Poly(N-isopropylacrylamide) and copolymers: a review on recent progresses in biomedical applications

The innate ability of poly(N-isopropylacrylamide) (PNIPAAm) thermo-responsive hydrogel to copolymerize and to graft synthetic polymers and biomolecules, in conjunction with the highly controlled methods of radical polymerization which are now available, have expedited the widespread number of papers published in the last decade—especially in the biomedical field. Therefore, PNIPAAm-based hydrogels are extensively investigated for applications on the controlled delivery of active molecules, in self-healing materials, tissue engineering, regenerative medicine, or in the smart encapsulation of cells. The most promising polymers for biodegradability enhancement of PNIPAAm hydrogels are probably poly(ethylene glycol) (PEG) and/or poly(e-caprolactone) (PCL), whereas the biocompatibility is mostly achieved with biopolymers. Ultimately, advances in three-dimensional bioprinting technology would contribute to the design of new devices and medical tools with thermal stimuli response needs, fabricated with PNIPAAm hydrogels.Peer ReviewedPostprint (published version

Smart Materials for Biomedical Applications: The Usefulness of Shape-Memory Polymers

Acknowledgments: This research work was supported by the Portuguese Foundation for Science & Technology (FCT) through the Project references CMUP-ERI/TIC/0021/2014 and UID/Multi/04044/2013. In addition, the authors would like to thank Portuguese National Innovation Agency (ANI) through the Project reference POCI-01-0247-FEDER-017963 and European Regional Development Fund (FEDER), through COMPETE2020 under the PT2020 program (POCI-01-0145-FEDER-023423). The authors (MYK and GBH) acknowledge the financial support of UGC, New Delhi under UPE-FAR-I Program [F. No. 14-3/2012 (NS/PE)] and DST, New Delhi under DST-PURSE-Phase-II Program [F. No. SR/PURSE Phase 2/13(G)].This review describes available smart biomaterials for biomedical applications. Biomaterials have gained special attention because of their characteristics, along with biocompatibility, biodegradability, renewability, and inexpensiveness. In addition, they are also sensitive towards various stimuli such as temperature, light, magnetic, electro, pH and can respond to two or more stimuli at the same time. In this manuscript, the suitability of stimuli-responsive smart polymers was examined, providing examples of its usefulness in the biomedical applications.info:eu-repo/semantics/publishedVersio

Responsive Polymers as Cell Surface Modifiers and 3D Healable Microenvironments

The interplay between cells and biomaterials constitutes a fertile ground to probe specific cellular functions and cues for therapeutic and research purposes. “Smart” materials encompass an extensive library that can lead to the design of dynamic multi-responsive constructs with great importance in the biomedical field. This work aims to describe diverse strategies on the modification of biological interfaces with synthetic polymers to promote the assembly of living cells and the design of multi-responsive healable cell-encapsulating constructs with interest in 3D in vitro modelling, drug delivery, cell-based therapies and tissue engineering. In the first part, cell membrane engineering approaches are introduced to create a responsive platform for the accelerated and simple formation of cellular aggregates/spheroids, and to study polymer-cell interactions by exploring biorthogonal ligand-receptor multivalent interactions under different conditions. Specifically, boronic acid- and succinimide-based copolymers were first synthesised and fully characterised by physicochemical methods, and found to bind covalently to natural moieties present on the membrane of several cell lines, which can regulate the development of cell spheroids and act as self-supporting “cellular glues”. The second part of the project is dedicated to the development of multi-responsive self-healing hydrogel nanocomposites for biomedical applications, where we further expanded the dynamic crosslinking nature of boronate ester bonds. The proposed gels could be prepared almost instantly, exhibited photo- and thermoreversible transient sol-gel type of transition with excellent healing properties, and no toxicity, which allows the system to be used as a versatile biologic delivery matrix. In summary, the results highlight novel and straightforward approaches that may pave the way to implement a biomaterial-cell platform with broad biotechnological applications

Establishment of dynamic culture conditions for the fabrication of advanced in vitro tissue models.

In this thesis, a system for the application of flow-induced, physiologically relevant shear stress on mammalian cells was established. The circulation of cell culture medium in culture circuits was generated by peristaltic pumps, which can be operated with a self-built control unit. Culture chambers for different applications were computer-assisted designed, characterized by computational fluid dynamics and subsequently manufactured either by CNC milling or 3D printing. Using this system, shear-induced changes on the cellular functionality of in vitro barrier models were investigated. The application of low, physiologically relevant shear forces to an established intestinal epithelial cell line, HT29-MTX, resulted in increased mucus production, as well as structural reorganization of the confluent cell layer towards 3-dimensional villi-like structures. The results could be transferred from solid cell culture substrates to commercially available cell culture inserts bearing membranes, allowing us to develop an improved in vitro model of the intestinal barrier with a physiologically relevant mucus layer. We then proceeded with the endothelial barrier. Here, we investigated the effects of flow-induced stress comparatively for HUVECs and iPSC-ECs. Both cell types showed the characteristic alignment upon application of flow, as well as increased layer thicknesses and improved functionality of the endothelial glycocalyx. In addition, for the first time, we were able to detach isotropic (HUVECs) and anisotropic (iPSC-ECs) cell monolayers from thermoresponsive surfaces and wrapped them around a 3D-printed scaffold while maintaining alignment, which represents an important step toward the development of blood vessels in vitro. Finally, we implemented a similar tubular construct into dynamic culture. The indirect coculture of HUVECs with fibroblasts allowed intercellular communication and resulted in the formation of stable vascular networks, while a second independent circuit allowed perfusion of the tubular blood vessel mimick. In summary, we established a versatile and reliable platform for the application of physiological shear forces to flat and lumenized tissues, enabling the development of improved in vitro barrier models.In dieser Arbeit wurde ein System zur Applikation fluss-induzierter, physiologisch relevanter Scherkräfte auf Säugerzellen etabliert. Die Zirkulation von Zellkulturmedium in Kulturkreisläufen wurde dabei über Peristaltikpumpen generiert, welche mit einer selbstgebauten Steuerung betrieben werden können. Kulturkammern für unterschiedliche Einsatzgebiete wurden computergestützt entworfen, mithilfe numerischer Strömungsmechanik charakterisiert und anschließend entweder durch CNC-Fräsen oder 3D-Druck hergestellt. Mithilfe dieses Systems wurde der Einfluss scherinduzierten Änderungen auf die zelluläre Funktionalität von in vitro Barrieremodellen untersucht. Die Applikation geringer, physiologisch relevanter Scherkräfte auf eine etablierte intestinale epitheliale Zelllinie, HT29-MTX, führte zu einer erhöhten Mucusproduktion, sowie der strukturellen Reorganisation der konfluenten Zellschicht hin zu 3-dimenionalen, Darmzotten-artigen Gebilden. Die Ergebnisse konnten von festen Zellkultursubstraten auf kommerziell erhältliche Zellkultureinsätze mit mikroporösen Membranen übertragen werden, sodass wir ein verbessertes in vitro Modell der intestinalen Barriere mit physiologisch relevanten Mucusschichten entwickeln konnten. Im Anschluss widmeten wir uns dem Studium der Barrierefunktion von Endothelzellen. Dabei untersuchten wir die Auswirkungen von fluss-induziertem Stress vergleichend für HUVECs und iPSCs-ECs. Beide Zellarten zeigten die charakteristische Ausrichtung mit dem Fluss, sowie erhöhte Schichtdicken und verbesserte Funktionalität der endothelialen Glykokalix. Zudem konnten wir erstmalig intakte isotrope (HUVECs) und anisotrope (iPSC-ECs) Zellmonolagen temperatur-gesteuert von thermoresponsiven Oberflächen ablösen und um ein 3D-gedrucktes Gerüst rollen, wobei die Zellausrichtung erhalten blieb, was einen wichtigen Schritt zur Entwicklung von Blutgefäßen in vitro darstellt. Abschließend implementierten wir ein ähnliches Konstrukt in die dynamische Kultur. Die Erweiterung des Systems durch eine indirekte Kokultur der HUVECs mit Fibroblasten ermöglichte die Ausbildung stabiler, vaskularer Netzwerke und ein zweiter unabhängiger Kreislauf erlaubte die Perfusion der künstlichen tubulären Blutgefäße. Zusammenfassend konnten wir eine vielseitige und verlässliche Plattform zur Applikation von physiologischen Scherkräften auf adhärenten Säugerzellen in 2D oder in 3D etablieren, die den Aufbau von verbesserten in vitro Barrieremodellen ermöglicht

Co-axial wet-spinning in 3D Bioprinting: state of the art and future perspective of microfluidic integration

Nowadays, 3D bioprinting technologies are rapidly emerging in the field of tissue engineering and regenerative medicine as effective tools enabling the fabrication of advanced tissue constructs that can recapitulate in vitro organ/tissue functions. Selecting the best strategy for bioink deposition is often challenging and time consuming process, as bioink properties-in the first instance, rheological and gelation-strongly influence the suitable paradigms for its deposition. In this short review, we critically discuss one of the available approaches used for bioprinting-namely co-axial wet-spinning extrusion. Such a deposition system, in fact, demonstrated to be promising in terms of printing resolution, shape fidelity and versatility when compared to other methods. An overview of the performances of co-axial technology in the deposition of cellularized hydrogel fibres is discussed, highlighting its main features. Furthermore, we show how this approach allows (i) to decouple the printing accuracy from bioink rheological behaviour-thus notably simplifying the development of new bioinks- A nd (ii) to build heterogeneous multi-materials and/or multicellular constructs that can better mimic the native tissues when combined with microfluidic systems. Finally, the ongoing challenges and the future perspectives for the ultimate fabrication of functional constructs for advanced research studies are highlighted. © 2018 IOP Publishing Ltd

Polypropylene mesh for hernia repair with controllable cell adhesion/de-adhesion properties

Herein, a versatile bilayersystem, composed by a polypropylene(PP)mesh and a covalently bonded poly(N-isopropylacrylamide) (PNIPAAm) hydrogel, is reported. The cell adhesion mechanism was successfully modulated by controlling the architecture of the hydrogel in terms of duration of PNIPAAm graftingtime, crosslinker content, and temperature of material exposure in PBS solutions (belowandabove the LCST of PNIPAAm). The best in vitroresults with fibroblast (COS-1) and epithelial (MCF-7) cells was obtained with a mesh modified with porous iPP-g-PNIPAAm bilayer system, prepared via PNIPAAm grafting for 2 h at the lowest N,N'-methylene bis(acrylamide) (MBA)concentration (1 mM). Under these conditions, the detachment of the fibroblast-like cells was 50% lower than that of the control, after 7 days of cell incubation, which represents a high de-adhesionof cellsin a short period. Moreover, the whole system showed an excellent stability in dry or wet media, proving that the thermosensitive hydrogel was well adhered to the polymer surface, after PP fibreactivation by cold plasma. This study opens new insights on the development of anti-adherent meshes for abdominal hernia repairs.Peer ReviewedPublished versio

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 TecnologiaOver the past three decades the use of supercritical carbon dioxide (scCO2) has received much attention as a green alternative in the synthesis and processing of polymers. The scope of this thesis is the development of biocompatible and “smart” porous structures using CO2-assisted processes. This thesis is organized in four main chapters. The first one reviews and highlights some potentialities of supercritical fluid technology and the following ones compile the experimental work developed. The work is divided in three main parts: in the first part (2nd chapter) a CO2-assisted phase inversion method was developed in order to prepare porous structures, namely membranes. In the second part (3rd chapter) the focus was the synthesis of “smart” polymers,especially thermo and pH sensitive polymers. Finally, these two areas were combined (4th chapter) for the preparation of “smart” porous structures. The common guide line was the preparation or processing of biodegradable and/or biocompatible materials with special emphasis on the preparation of porous matrices, namely membranes and scaffolds, with controlled morphology. For membrane preparation a new high pressure apparatus and a new high pressure cell were developed. Polysulfone membranes (a biocompatible polymer with numerous applications in the medical field) were prepared and the effect of the solvent affinity and depressurization rate in the morphology and in the performance in terms of pure water flux of the membranes was investigated. The incorporation of a foaming agent was also analyzed and the high pressure CO2 capability to swell and melt polycaprolactone (PCL) was used to produce and control the porosity and the properties of the membranes. Finally, a natural and water soluble polymer (chitosan) was processed. The presence of water in the casting solution introduced extraordinary difficulties due to the low affinity between water and CO2. To induce the phase inversion a co-solvent (ethanol)was introduced in the CO2 stream. The obtained devices (membranes and beads) were fabricated using moderate temperatures and “green” solvents (ethanol, water and CO2). The morphology and the three dimensional (3D) structures were controlled by altering the co-solvent (ethanol) composition in the CO2 non-solvent stream during the demixing induced process. Microarchitectural analysis by scanning electron microscopy identified the formation of particulate agglomerates when 10% of ethanol in the scCO2 stream was used and detected the development of porous membranes with different morphologies and mechanical properties depending on the programmed gradient mode and the entrainer percentage (2.5-5%) added to the scCO2 stream. These chitosan matrices exhibited low solubility at neutral pH conditions, with no further modifications, demonstrating their applicability in bioreactors as static (membranes) or stirred (beads) culture devices. It was also demonstrated that the current method is able to prepare, in a single-step, an implantable antibiotic release system by co-dissolving gentamicin with chitosan and the solvent. In addition, the cytotoxicity as well as the ability of these structures to support the adhesion and proliferation of human mesenchymal stem cells (hMSC) in vitro were also addressed. After 2 weeks in culture, a 9-fold increase was obtained (versus 6 of the control). More importantly, cells maintained their clonogenic potential and immunophenotype (>95% CD 105+ Cells after 7 days of culture). In this chapter, a hypothetical schematic ternary diagram for the systems polymer–solvent–CO2 is used to discuss and explain the results. Another goal of this thesis was the synthesis of “smart” polymers. Chapter 3, addresses the precipitation polymerization of a thermoresponsive hydrogel, poly(N-isopropylacrylamide)(PNIPAAm), in scCO2. This hydrogel has a transition temperature, hereinafter called low critical solution temperature (LCST), around 32 ºC in an aqueous solution, close to body temperature. A strategy of solvent-free impregnation/coating of polymeric surfaces with PNIPAAm was suggested, in order to further extend the applications of membranes or porous bulky systems. The in situ synthesis of PNIPAAm within a chitosan scaffold was tested as a proof of concept, in order to produce smart partially-biodegradable scaffolds for tissue engineering applications. The LCST was tuned by copolymerization or graft polymerization of NIPAAm with other monomers. Copolymerization with hydroxyethyl methacrylate (HEMA) was used to decrease the LCST temperature from 32.2 ºC to approximately 27.7 ºC. Cloud point measurements of CO2 + HEMA system were used to optimize the polymerization temperature. Experimental data were obtained at 40 ºC, 50 ºC and 65 ºC and pressures up to 21.1 MPa. Soave-Redlich-Kwong equation of state with Mathias-Klotz-Prausnitz mixing rule was used to model experimental results and a good correlation was achieved. To increase the LCST, polyethylene oxide (an hydrophilic polymer) was grafted to PNIPPAAm. Dual stimulus (thermo and pH responsive) hydrogels were also prepared by copolymerizing methacrylic acid with PNIPAAm. As a proof of concept fluorouracil was incorporated in the hydrogels network and their release was controlled by temperature and pH stimulus. In chapter 4 the concepts of the previous chapters were put together envisaging the preparation of“smart” functional polymeric devices with targeted physical and chemical properties namely: (i) chitosan-based dual stimulus scaffolds (temperature and pH responsive); (ii) polysulfone-based thermoresponsive membranes and (iii) polymethylmethacrylate-based membranes. The chitosan scaffolds (pH sensitive) were coated/impregnated with a thermoresponsive polymer,poly(N-isopropylacrylamide) (PNIPAAm), using scCO2 as a carrier to homogeneously distribute the hydrogels monomer within the chitosan scaffolds and as a solvent to perform the polymerization reaction.Fundação para a Ciência e Tecnologia através da bolsa de Doutoramento (SFRH/BD/16908/2004) e do projecto PTDC/CTM/70513/200

Development of in situ forming, polysaccharide-based, self-healable and printable hydrogels por soft actuators and biomedical applications.

316 p.La impresión 4D se presenta como una alternativa prometedora para el desarrollo de materiales para la biomedicina. Hidrogeles preparados mediante impresión 3D capaces de variar su forma en respuesta a estímulos externos constituyen este tipo de sistemas. Estos hidrogeles 4D son sustratos con la capacidad única de adaptarse e imitar los complejos microambientes dinámicos existentes durante los procesos naturales de crecimientos y diferenciación celular. Este carácter dinámico está basado en interacciones físicas/químicas específicas que a su vez son la base para la formación de los denominados hidrogeles in situ, capaces de formarse únicamente ante variaciones específicas del medio. Estos geles in situ, se conocen como los sustratos más eficaces dentro de las nuevas terapias personalizadas de medicina regenerativa.Es por eso que, esta tesis pretende desarrollar hidrogeles in situ adaptables a la tecnología de impresión 3D que sean biodegradables y que muestren adecuadas propiedades mecánicas y capacidad de responder variando su forma ante estímulos externos (pH/iones, temperatura, luz, campo eléctrico y/o magnético) así como habilidad para auto-repararse. Para el desarrollo de estos nuevos materiales con propiedades avanzadas se han seleccionado 3 polisacáridos, el quitosano, ácido hialurónico y alginato, como materiales para la formación de todos los hidrogeles presentados a lo largo de la tesis doctoral. La impresión 4D se presenta como una alternativa prometedora para el desarrollo de materiales para la biomedicina. Hidrogeles preparados mediante impresión 3D capaces de variar su forma en respuesta a estímulos externos constituyen este tipo de sistemas. Estos hidrogeles 4D son sustratos con la capacidad única de adaptarse e imitar los complejos microambientes dinámicos existentes durante los procesos naturales de crecimientos y diferenciación celular. Este carácter dinámico está basado en interacciones físicas/químicas específicas que a su vez son la base para la formación de los denominados hidrogeles in situ, capaces de formarse únicamente ante variaciones específicas del medio. Estos geles in situ, se conocen como los sustratos más eficaces dentro de las nuevas terapias personalizadas de medicina regenerativa.Es por eso que, esta tesis pretende desarrollar hidrogeles in situ adaptables a la tecnología de impresión 3D que sean biodegradables y que muestren adecuadas propiedades mecánicas y capacidad de responder variando su forma ante estímulos externos (pH/iones, temperatura, luz, campo eléctrico y/o magnético) así como habilidad para auto-repararse. Para el desarrollo de estos nuevos materiales con propiedades avanzadas se han seleccionado 3 polisacáridos, el quitosano, ácido hialurónico y alginato, como materiales para la formación de todos los hidrogeles presentados a lo largo de la tesis doctoral

Active Stimuli-Responsive Polymer Surfaces and Thin Films: Design, Properties and Applications: Active Stimuli-Responsive Polymer Surfaces and Thin Films: Design, Properties and Applications

Design of 2D and 3D micropatterned materials is highly important for printing technology, microfluidics, microanalytics, information storage, microelectronics and biotechnology. Biotechnology deserves particular interest among the diversity of possible applications because its opens perspectives for regeneration of tissues and organs that can considerably improve our life. In fact, biotechnology is in constant need for development of microstructured materials with controlled architecture. Such materials can serve either as scaffolds or as microanalytical platforms, where cells are able to self-organize in a programmed manner. Microstructured materials, for example, allow in vitro investigation of complex cell-cell interactions, interactions between cells and engineered materials. With the help of patterned surfaces it was demonstrated that cell adhesion and viability as well as differentiation of stem cells1 depend of on the character of nano- and micro- structures 2 as well as their size. There are number of methods based on optical lithography, atomic force microscopy, printing techniques, chemical vapor deposition, which have been developed and successfully applied for 2D patterning. While each of these methods provides particular advantages, a general trade-off between spatial resolution, throughput, “biocompatibility of method” and usability of fabricated patterned surfaces exists. For example, AFM-based techniques allow very high nanometer resolution and can be used to place small numbers of functional proteins with nanometer lateral resolution, but are limited to low writing speeds and small pattern sizes. Albeit, the resolution of photolithography is lower, while it is much faster and cheaper. Therefore, it is highly desirable to develop methods for high-resolution patterning at reasonably low cost and high throughput. Although many approaches to fabricate sophisticated surface patterns exist, they are almost entirely limited to producing fixed patterns that cannot be intentionally modified or switched on the fly in physiologic environment. This limits the usability of a patterned surface to a single specific application and new microstructures have to be fabricated for new applications. Therefore, it is desirable to develop methods for design of switchable and rewritable patterns. Next, the high-energy of the ultraviolet radiation, which is typically used for photolithography, can be harmful for biological species. It is also highly important to develop an approach for photopatterning where visible light is used instead of UV light. Therefore, it is very important for biotechnological applications to achieve good resolution at low costs, create surface with switchable and reconfigurable patterns, perform patterning in mild physiologic conditions and avoid use of harmful UV light. 3D patterning is experimentally more complicated than 2D one and the applicability of available techniques is substantially limited. For example, interference photolithography allows fabrication of 3D structures with limited thickness. Two-photon photolithography, which allows nanoscale resolution, is very slow and highly expensive. Assembling of 3D structures by stacking of 2D ones is time consuming and does not allow fabrication of fine hollow structures. At the same time, nature offers an enormous arsenal of ideas for the design of novel materials with superior properties. In particular, self-assembly and self-organization being the driving principles of structure formation in nature attract significant interest as promising concepts for the design of intelligent materials 3. Self-folding films are the examples of biomimetic materials4. Such films mimic movement mechanisms of plants 5-7 and are able to self-organize and form complex 3D structures. The self-folding films consist of two materials with different properties. At least one of these materials, active one, can change its volume. Because of non-equal expansion of the materials, the self-folding films are able to form a tubes, capsules or more complex structure. Similar to origami, the self-folding films provide unique possibilities for the straightforward fabrication of highly complex 3D micro-structures with patterned inner and outer walls that cannot be achieved using other currently available technologies. The self-folded micro-objects can be assembled into sophisticated, hierarchically-organized 3D super-constructs with structural anisotropy and highly complex surface patterns. Till now most of the research in the field of self-folding films was focused on inorganic materials. Due to their rigidity, limited biocompatibility and non-biodegradability, application of inorganic self-folding materials for biomedical purposes is limited. Polymers are more suitable for these purposes. There are many factors, which make polymer-based self-folding films particularly attractive. There is a variety of polymers sensitive to different stimuli that allows design of self-folding films, which are able to fold in response to various external signals. There are many polymers changing their properties in physiological ranges of pH and temperature as well as polymers sensitive to biochemical processes. There is a variety of biocompatible and biodegradable polymers. These properties make self-folding polymer highly attractive for biological applications. Polymers undergo considerable and reversible changes of volume that allows design of systems with reversible folding. Fabrication of 3D structures with the size ranging from hundreds of nanometers to centimeters is possible. In spite of their attractive properties, the polymer-based systems remained almost out of focus – ca 15 papers including own ones were published on this topic (see own review 8, state October 2011). Thereby the development of biomimetic materials based on self-folding polymer films is highly desired and can open new horizons for the design of unique 3D materials with advanced properties for lab-on-chip applications, smart materials for everyday life and regenerative medicine

Thermosensitive and photopolymerizable hydrogels based on Pluronic F127

Here it is reported the design, synthesis and characterization of new Pluronic® F127 derivatives, with the ability to form thermosensitive and photopolymerizable hydrogels. These active hydrogels undergo a sol-to-gel transition by increasing the temperature in a physiologically important temperature range thus resulting attractive for biomedical applications and drug delivery systems. Pluronic® F127 has been functionalized with photoreactive groups and the obtained derivatives and their precursors have been fully characterized by conventional techniques. Then the aimed compounds have been processed as macroscopically molded hydrogels and as nanostructured hydrogels (nanogels). A highly crosslinked internal structure has been reached by the photopolymerization technique for the thermosensitive macroscopic hydrogels designed to act as cell scaffolds for cartilage repair. Swelling and degradation studies as well as their morphological characterization by SEM have been carried out. Concerning the nanostructured hydrogels (nanogels), after determining its critical micellar concentration and applying a photopolymerization process to fix the nanostructure, they have been characterized by TEM, SEM and DLS. Cell viability assays have been carried out for both types of system, the macroscopic hydrogel and the nanogel