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.

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.

NMR metabolomic investigation of astrocytes interacted with Aß42 or its complexes with either copper(II) or zinc(II).

Alzheimer's disease (AD) is the leading cause of senile dementia. One of the main hallmarks of AD is the presence of amyloid plaques in the brain, primarily formed by fibrils of the amyloid-ß (Aß) peptide. Transition metal ions, such as Cu(2+) and Zn(2+) have been found at high concentrations in senile plaques isolated from AD patients and evidence have been reached that (i) Aß aggregation is greatly affected by Cu(2+) and Zn(2+) and (ii) Cu(2+), implicated in the formation of reactive oxygen species, leads to mitochondrial dysfunctions ultimately leading to neuronal cells death. Aß, apart from being toxic to neural cells, induces reactive astrocytosis in cell culture. Astrocytes play many crucial roles to sustain normal brain function by maintaining the cerebral homeostasis, modulating the synaptic transmission, and providing a metabolic support for neuronal growth. Although many studies have shown that Aß fibrils interfere in the main astrocytic functions aimed at supporting the neuronal activity, nothing is known about the effects of Zn(2+)- and Cu(2+)-induced Aß aggregates on astrocyte functions. In this study the effects of treatments with Aß(42), either in absence or in the presence of Cu(2+) and Zn(2+), on astrocyte cell cultures were evaluated by using classical cellular assay and by looking at changes in metabolic profiles in the cellular medium by using nuclear magnetic resonance spectroscopy (NMR). Our results indicate that metal induced Aß aggregation strongly affects the metabolites involved in the neurotransmission activity supporting a deleterious impact of Cu(2+) and Zn(2+) Aß amyloidogenesis on astrocyte functions.

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.

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.

A new amido phosphonate derivative of carboxymethylcellulose with an osteogenic activity and which is capable of interacting with any Ti surface.

A new phosphonate derivative of carboxymethylcellulose (CMC) was recently synthesized (CMCAPh). The phosphonate polysaccharide was obtained by using a carbodiimide-like activating agent for carboxylic groups and 2-aminoethyl-phosphonic acid to create an amide bond between the amine of the phosphonate agent and the carboxylic acids of CMC. The polymer was characterized by (31)P NMR, FT-IR, and potentiometric titration. CMCAPh showed different properties from CMC and its amidated derivative polymer CMCA. The behavior in solution of CMCAPh polymer towards normal human osteoblasts (NHOst) was studied in vitro, monitoring the cell proliferation, cell differentiation, and osteogenic activity and was then compared with the amidic derivative of carboxymethylcellulose (CMCA). Furthermore, CMCAPh was used to coat titania disks with the aim of increasing the osteogenic activity of implant surfaces. The polymer film on the titania surface was characterized by AFM and TOF-SIMS analysis. An ATR FT-IR study was carried out to evaluate the polymer bonding mode onto the titanium surface. Osteoblast morphology was evaluated by SEM. Adhesion analysis of NHOst demonstrated a better adhesion on the titanium surface coated with CMCAPh than on the bare titanium surface.

The effects of the electric fields on hydrogels to achieve antitumoral drug release.

The achievement of electrochemotherapy was obtained using electrodes covered with guar gum (GG) hydrogel swollen in a sulfate bleomycin solution. The bleomycin delivery into the plasma membranes of cancer cells occurs only when field strength (V/cm) was applied, decreasing the drug contact with healthy tissues. The effect of the delivered bleomycin at different concentrations was examined on tumoral mouse fibroblast (NIH3T3) and human coronary artery endothelial cells. The GG hydrogel released the drug only when the field strength was applied and the amount depended on the electromotive force. Consequently, cellular survival depended on the field strength. Moreover in vitro results showed a bigger cellular mortality of the NIH3T3 compared with endothelial cells. The best parameters to be utilized in electrochemotherapy were ascertained.

Spiral and square microstructured surfaces: the effect of the decreasing size of photo-immobilized hyaluronan domains on cell growth.

Spiral and squared micropatterned surfaces of decreasing dimensions were realized by photo-immobilizing a photoreactive hyaluronan (Hyal) derivative on silanized glass substrates. The microstructured surfaces were observed by atomic force microscope and scanning electron microscope. Scanning electron microscope analysis revealed the presence of a spiral (ranging from 100 microm down to 1 microm in the central part) and a square pattern consisting of a central square of 100 microm x 100 microm and squares of different dimensions decreasing from the centre to the edges of the micropatterned area (2 microm x 1 microm). Three cell types were tested on all the microstructured surfaces: human coronary artery endothelial cells (HCAEC), human dermal fibroblasts (C54), and NIH 3T3 fibroblasts. Cell adhesion analysis demonstrated that HCAEC and C54 did not adhere to the immobilized Hyal on silanized glass but adapted their shape to the different sizes of the square and spiral patterns. Also, in both geometric patterns, the reduction of the adhesive glass width induced C54 to create bonds amongst themselves. NIH 3T3 cells adhered inside the squares and the spiral but reducing the adhesive glass width induced NIH 3T3 to adhere to immobilized Hyal. This fact is explained by the interactions between the cells and the immobilized Hyal as a consequence of the CD44/Hyal binding.

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 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.

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.

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.

An amidated carboxymethylcellulose hydrogel for cartilage regeneration.

An amidic derivative of carboxymethylcellulose was synthesized (CMCA). The new polysaccharide was obtained by converting a large percentage of carboxylic groups ( approximately 50%) of carboxymethylcellulose into amidic groups rendering the macromolecule quite similar to hyaluronan. Then, the polysaccharide (CMCA) was crosslinked. The behavior of CMCA hydrogel towards normal human articular chondrocytes (NHAC) was in vitro studied monitoring the cell proliferation and synthesis of extra cellular matrix (ECM) components and compared with a hyaluronan based hydrogel (Hyal). An extracellular matrix rich in cartilage-specific collagen and proteoglycans was secreted in the presence of hydrogels. The injectability of the new hydrogels was also analysed. An experimental in vivo model was realized to study the effect of CMCA and Hyal hydrogels in the treatment of surgically created partial thickness chondral defects in the rabbit knee. The preliminary results pointed out that CMCA hydrogel could be considered as a potential compound for cartilage regeneration.

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.

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.