Synthesis of N, N,O and O-carboxymethyl chitosan derivatives of controllable substitution degrees and their utilization as electrospun scaffolds for bone tissue engineering.

Most chitosan (CS) carboxymethylation approaches are weighed down by insufficient description protocols regarding the reaction specificity and the degree of substitution (DS). Here, we provide three carboxymethylation protocols of enhanced specificity towards the amine (N-), amine/hydroxy (N,O-) and hydroxy (O-) groups of CS. The DS for all samples was found to be ∼70 %, as confirmed by NMR analysis. To illustrate the modified materials' potential in bone tissue regeneration, each derivative was blended with poly(vinyl alcohol) to prepare scaffolds via electrospinning. Both materials and electrospun membranes were characterized in terms of their physicochemical properties by Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry and water contact angle measurements. The electrospun membranes' swelling ratio, degradation, tensile strength, as well as morphology were also examined through scanning electron microscopy before and after heat treatment at 120 °C, which was used as a method of physical stabilization, leading to significantly enhanced degradation rate and mechanical strength. The three electrospun scaffold types were loaded with MC3T3-E1 pre-osteoblastic cells and their cell adhesion, viability and growth rate were assessed. Finally, osteogenic differentiation was examined by means of alkaline phosphatase activity measurement, calcium mineralization and formation of extracellular matrix markers, with all materials showing promising prospects.