Injectable and Gellable Chitosan Formulations Filled with Cellulose Nanofibers for Intervertebral Disc Tissue Engineering.

The development of non-cellularized injectable suspensions of viscous chitosan (CHI) solutions (1.7⁻3.3% (w/w)), filled with cellulose nanofibers (CNF) (0.02⁻0.6% (w/w)) of the type nanofibrillated cellulose, was proposed for viscosupplementation of the intervertebral disc nucleus pulposus tissue. The achievement of CNF/CHI formulations which can gel in situ at the disc injection site constitutes a minimally-invasive approach to restore damaged/degenerated discs. We studied physico-chemical aspects of the sol and gel states of the CNF/CHI formulations, including the rheological behavior in relation to injectability (sol state) and fiber mechanical reinforcement (gel state). CNF-CHI interactions could be evidenced by a double flow behavior due to the relaxation of the CHI polymer chains and those interacting with the CNFs. At high shear rates resembling the injection conditions with needles commonly used in surgical treatments, both the reference CHI viscous solutions and those filled with CNFs exhibited similar rheological behavior. The neutralization of the flowing and weakly acidic CNF/CHI suspensions yielded composite hydrogels in which the nanofibers reinforced the CHI matrix. We performed evaluations in relation to the biomedical application, such as the effect of the intradiscal injection of the CNF/CHI formulation in pig and rabbit spine models on disc biomechanics. We showed that the injectable formulations became hydrogels in situ after intradiscal gelation, due to CHI neutralization occurring in contact with the body fluids. No leakage of the injectate through the injection canal was observed and the gelled formulation restored the disc height and loss of mechanical properties, which is commonly related to disc degeneration.

C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-O-sulfated heparan sulfate domains.

Heparan sulfate (HS), a complex polysaccharide of the cell surface, is endowed with the remarkable ability to bind numerous proteins and, as such, regulates a large variety of biological processes. Protein binding depends on HS structure; however, in the absence of a template driving its biosynthesis, the mechanism by which protein binding sequences are assembled remains poorly known. Here, we developed a chemically defined 13C-labeled substrate and NMR based experiments to simultaneously follow in real time the activity of HS biosynthetic enzymes and characterize the reaction products. Using this new approach, we report that the association of C5-epimerase and 2-O-sulfotransferase, which catalyze the production of iduronic acid and its 2-O-sulfation, respectively, is necessary to processively generate extended sequences of contiguous IdoA2S-containing disaccharides, whereas modifications are randomly introduced when the enzymes are uncoupled. These data shed light on the mechanisms by which HS motifs are generated during biosynthesis. They support the view that HS structure assembly is controlled not only by the availability of the biosynthetic enzymes but also by their physical association, which in the case of the C5-epimerase and 2-O-sulfotransferase was characterized by an affinity of 80 nM as demonstrated by surface plasmon resonance experiments.