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.

Chemical and biological properties of polysaccharide-coated titania nanoparticles: the key role of proteins.

In this study, we investigated the physicochemical and biological properties of naked and coated titania nanoparticles. The aim of this study was to verify the effect of the biopolymer coatings (hyaluronic acid and its biphosphonated derivative) and the role of protein adsorption from a cell culture medium on the citotoxicity of nanoparticles. Infrared spectroscopy (FT-IR) was used to investigate the interactions between the nanoparticles and the polysaccharides. The ζ potentials and the average hydrodynamic diameters of naked and coated nanoparticles dispersed in deionized water, medium with and without fetal bovine serum, were measured by means of dynamic light scattering (DLS). FT-IR and DLS measurements indicate that serum proteins are adsorbed on the NPs' surface. The biological tests show that naked and coated TiO(2) NPs do not induce an acute toxic effect on fibroblast cell cultures. This result shows that protein adsorption on NPs is an important factor in explaining the effect of NPs on cellular behavior.

The immobilization of titania nanoparticles on hyaluronan films and their photocatalytic properties.

We have developed a method to bind titania nanoparticles onto hyaluronic films (HA) photoimmobilized on silanized glass. Titania nanoparticles were deposited on the HA films from commercially available dispersions by casting and dip-coating methods at various pH values. XPS was used to monitor the deposition of titania and to estimate the surface coverage of the nanoparticles. The topography of the titania-modified HA films was investigated by means of AFM. XPS results indicate that the titania surface coverage depends on the preparation method and the pH of the dispersion. We found that the maximum titania nanoparticle surface coverage was obtained by the casting method with the formation of aggregates and multilayers of particles. The titania surface coverage for the surfaces prepared by the dip-coating method is pH-dependent. The surfaces prepared at pH 2 show a surface coverage of 65% and a rather uniform distribution of particles. We found that titania nanoparticles are anchored in a stable way to the HA substrate in a phosphate buffer solution (PBS) and that the interaction between the HA and the titania is through the carbonyl group of carboxylates and amidic groups of the polymer. AFM images clearly show that titania nanoparticles are uniformly distributed over the HA films. By measuring the average diameter and the average height of the nanoparticles deposited on HA films it appears that the particles are partially embedded in the polysaccharide films. The results of the study on the photobleaching of methylene blue indicate that the characteristic photocatalytic activity of titania is maintained when the nanoparticles are anchored to the HA substrate.

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.

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.

Functionalized titanium oxide surfaces with phosphated carboxymethyl cellulose: characterization and bonelike cell behavior.

The performance of dental or orthopedic implants is closely dependent on surface properties in terms of topography and chemistry. A phosphated carboxymethylcellulose containing one phosphate group for each disaccharide unit was synthesized and used to functionalize titanium oxide surfaces with the aim to improve osseointegration with the host tissue. The modified surfaces were chemically characterized by means of X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The investigation of the surface topography was performed by atomic force microscopy measurements before and after the polysaccharide coating. In vitro biological tests using osteoblastlike cells demonstrated that functionalized TiO(2) surfaces modulated cell response, in terms of adhesion, proliferation,and morphology. Phosphated carboxymethylcellulose promoted better cell adhesion and significantly enhanced their proliferation. The morphology of cells was polygonal and more spread on this type of modified surface.These findings suggest that the presence of a phosphate polysaccharide coating promotes osteoblast growth on the surface potentially improving biomaterial osseointegration.

A novel strategy to obtain a hyaluronan monolayer on solid substrates.

The aim of this study was to find a novel simple method to obtain polysaccharide ultrathin layers on solid substrates to investigate the interaction between the surface and the biological environment. A Hyaluronan (Hyal) monolayer with a well-defined chemistry was obtained by exploiting the capability of organosilanes to spontaneously adhere onto glass surfaces. A silane alkylic chain was conjugated with Hyal, and the derivatized polysaccharide was allowed to spontaneously adhere onto a glass surface. The elemental analysis of the modified polysaccharide demonstrated that one out of five disaccharide units was conjugated with the alkyl silane chain, corresponding to a substitution degree of the carboxylate groups of approximately 20%. The film of the modified polysaccharide was characterized by means of X-ray photoelectron spectroscopy (XPS), water contact angle, and atomic force microscopy (AFM) measurements. XPS analysis demonstrated that we obtained a Hyal layer with a thickness of about 2.0 nm corresponding to a Hyal monolayer. The Hyal-coated surfaces appeared to be rather smooth and highly hydrophilic and showed significant resistance to nonspecific cell adhesion.

Proliferative and re-defferentiative effects of photo-immobilized micro-patterned hyaluronan surfaces on chondrocyte cells.

A photo-immobilisation procedure was utilised to create two different micro-patterned surfaces (tracks 25 and 5 microm wide) of hyaluronan (Hyal) on polyethylene-terephthalate (PET) previously plasma activated. Aim of the study was to investigate the proliferation and re-differentiation capacity of articular chondrocytes cultured on micro-patterned Hyal, compared to homogeneous Hyal and plain plasma-treated (pt-)PET substrates. Cytotoxicity, cell proliferation, activation and differentiation of articular knee cartilage chondrocytes (Mongrel sheep) were evaluated after 14 days of culture. It was found that micro-patterned Hyal surfaces induced the adhesion, migration and alignment of chondrocytes, as shown by light and scanning electron microscopy. Furthermore, the same surfaces induced chondrocyte differentiation, with a significant increase of aggrecan and collagen type II production, while homogeneous Hyal and pt-PET surfaces did not.

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.