Daniela Pasqui

A novel strategy for engineering hydrogels with ferromagnetic nanoparticles as crosslinkers of the polymer chains. Potential applications as a targeted drug delivery system

An efficient strategy is employed for the preparation of magnetic hybrid hydrogels consisting of functionalized CoFe2O4 magnetic nanoparticles covalently bound to a carboxymethylcellulose (CMC) polymer. The method involves the formation of an amide bond between the carboxylic groups of CMC and the amine groups of functionalized nanoparticles, which work as the crosslinking agents of the polymer chains. The hybrid hydrogels were chemically and morphologically characterized. The rheological properties of the hydrogels were also investigated with the aim to verify their behavior under an applied mechanical stress. The hybrid hydrogel turns out to be thixotropic. Thanks to the presence of magnetic nanoparticles, the hydrogel is capable of responding to an external magnetic field. Preliminary data show the possibility of loading the hydrogel with a dye, which can be considered as a drug model, to squeeze it through a syringe and to drive the material by the application of an external magnetic field.

A thixotropic hydrogel from chemically cross-linked guar gum: synthesis, characterization and rheological behaviour.

Polysaccharide guar gum (GG) was cross-linked in an alkaline solution with polyethylene glycol diglycidyl ether (PEGDGE) to create a new hydrogel. The GG hydrogel was examined by FT-IR spectroscopy, AFM analysis and SEM analysis. The water uptake of the GG hydrogel was measured at different pHs, and rheological studies were performed to verify the thixotropic nature of the material. Rheological studies revealed the pseudoplastic behaviour of the GG hydrogel and its thixotropic nature. AFM analysis on a sample which was subjected to shear stress showed the presence of nanoparticles in the hydrogel. When the sample was left to settle, the gel surface returned to its original homogenous morphology. The thixotropic and injectable nature of the GG hydrogel suggest its possible use in biomedical applications.

Micropatterned surfaces for the control of endothelial cell behaviour.

Micropatterned materials were synthesised by photoimmobilising the sulphated hyaluronic acid, adequately functionalised with a photoreactive moiety, on glass substrates. Four different patterns (10, 25, 50 and 100 microns) were obtained. The spectroscopic and microscopic analysis of the microstructured surfaces revealed that the photoimmobilisation process was successful, demonstrating that the photomask was well reproduced on the sample surface. Analysis of endothelial cell behaviour on these micropatterned materials was performed in terms of adhesion, locomotion and orientation. Decreasing the stripe dimensions a more fusiform shape of the adhered endothelial cells was observed. At the same time the cell locomotion and orientation were increased. Furthermore, a photoimmobilisation of stripes of HyalS (10 and 100 microns) was performed on a continuous HyalS layer, in turn immobilised on glass substrate. Being excluded a different chemistry between the stripe and the substrate, the influence of topography on the behaviour of endothelia cells was thus envisaged.

Carboxymethyl cellulose—hydroxyapatite hybrid hydrogel as a composite material for bone tissue engineering applications

Natural bone is a complex inorganic-organic nanocomposite material, in which hydroxyapatite (HA) nanocrystals and collagen fibrils are well organized into hierarchical architecture over several length scales. In this work, we reported a new hybrid material (CMC-HA) containing HA drown in a carboxymethylcellulose (CMC)-based hydrogel. The strategy for inserting HA nanocrystals within the hydrogel matrix consists of making the freeze-dried hydrogel to swell in a solution containing HA microcrystals. The composite CMC-HA hydrogel has been characterized from a physicochemical and morphological point of view by means of FTIR spectroscopy, rheological measurements, and field emission scanning electron microscopy (FESEM). No release of HA was measured in water or NaCl solution. The distribution of HA crystal on the surface and inside the hydrogel was determined by time of flight secondary ion mass spectrometry (ToF-SIMS) and FESEM. The biological performance of CMC-HA hydrogel were tested by using osteoblast MG63 line and compared with a CMC-based hydrogel without HA. The evaluation of osteoblast markers and gene expression showed that the addition of HA to CMC hydrogel enhanced cell proliferation and metabolic activity and promoted the production of mineralized extracellular matrix.

Cell behaviour on chemically microstructured surfaces

Abstract Micropatterned surfaces with different chemical topographies were synthesised in order to investigate the influence of surface chemistry and topography on cell behaviour. The microstructured materials were synthesised by photoimmobilising natural Hyaluronan (Hyal) and its sulphated derivative (HyalS), both adequately functionalised with a photorective moiety, on glass substrates. Four different grating patterns (10, 25, 50 and 100 μm) were used to pattern the hyaluronan. The micropatterned samples were analysed by Secondary Ions Mass Spectrometry, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy to investigate the chemistry and the topography of the surfaces. The spectroscopic and microscopic analysis of the microstructured surfaces revealed that the photoimmobilisation process was successful, demonstrating that the photomask patterns were well reproduced on the sample surface. The influence of chemical topographies on the cell behaviour was then analysed. Human and 3T3 fibroblasts, bovine aortic and human (HGTFN line) endothelial cells were used and their behaviour on the micropatterned surfaces was analysed in terms of adhesion, proliferation, locomotion and orientation. Both chemical and topographical controls were found to be important for cell guidance. By decreasing the stripe dimensions, a more fusiform shape of cell was observed. At the same time, the cell locomotion and orientation parallel to the structure increased. However, differences in cell behaviour were detected according to both cell type and micropattern dimensions.

The use of hyaluronan and its sulphated derivative patterned with micrometric scale on glass substrate in melanocyte cell behaviour

Surface microfabrication techniques were widely utilised for the spatial control of in vitro cell behaviour. A photo-immobilisation procedure was utilised to create micropatterned surfaces: four different stripe patterns (100, 50, 25 and 10 microm) of hyaluronan (Hyal) and its sulphated derivative (HyalS) on silanised glass substrate were obtained.The morphological analysis showed that the surface topography showed regular stripes of 100, 50, 25 and 10 microm wide and ranging from 300 nm up to 1 microm in thickness. They reproduced the exact photo-mask pattern: glass stripes alternating with polysaccharide ones. On the contrary, Hyal microstructures showed just a topographic pattern as the glass stripes appeared to be covered by a thin layer of the macromolecule by TOF-SIMS. Cell adhesion studies demonstrated that melanocytes adhered and oriented within the first 2h of culture on HyalS microdomains and not on Hyal microstructures where they spread on glass substrate around the patterned area. Double photo-immobilised samples characterised by a 100 microm stripe pattern of Hyal or HyalS on the top of a continuous layer of the two polysaccharides were also created in order to investigate the effect of the topography on cell behaviour. The obtained results demonstrated that melanocytes adhered on HyalS stripes while on the Hyal micropatterned surfaces they spread on silanised glass substrate around the structured area, resulting in the exclusion of the topographic pattern.

Inter-penetrating hydrogels (IPHs) as a new class of injectable polysaccharide hydrogels with thixotropic nature and interesting mechanical and biological properties

Hydrogels are important and very promising materials for biomedical applications, such as cell scaffolds and drug delivery systems. A new class of injectable materials was obtained by mixing polysaccharide chemically crosslinked hydrogels. The mixing was performed by exploiting the thixotropic behaviour of the polysaccharide hydrogels: in other words they were passed through a syringe to subject them to shear stress and render them fluid, then they were mixed together in the fluid state. Once the material regained its consistency, a new hydrogel was obtained, called an inter-penetrating hydrogel (IPH), containing both the native hydrogels. This new class of materials showed different physical-chemical, mechanical and biological properties from the native hydrogels utilized for the mixing. The hydrogels taken into consideration were: negatively charged carboxy methyl cellulose (CMC) and hyaluronic acid (Hyal), uncharged guar gum (GG), and chitosan (CHT), which bore positive charges at physiological pH. The IPHs obtained – i.e. CMC-CHT, CMC-GG and CMC-Hyal – were characterized by means of Fourier transform infrared spectroscopy (FT-IR) and swelling measurements. The mechanical properties of the IPHs were investigated by rheometric analyses, while their morphology was studied by scanning electronic microscopy (SEM). The biological performance of the IPHs was evaluated using fibroblasts (NIH 3T3 cell line) and compared with that of the native hydrogels.

Protein adsorption on derivatives of hyaluronic acid and subsequent cellular response.

The modulation of biological interactions with artificial surfaces is a vital aspect of biomaterials research. Serum protein adsorption onto photoreactive hyaluronic acid (Hyal-N(3)) and its sulfated derivative (HyalS-N(3)) was analyzed to determine extent of protein interaction and protein conformation as well as subsequent cell adhesion. There were no significant (p < 0.01) differences in the amount of protein adsorbed to the two polymers; however, proteins were found to be more loosely bound on HyalS-N(3) compared with Hyal-N(3). Fibronectin was adsorbed onto HyalS-N(3) in such an orientation as to allow the availability of the cell binding region, while there was more restricted access to this region on fibronectin adsorbed onto Hyal-N(3). This was confirmed by reduced cell adhesion on fibronectin precoated Hyal-N(3) compared with fibronectin precoated HyalS-N(3). Minimal cell adhesion was observed on albumin and serum precoated Hyal-N(3). The quartz crystal microbalance confirmed that specific cell-surface interactions were experienced by cells interacting with the fibronectin precoated polymers and serum precoated HyalS-N(3).

The Topography of Microstructured Surfaces Differently Affects Fibrillin Deposition by Blood and Lymphatic Endothelial Cells in Culture

While tissue-engineered blood vessels have already been successfully used in surgical practice, artificially restoring lymphatic circulation when needed is still far to be realized. Stability of arterial vessel wall depends on proper fibrillin deposition; fibrillin in fact is the scaffold for elastic fiber formation. In lymphatic vessels fibrillin is probably implied in lymph formation in response to interstitial requirements. This study was designed to verify whether fibrillin deposition is influenced by the topography of the substrate on which blood and lymphatic endothelial cells grow. Blood and lymphatic endothelial cells were cultured on microstructured surfaces with different topography: stripes of different widths (25, 50, and 100 microm), squares and rectangles, and spiral geometry, obtained by the photoimmobilization of Hyaluronan (Hyal) on aminosilanized glass. Cell orientation and fibrillin deposition were influenced by the topography of the microstructure. Blood endothelial cells deposited fibrillin as a bundle running parallel to the major axis of stripes and spirals, whereas the irregular network of fibrillin deposited by lymphatic endothelial cells was affected by the topography of the substrate only in the smallest stripes. These data bring a contribution to the basic knowledge required to design tissue-engineered blood and lymphatic vessels capable of adapting to the functional requirements of the surrounding environment.

Micropatterned polysaccharide surfaces via laser ablation for cell guidance

Micropatterned materials were obtained by a controlled laser ablation of a photoimmobilised homogeneous layer of hyaluronic acid (Hyal) and its sulphated derivative (HyalS). The photoimmobilisation was performed by coating the polysaccharide, adequately functionalised with a photoreactive group, on aminosilanised glass substrate and immobilising it on the surface under UV light. Hyal or HyalS photoimmobilised samples were then subjected to laser ablation with wavelengths in the UV regions in order to drill the pattern. Four different patterns with stripes of 100, 50, 25 and 10 μm were generated. A chemical characterisation by attenuated total reflection/Fourier transform infrared (ATR/FT-IR) and time of flight-secondary ions mass spectrometry (TOF-SIMS) confirmed the success of the laser ablation procedure and the presence of alternating stripes of polysaccharide and native glass. The exact dimensions of the stripes were determined by atomic force microscopy. The analysis of cell behaviour in terms of adhesion, proliferation and movement using mouse fibroblasts (3T3 line) and bovine aortic endothelial cells (BAEC) was also performed.