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 effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn.

Platelet adhesion and activation induced by fibrinogen (Fbg) coating on polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) were analyzed. Hyal or HyalS was coated and grafted on the glass substrate using a photolithographic method. The Fbg coating was achieved by two different routes: the immobilization of Fbg by means of covalent bond to the polysaccharide layers and the mere adsorption of Fbg to Hyal and HyalS surfaces. Platelet adhesion and activation to the surfaces were evaluated using, respectively, scanning electron microscopy (SEM) and quantifying the release of Platelet Factor 4 by ELISA. The method used for the coating of the surfaces with the Fbg influenced the platelet response. In fact, platelet adhesion and activation took place on surfaces covered by bound Fbg but not on those containing adsorbed Fbg. To explain this difference, the molecular mechanism involved in the Fbg--platelet interaction was investigated blocking platelet membrane receptors by monoclonal antibodies. Because the interaction between Fbg and the GPIIb/IIIa platelet membrane receptor was the only molecular pathway involved, Fbg conformation after the interaction (adsorption or binding) with the Hyal and the HyalS chains and the role of serum proteins adsorbed on the Fbg containing surfaces were accurately analyzed. Both adsorbed and bound Fbg prevented the adsorption of further serum proteins; consequently, a direct interaction between Fbg and platelets was supposed and the different platelet behavior was ascribed to the different conformational changes that occurred after the adsorption and the chemical binding of the Fbg to the Hyal and HyalS surfaces.

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Superoxide dismutase (SOD) was chemically bound to carboxymethyl-cellulose (CMC) polymer.Furthermore, SOD was also trapped into two hydrogels of CMC with 50% and 90% crosslinking degree. The ability of the two SOD-CMC hydrogels to capture SOD and their release kinetics were investigated. ATR FT-IR spectrometry was used to study the conformation of SOD interacting with both CMC polymer and hydrogels. The effect of SOD-CMC polymer conjugate and SOD-CMC hydrogel systems upon human fibroblasts was studied in vitro measuring the cell proliferation inhibition index and evaluating cell morphology. Using the xanthine oxidase-nitroblue tetrazolium assay, the specific activity of bound SOD to CMC polymer or trapped into hydrogels was evaluated. The specific activity of the enzyme was higher in SOD-CMC hydrogels than in SOD-CMC polymer conjugates.