthermore, its intrinsic osteogenic properties due to the glass composition provided the adequate conditions for promoting the differentiation of MSCs without osteogenic stimulation. The combined properties achieved indicate that the biocomposites prepared are suitable candidate cellularized biomaterials for bone tissue engineering applications.
O osso é um tecido conjuntivo de extrema importância no organismo humano, tendo funções como suporte ou proteção de órgãos internos, sendo também metabolicamente relevante como o principal reservatório de minerais e assegurando a hematopoiese com a medula óssea. Dado o envelhecimento da população, tem-se verificado um aumento da incidência de doenças degenerativas deste tecido, sendo assim essencial aplicar terapias altamente eficientes para o tratamento dessas patologias. A Engenharia de Tecidos surge como uma tecnologia promissora no tratamento destes problemas, como a perda de massa óssea e problemas nas articulações. Neste trabalho, foram produzidos biomateriais compósitos, baseados numa matriz polimérica sob a forma de hidrogel reforçada com partículas de vidro bioativo. Individualmente, estes materiais apresentam um elevado teor em água favorável ao transporte de nutrientes, e propriedades osteogénicas, respetivamente. O polímero selecionado foi a pectina funcionalizada com RGD, dadas as suas propriedades interessantes como a biocompatibilidade, capacidade de promover a adesão celular e adequabilidade para o encapsulamento de células, e o vidro bioativo apresenta uma composição de 70% de diópsido e 30% de fosfato tricálcico (Di-70) isento de alcalinos e sendo composto por SiO2, CaO, MgO e P2O5. Diferentes formulações de hidrogéis compósitos foram testadas, em que se variou a concentração de polímero, a concentração de biovidro e o seu tamanho de partícula. Analisaram-se as propriedades viscoelásticas dos biocompósitos, bem como o seu comportamento biológico, com ensaios de citotoxicidade, e ainda as propriedades osteogénicas do material, pela incubação de hidrogéis contendo células estaminais mesenquimais (MSCs) em meio basal e osteogénico durante 21 dias. Os resultados deste trabalho indicam que foi possível preparar um biomaterial compósito de propriedades mecânicas ajustáveis, com capacidade de reticular in situ em tempos clinicamente desejáveis sem necessitar agentes reticulantes externos. Para além disso, as propriedades osteogénicas intrínsecas do biovidro forneceram as condições adequadas para a promoção da diferenciação de MSCs sem estimulação osteogénica adicional. As propriedades combinadas alcançadas indicam que os biocompósitos preparados têm potencial para ser aplicados em engenharia de tecido ósseo.