Azobenzene-based Biomaterials as Dynamic Cell Culture Systems

The aim of this Thesis is to fabricate dynamic light-switchable biomaterials as scaffolds to study cell behavior in a more complex environment than the one generated by the use of static systems. We take advantage of compelling properties of azobenzenes to engineer photoresponsive 2D and semi-3D platforms to investigate different biological processes from adhesion up to differentiation. In vivo, in addition to the chemical and mechanical properties, the topographic cues play a main role to guide cell response. The ECM is filled with nano- to micro-meter scale landscapes (e.g., ridges, pores and fibers) in continuous remodeling. However, the topography of the most widely implemented in vitro platforms is static and difficulty mimics the dynamicity of ECM. Thus, we propose platforms, which can dynamically tune on demand their topographic properties upon external stimulation. In particular, azobenzene-containing systems can tune their properties under light illumination, recapitulating the spatial-temporal changes of the physiological cell environment. In Chapter 1, we will discuss about important properties of azobenzene molecules and present different applications of a variety of materials containing azobenzenes from amorphous materials to highly organize liquid crystal polymers. In particular, we will focus on the recent use of azopolymers as dynamic cell instructive materials. As of now, there is a lack of knowledge on the role of dynamic topography and, even more on its effect on stem cell differentiation. In the light of this, in Chapter 2 we will present a technique to photo patterning azopolymer thin films in situ by means of a laser-based confocal microscopy. Further, we will analyze the human mesenchymal stem cell (hMSC) response after the spatial-temporal dynamic topographic changes. In more details, a mass migration phenomenon of azopolymers elicited under light irradiation allows to emboss a variety of patterns on cell-populated azopolymer films. We will investigate the stem cell response on a switchable topography from a linear pattern to a grid both in term of cell cytoskeletal re-organization and cell differentiation. Our aim is to investigate the impact of dynamic remodeling of cell environment on hMSCs gene expression profile, in comparison to static surfaces. In order to achieve our goal, we will investigate the cell behavior over time, changing the topographic aspects of the substrate and analyzing the effect of dynamic cues in modulating cell morphology and osteogenic gene expression profile. In particular, we will investigate whether epigenetic effect induced by changes in the biophysical properties of the substrate over time would redirect the expression of lineage specific markers. In Chapter 3, we will discuss about an athermal photofluidization process that can directly reshape an azopolymer pillar array in the presence of cells to investigate the dynamic reassemble of F-Actin on deformed pillars. We will show that pillar arrays can be reshaped along the direction of laser polarization, resulting in elongated structures with controllable eccentricity. This light-driven phenomenon, permits to usesuch type of systems as platforms to analyze cell membrane curvature remodeling in respond to dynamic pillar reshaping. The plasma membrane wraps around the pillars, which generate local curvatures on cell membrane and trigger the F-actin accumulation. Human bone osteosarcoma epithelial cells (U2OS) will be used to investigate the reorganization of F-Actin during the platform transition from pillar to ellipsoidal-shape structures over time. In Chapter 4, we will focus on designing semi-3D hydrogel platforms containing azobenzene to engineer and manipulate culture systems in order to develop photoactuable cell confining systems. Acrylamide-modified gelatin containing azobenzene-based cross linkers will be used to microfabricate well-defined semi-3D photo-responsive structures by means of two-photon lithography (2PP). As proof of concept, we will show an example of an array of squared structures, where cells are physically confined between the adjacent gelatin blocks, which can be remotely stimulated. The light irradiation can be converted in a local mechanical stimulation able to deform the nucleus at a single-cell level. In Conclusion and Future Perspectives, a summary of the main results achieved in this thesis is presented and future applications are proposed

Screening Platform for Cell Contact Guidance Based on Inorganic Biomaterial Micro/nanotopographical Gradients

High -throughput screening (HTS) methods based on topography gradients or arrays have been extensively used to investigate cell material interactions. However, it is a huge technological challenge to cost efficiently prepare topographical gradients of inorganic biomaterials due to their inherent material properties. Here, we developed a novel strategy translating PDMS-based wrinkled topography gradients with amplitudes from 49 to 2561 nm and wavelengths between 464 and 7121 nm to inorganic biomaterials (Sio(2), Ti/Tio(2), Cr/Cro(3), and AL(2)O(3)) which are frequently used clinical materials. Optimal substratum conditions promoted human bone-marrow derived mesenchymal stem cell alignment, elongation, cytoskeleton arrangement, filopodia development as well as cell adhesion in vitro, which depended both on topography and interface material. This study displays a positive correlation between cell alignment and the orientation of cytoskeleton, filopodia, and focal adhesions. This platform vastly minimizes the experimental efforts both for inorganic material interface engineering and cell biological assessments in a facile and effective approach. The practical application of the HTS technology is expected to aid in the acceleration of developments of inorganic clinical biomaterials

Biomimetic Composite Scaffold With Phosphoserine Signaling for Bone Tissue Engineering Application

In guided bone tissue engineering, successful ingrowth of MSCs depends primarily on the nature of the scaffold. It is well-known that only seconds after implantation, biomaterials are coated by a layer of adsorbed proteins/peptides which modulates the subsequent cell/scaffold interactions, especially at early times after implantation. In this work, nanohydroxyapatite and collagen based composite materials (Coll/nanoHA) were modified with phosphorylated amino acid (O-phospho-L-serine–OPS) to mimic bone tissue, and induce cell differentiation. The choice for this phosphorylated amino acid is due to the fact that osteopontin is a serine-rich glycol-phosphoprotein and has been associated to the early stages of bone formation, and regeneration. Several concentrations of OPS were added to the Coll/nanoHA scaffold and physico-chemical, mechanical, and in vitro cell behavior were evaluated. Afterwards, the composite scaffold with stronger mechanical and best cellular behavior was tested in vivo, with or without previous in vitro culture of human MSC's (bone tissue engineering). The OPS signaling of the biocomposite scaffolds showed similar cellular adhesion and proliferation, but higher ALP enzyme activity (HBMSC). In vivo bone ectopic formation studies allowed for a thorough evaluation of the materials for MSC's osteogenic differentiation. The OPS-scaffolds results showed that the material could modulated mesenchymal cells behavior in favor of osteogenic differentiation into late osteoblasts that gave raised to their ECM with human bone proteins (osteopontin) and calcium deposits. Finally, OPS-modified scaffolds enhanced cell survival, engraftment, migration, and spatial distribution within the 3D matrix that could be used as a cell-loaded scaffold for tissue engineering applications and accelerate bone regeneration processes.This article is a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). In addition, it was supported by Portuguese funds through FCT/MCTES in the framework of the project UID/BIM/04293/2019 and Christiane Salgado contract (DL 57/2016/CP1360/CT0001). Microscopy imaging was performed at the Bioimaging Center for Biomaterials and Regenerative Therapies (b.IMAGE) with the assistance of Maria L?zaro at i3S. The authors also thank Paula Magalh?es and T?nia Meireles (CCGEN), Rossana Correia (HEMS), Cl?udia Machado (i3S), Rui Rocha (CEMUP), Paula Sampaio (ALM) and Lu?s Carlos Matos (FEUP) for the assistance in this work. FT-IR was performed at the Biointerfaces and Nanotechnology (BN) core facility (i3S) with the assistance of Ricardo Vidal. We also thank FLUIDINOVA, S.A for the provision of nanohydroxyapatite (nanoXIM.HAp202)

Biomimetic composite scaffold with phosphoserine signaling for bone tissue engineering application

In guided bone tissue engineering, successful ingrowth of MSCs depends primarily on the nature of the scaffold. It is well-known that only seconds after implantation, biomaterials are coated by a layer of adsorbed proteins/peptides which modulates the subsequent cell/scaffold interactions, especially at early times after implantation. In this work, nanohydroxyapatite and collagen based composite materials (Coll/nanoHA) were modified with phosphorylated amino acid (O-phospho-L-serine–OPS) to mimic bone tissue, and induce cell differentiation. The choice for this phosphorylated amino acid is due to the fact that osteopontin is a serine-rich glycol-phosphoprotein and has been associated to the early stages of bone formation, and regeneration. Several concentrations of OPS were added to the Coll/nanoHA scaffold and physico-chemical, mechanical, and in vitro cell behavior were evaluated. Afterwards, the composite scaffold with stronger mechanical and best cellular behavior was tested in vivo, with or without previous in vitro culture of human MSC's (bone tissue engineering). The OPS signaling of the biocomposite scaffolds showed similar cellular adhesion and proliferation, but higher ALP enzyme activity (HBMSC). In vivo bone ectopic formation studies allowed for a thorough evaluation of the materials for MSC's osteogenic differentiation. The OPS-scaffolds results showed that the material could modulated mesenchymal cells behavior in favor of osteogenic differentiation into late osteoblasts that gave raised to their ECM with human bone proteins (osteopontin) and calcium deposits. Finally, OPS-modified scaffolds enhanced cell survival, engraftment, migration, and spatial distribution within the 3D matrix that could be used as a cell-loaded scaffold for tissue engineering applications and accelerate bone regeneration processes.info:eu-repo/semantics/publishedVersio

On the Interaction between 1D Materials and Living Cells

One-dimensional (1D) materials allow for cutting-edge applications in biology, such as single-cell bioelectronics investigations, stimulation of the cellular membrane or the cytosol, cellular capture, tissue regeneration, antibacterial action, traction force investigation, and cellular lysis among others. The extraordinary development of this research field in the last ten years has been promoted by the possibility to engineer new classes of biointerfaces that integrate 1D materials as tools to trigger reconfigurable stimuli/probes at the sub-cellular resolution, mimicking the in vivo protein fibres organization of the extracellular matrix. After a brief overview of the theoretical models relevant for a quantitative description of the 1D material/cell interface, this work offers an unprecedented review of 1D nano- and microscale materials (inorganic, organic, biomolecular) explored so far in this vibrant research field, highlighting their emerging biological applications. The correlation between each 1D material chemistry and the resulting biological response is investigated, allowing to emphasize the advantages and the issues that each class presents. Finally, current challenges and future perspectives are discussed

Femtosecond laser microstructuring of alumina toughened zirconia for surface functionalization of dental implants

The continuous need for high-performance implants that can withstand mechanical loads while promoting implant integration into bone has focused recent research on the surface modification of hard ceramics. Their properties of biocompatibility, high mechanical and fatigue resistance and aesthetic color have contributed to its succefull applications in dentistry. Alumina toughened Zirconia (ATZ) has been gaining attention as a material for dental implants applications due to its advanced mechanical properties and minimal degradation at body temperature. Still, in order to improve tissue response to this bioinert material, additional modifications are desirable. Improving the surface functionality of this ceramic could lead to enhanced implant-tissue interaction and subsequently, a successful implant integration.In this work, microtopographies were developed on the surface of Alumina toughened Zirconia using an ultrafast laser methodology, aiming at improving the cellular response to this ceramic. Microscale grooves and grid-like geometries were produced on ATZ ceramics by femtosecond laser ablation, with a pulse width of 150 fs, wavelength of 800 nm and repetition rate of 1 kHz. The variation of surface topography, roughness, chemistry and wettability with different laser processing parameters was examined.Cell-surface interactions were evaluated for 7 days on both microstructured surfaces and a non-treated control with pre-osteoblasts, MC3T3-E1 cells. Both surface topographies showed to improve cell response, with increased metabolic activity when compared to the untreated control and modulating cell morphology up to 7 days.The obtained results suggest that femtosecond laser texturing may be a suitable non-contact methodology for creating tunable micro-scale surface topography on ATZ ceramics to enhance the biological response

Influence of nanosecond laser surface patterning on dental 3Y-TZP: Effects on the topography, hydrothermal degradation and cell response

Objectives Laser surface micropatterning of dental-grade zirconia (3Y-TZP) was explored with the objective of providing defined linear patterns capable of guiding bone-cell response. Methods A nanosecond (ns-) laser was employed to fabricate microgrooves on the surface of 3Y-TZP discs, yielding three different groove periodicities (i.e., 30, 50 and 100 µm). The resulting topography and surface damage were characterized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). X-Ray diffraction (XRD) and Raman spectroscopy techniques were employed to assess the hydrothermal degradation resistance of the modified topographies. Preliminary biological studies were conducted to evaluate adhesion (6 h) of human mesenchymal stem cells (hMSC) to the patterns in terms of cell number and morphology. Finally, Staphylococcus aureus adhesion (4 h) to the microgrooves was investigated. Results The surface analysis showed grooves of approximately 1.8 µm height that exhibited surface damage in the form of pile-up at the edge of the microgrooves, microcracks and cavities. Accelerated aging tests revealed a slight decrease of the hydrothermal degradation resistance after laser patterning, and the Raman mapping showed the presence of monoclinic phase heterogeneously distributed along the patterned surfaces. An increase of the hMSC area was identified on all the microgrooved surfaces, although only the 50 µm periodicity, which is closer to the cell size, significantly favored cell elongation and alignment along the grooves. A decrease in Staphylococcus aureus adhesion was observed on the investigated micropatterns. Significance The study suggests that linear microgrooves of 50 µm periodicity may help in promoting hMSC adhesion and alignment, while reducing bacterial cell attachment.Peer ReviewedPostprint (published version

PHYSICAL RESPONSES OF CELLS TO GRATING TOPOGRAPHY VIA MECHANO-SENSING

Ph.DDOCTOR OF PHILOSOPH