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.

Heterotypic cell-cell interaction on micropatterned surfaces.

PURPOSE: The aim of this paper was to study the influence of chemical and topographical signals on cell behavior and to obtain a heterotypic cell-cell interaction on microstructured domains. METHODS: The polysaccharide hyaluronic acid (Hyal) was photoimmobilized on glass surfaces in order to obtain a pattern with squares and rectangles of different dimensions and chemistry. The microstructured surfaces were characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The behavior of human coronary artery endothelial cells (HCAEC) and human tumoral dermal fibroblasts (C54) was investigated on these micropatterned surfaces by adhesion studies. Moreover heterotypic interaction among C54 and HCAEC adherent on patterned surfaces was evaluated by time-lapse video microscopy RESULTS: Surface analysis revealed the presence of a pattern consisting of alternating glass and Hyal microstructures whose dimensions decreased from the center to the edge of the sample. Neither HCAEC nor C54 adhered to the immobilized Hyal but both adapted their shape to the different sizes of the glass squares and rectangles. The number of adherent cells depended on the dimensions of both the glass domains and the nuclei of the cells. Co-cultured C54 on HCAEC patterned surfaces showed a heterotypic cell-cell interaction in the same chemical and topographic domain. CONCLUSIONS: A heterotypic cell-cell interaction occurred in the same chemical and topographic micro-domains but in narrow areas only. Moreover, the number of cells adhering to the glass domains and cell morphology depended on the dimensions of both adhesive areas and cell nuclei.

The role of fibronectin in cell adhesion to spiral patterned TiO2 nanoparticles.

Spiral micropatterned surfaces of decreasing dimensions were produced by photo-immobilizing a photo-reactive hyaluronan (Hyal) derivative on TiO2 nanoparticles. The microstructured surfaces were characterized by both scanning electron microscopy and atomic force microscopy analysis. The behavior, of both endothelial cells (HCAEC) and tumoral mouse fibroblasts (NIH3T3) on the patterned surfaces was evaluated. HCAEC adhered only to the TiO2 nanoparticles avoiding contact with the Hyal. NIH3T3 adhered to and completely covered the TiO2 spiral but prolonging the culture time, it also covered the external photo-immobilized Hyal surface. The role of fibronectin to mediate cell adhesion to the TiO2 pattern surfaces was evaluated by experiments with blocked fibronectin membrane receptors on both HCAEC and NIH3T3. The results showed the absence of any adhering cells. Therefore, fibronectin seemed to be the only key protein in mediating cell adhesion to these TiO2 substrates.

Amidic alginate hydrogel for nucleus pulposus replacement.

Degeneration of intervertebral discs is the most common cause of back pain. The first phase of this degenerative process involves the nucleus pulposus (NP). A rapid recovery of this structure can prevent further degradation of the annulus fibrosus. A new amidic derivative of alginate (AAA) was developed to obtain a polysaccharide possessing some of the physical-chemical properties of Hyal (i.e. viscosity) without losing the rigidity of the native alginate structure. The modified polysaccharide was crosslinked using 1.3 diaminopropane as crosslinking agent. The hydrogel obtained was characterized in terms of water uptake and rheological behavior. In particular, the viscoelastic behavior of the hydrogel was determined in shear stress under dynamic conditions and compared with the behavior of nondegenerated human lumbar NP. We then assessed the effect of the AAA hydrogel on NHC (Normal Human Chondrocyte) cell viability and on the production of important extracellular matrix factors, such as glycosaminoglycans and Type II collagen. In conclusion, the results achieved in this study demonstrated that the amidic alginate-based scaffold is a promising material to be utilized in the replacement of NP.

The applicability of an amidated polysaccharide hydrogel as a cartilage substitute: structural and rheological characterization.

An amidic derivative of a carboxymethylcellulose-based hydrogel was obtained and characterized in terms of amidation degree. NMR studies and FT-IR imaging spectroscopy demonstrated that the reaction allowed a polymer to be obtained that was characterized by a regular distribution of amidic groups along the polysaccharide chains. Through this regularity, a homogenous three-dimensional scaffold was obtained, which maintained the thixotropic property of the linear polysaccharide.

Competitive protein adsorption on micro patterned polymeric biomaterials, and viscoelastic properties of tailor made extracellular matrices.

Cell adhesion on biomaterial surfaces and the vitality of anchorage dependent cells is affected by several parameters of an adsorbate layer which is intentionally or spontaneously formed. Surface pre-treatments and several conditioning steps prior and during to the cell/biomaterial contact affect the composition, orientation, quantity and viscoelasticity of the interfacing layer between cells and biomaterial. This work was performed to elucidate the response of cells on two modified biomaterial surfaces based on protein or carbohydrate adsorbates: (a) Masked UV irradiations opened a simple route to obtain chemically patterned substrates controlling serum protein adsorption and cell adhesion. It is possible to achieve structures of subcellular size and to produce immobilized gradients. In order to examine the protein matrix deposited on these substrates we applied a quartz microbalance technique (QCM-D) capable to extract viscoelastic data in addition to the mass uptake during plasma protein deposition. It was found that the quantity and viscosity of surface bound albumin is lowered when the surface is modified (patterned) by UV exposure. Hence, the UV modification promotes the competitive adsorption of cell adhesion proteins from the media or upon secretion by the cells and yields to the observed cell patterns. (b) Another tissue engineering technique, using immobilized, modified and/or cross linked hyaluronic acid (HA), an important extra cellular matrix component in vivo, is also examined by QCM-D. Our data demonstrate that HA can be modified by an activation with a carbodiimide, followed by the application of an alpha,omega-bisamino polyethyleneglycol. The QCM-D data can be interpreted as a stiffening of the HA layer combined with the release of hydration water. Further, the hydration state and the viscoelastic behaviour of surface bound ultrathin HA hydrogels was examined. Quantification of viscoelastic parameters of thin films of ECM by QCM-D is valuable for the interpretation of durotaxis, describing effects of mechanical substrate parameters on the adhesion and motility of cells.

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.

Low- and high-resolution nuclear magnetic resonance (NMR) characterisation of hyaluronan-based native and sulfated hydrogels.

Hyaluronan-based hydrogels were synthesised using different crosslinking agents, such as 1,3-diaminopropane (1,3-DAP) and 1,6-diaminohexane (1,6-DAE). The hydrogels were sulfated to provide materials (Hyal-1,3-DAP, Hyal-1,6-DAE, HyalS-1,3-DAP and HyalS-1,6-DAE) that were characterised by both high- and low-resolution nuclear magnetic resonance (NMR) spectroscopy. The (13)C NMR spectra of the materials were analysed to identify, characterise and study the crosslinking degree of the hydrogels. The crosslinking degree was also determined by potentiometric titration and the effectiveness of the two techniques was compared. Measurements of longitudinal relaxation times (spin-lattice) and of NOE enhancement were used to study the mobility of the hydrogels. Low-resolution NMR studies allowed the determination of the water transport properties in the hydrogels. In addition, the swelling degree for the various hydrogels was calculated as a function of the longitudinal and transversal relaxation times of the water molecules. Lastly, the self-diffusion coefficients of the water in interaction with the four polysaccharides were measured by the pulsed field gradient spin echo (PFGSE) sequence.

Thixotrophy property of hydrogels to evaluate the cell growing on the inside of the material bulk (Amber effect).

The realization of injectable hydrogels is one of the most challenging aims of biomedical research. Most injectable hydrogels are obtained by photopolymerization in situ. A hydrogel which is able to pass through a needle without losing its structure once crosslinked would be of great interest for several biomedical applications. In this work, hyaluronane and alginate-based 50% hydrogels were synthesized, their thixotrophic behavior was verified and their mechanical properties (G' and G'') were determined before and after the passage through the needle. A morphological analysis by scanning electron microscopy (SEM) was also performed in order to evaluate the effect of the passage of the hydrogel through the needle on the morphological structure of the material. The thixotrophic property of these hydrogels is used to realize a cell-containing material that supports cell proliferation and growth permitting in vivo engineering of new tissues. In fact, thixotrophic hydrogel can be sucked together with cell suspension and in this way cells remain entrapped inside the hydrogel structure (Amber effect). A comparison of the cell growth using this technique with the classical seeding technique (cell seeding onto the hydrogel) was performed. A different behavior was found between hyaluronane and alginate-based hydrogels.

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.

Hyaluronic acid hydrogel added with ibuprofen-lysine for the local treatment of chondral lesions in the knee: in vitro and in vivo investigations.

The effect of hyaluronan-based hydrogel with a cross-linking degree of 50% (i.e., the amount of carboxylate groups involved in the cross-lining reaction) (Hyal 50%), loaded with an anti-inflammatory drug (ibuprofen-lysine) for local administration in the osteoarthritic knee was evaluated. The kinetic of drug release from Hyal 50% hydrogel was performed in vitro by a continuous flow system. The release was studied at three different rates (0.375, 0.073, and 0.005 mL/s), and for the slowest one a period of 8 days was necessary to receive the complete release of the drug. A chondral defect was caused in the right femoral medial condyle of 24 rabbits. Twelve animals were treated with Hyal 50%+ibuprofen-lysine and 12 with Hyal 50% by percutaneous injection. The treatments were repeated every 10 days, for three or five times. At 30 and 50 days, no significant differences were observed between the groups (mean score at 30 days: 4.4+/-0.6 in Hyal 50%-treated group and 5.1+/-0.7 in Hyal 50% +Ibuprofen-lysine-treated group; mean score at 50 days: 6.9+/-0.6 in Hyal 50%-treated group and 7.1+/-0.6 in Hyal 50%+ Ibuprofen-lysine-treated group). A significant difference was observed in bone mineral density of the total tibia of rabbits treated by Hyal 50% + Ibuprofen-lysine in comparison with those treated only by Hyal 50%. The positive effects of Hyal 50% on chondral lesions were maintained in the presence of ibuprofen-lysine with the adjunctive effect of an increased limb usage, most likely due to less pain and discomfort.

New Formulations of Polysaccharide-Based Hydrogels for Drug Release and Tissue Engineering.

Polysaccharide-based hydrogels are very promising materials for a wide range of medical applications, ranging from tissue engineering to controlled drug delivery for local therapy. The most interesting property of this class of materials is the ability to be injected without any alteration of their chemical, mechanical and biological properties, by taking advantage of their thixotropic behavior. It is possible to modulate the rheological and chemical-physical properties of polysaccharide hydrogels by varying the cross-linking agents and exploiting their thixotropic behavior. We present here an overview of our synthetic strategies and applications of innovative polysaccharide-based hydrogels: hyaluronan-based hydrogel and new derivatives of carboxymethylcellulose have been used as matrices in the field of tissue engineering; while guar gum-based hydrogel and hybrid magnetic hydrogels, have been used as promising systems for targeted controlled drug release. Moreover, a new class of materials, interpenetrating hydrogels (IPH), have been obtained by mixing various native thixotropic hydrogels.

On the Mechanism of Drug Release from Polysaccharide Hydrogels Cross-Linked with Magnetite Nanoparticles by Applying Alternating Magnetic Fields: the Case of DOXO Delivery.

The chemical, biological and physical properties of carboxymethylcellulose (CMC) hydrogels with silanized magnetite (Fe3O4) nanoparticles (NPs) as cross-linker were investigated and compared with the analogous hydrogel obtained by using 1,3-diaminopropane (DAP) as cross-linker. The magnetic hydrogel was characterized from the chemical point of view by FT-IR, whereas the morphology of the hydrogel was investigated by FESEM and STEM. The water uptake and rheological measurements reveal how much the swelling and mechanical properties change when CMC is cross-linked with silanized magnetite NPs instead of with DAP. As far as the biological properties, the hybrid hydrogel neither exerts any adverse effect nor any alteration on the cells. The magnetic hydrogels show magnetic hysteresis at 2.5 K as well as at 300 K. Magnetic measurements show that the saturation magnetization, remanent magnetization and coercive field of the NPs are not influenced significantly by the silanization treatment. The magnetic hydrogel was tested as controlled drug delivery system. The release of DOXO from the hydrogel is significantly enhanced by exposing it to an alternating magnetic field. Under our experimental conditions (2 mT and 40 kHz), no temperature increase of the hydrogel was measured, testifying that the mechanism for the enhancement of drug release under the AMF involves the twisting of the polymeric chains. A static magnetic field (0.5 T) does not influence the drug release from the hydrogel, compared with that without magnetic field.

Platelet adhesion to commercial and modified polymer materials in animals under psychological stress and in a no-stress condition.

It is well known that stressful stimuli change blood functions and that protein and platelet parameters are altered in humans and animals subjected to stress. We have examined the influence of psychological stress on the morphological responses of platelets on commercially available materials [polyester (VP), fluoropassivated polyester (VPF), non-woven benzylic ester of hyaluronic acid (Hyaff11)] and on materials synthesised (PUPA) and/or surface modified by sulphation (Hyaff11S) or by immobilisation of the anticoagulant molecules heparin and sulphated hyaluronic acid (PUPA-Heparin, PUPA-HyalS, HyalS-PET). Moreover, the anticoagulant activity (i.e. thrombin inactivation) of the materials was analysed. In the no-stress condition, the surfaces with a low degree of platelet adhesion were Hyaff11S, HyalS-PET, PUPA-Heparin and PUPA-HyalS. Hyaff11, PET and PUPA had the highest number of adherent platelets within the series. VP and VPF exhibited an intermediate behaviour. The exposure of animals to stress induced a dramatic change in platelet number and morphology on PET, HyalS-PET, PUPA, PUPA-HyalS and Hyaff11: there was a higher degree of platelet adhesion, increased platelet spreading and the appearance of pseudopodia. In VP, VPF, Hyaff11S and PUPA-Heparin, there were no changes in platelet adhesion in stress conditions with respect to the no-stress condition; the latter two materials, the only ones able to prolong thrombin time, had a very low number of adherent platelets.

Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds.

The re-differentiation capacities of human articular and chick embryo sternal chondrocytes were evaluated by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-11-S material promoted the highest rate of chondrocyte proliferation. RNA isolated from human and chick embryo chondrocytes cultured in Petri dishes, HYAFF-11 or HYAFF-11-S were subjected to semi-quantitative RT-PCR analyses. Human collagen types I, II, X, human Sox9 and aggrecan, chick collagen types I, II, IX and X were analysed. Results showed that human collagen type II mRNA expression was upregulated on HYAFF-11 biomaterials. In particular, a high level of collagen type IIB expression was associated with three-dimensional culture conditions, and the HYAFF-11 material was the most supportive for human collagen type X mRNA expression. Human Sox9 mRNA levels were constantly maintained in monolayer cell culture conditions over a period of 21 days, while these were upregulated when chondrocytes were cultured on HYAFF-11 and HYAFF-11S. Furthermore, chick collagen type IIA and IIB mRNA expression was detected after only 7 days of HYAFF-11 culture. Chick collagen type IX mRNA expression decreased in scaffold cultures over time. Histochemical staining performed in engineered cartilage revealed the presence of a de novo synthesized glycosaminoglycan-rich extracellular matrix; immunohistochemistry confirmed the deposition of collagen type II. This study showed that the three-dimensional HYAFF-11 culture system is both an effective chondrocyte delivery system for the treatment of articular cartilage defects, and an excellent in vitro model for studying cartilage differentiation.

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.

Formation of defined microporous 3D structures starting from cross-linked hydrogels.

A new and simple technique was developed to obtain polysaccharide (hyaluronane, alginate and carboxymethylcellulose) -based hydrogels with a defined porous morphology. The technique consists of stratifying a cross-linked hydrogel on a filter with known pore diameter. CO(2) bubbles, produced by the addition of HCl to a porogen salt NaHCO(3), are forced to pass through the filter, and they induce the hydrogel to assume a porous morphology. The presence and distribution of pores was confirmed by scanning-electron microscopy (SEM). A strict correspondence was found between the porosity of the filter and the pore diameter in the hydrogels. Water uptake measurements showed a decreased amount of water taken up by the porous hydrogels compared with the native hydrogels, due to a compacting of the material. An explanation of the porous material properties of Hyal hydrogel was given on the basis of FTIR spectra.

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.

Biohydrogels with magnetic nanoparticles as crosslinker: characteristics and potential use for controlled antitumor drug-delivery.

Hybrid magnetic hydrogels are of interest for applications in biomedical science as controlled drug-delivery systems. We have developed a strategy to obtain novel hybrid hydrogels with magnetic nanoparticles (NPs) of CoFe(2)O(3) and Fe(3)O(4) as crosslinker agents of carboxymethylcellulose (CMC) or hyaluronic acid (HYAL) polymers and we have tested these systems for controlled doxorubicin release. The magnetic NPs are functionalized with (3-aminopropyl)trimethoxysilane (APTMS) in order to introduce amino groups on the surface. The amino coating is determined and quantified by standard Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy methods, and by cyclic voltammetry, a novel approach that permits us to look at the solution properties of the functionalized NPs. The gel formation involves the creation of an amide bond between the carboxylic groups of CMC or HYAL and the amine groups of functionalized NPs, which work as crosslinking agents of the polymer chains. The hybrid hydrogels are chemically and morphologically characterized. The rheological and the water uptake properties of the hydrogels are also investigated. Under the application of an alternating magnetic field, the CMC-HYAL hybrid hydrogel previously loaded with doxorubicin shows a drug release greater than that showed by the CMC-HYAL hydrogel crosslinked with 1,3-diaminopropane. In conclusion, the presence of magnetic NPs makes the synthesized hybrid hydrogels suitable for application as a drug-delivery system by means of alternating magnetic fields.