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Up till now, chitosan has confirmed its versatile application in skin, cartilage and bone tissue engineering, as well as in drug delivery applications. This study is focused on enzymatic degradation of porous chitosan structures usually designed for mentioned purposes. In vitro degradation was monitored during four weeks of incubation at physiological temperature and in two different media, phosphate buffer saline solution and water.The scaffolds were characterised before and after enzymatic degradation using scanning electron microscopy and infrared spectroscopy with Fourier transformations (FTIR). According to the gravimetric analysis, higher weight loss of chitosan scaffolds was observed in buffered medium with respect to the water. The results implied that the total weight loss obtained in buffer involves physical dissolution of chitosan and lysozyme cleavage of glycoside bond. Importantly, FTIR identification of chitosan scaffolds after enzymatic degradation indicated the absence of lysozyme activity in water, indicating that weight loss is a result of the chitosan dissolution. This finding greatly impacts design of degradation experiments and characterisation of degradation behaviour of chitosan-based materials utilised as implants or drug delivery systems.

The development of bioactive injectable system as cell carrier with minimal impact on viability of encapsulated cells represents a great challenge. In the present work, we propose a new pH-responsive chitosan-hydroxyapatite-based hydrogel with sodium bicarbonate (NaHCO) as the gelling agent. The in situ synthesis of hydroxyapatite phase has resulted in stable composite suspension and final homogeneous hydrogel. The application of sodium bicarbonate has allowed non-cytotoxic fast gelation of chitosan-hydroxyapatite within 4min, and without excess of sodium ions concentration. Rheological properties of crosslinked hydrogel have demonstrated possible behaviour as 'strong physical hydrogel'. The live dead staining has confirmed good viability and dispersion, as well as proliferation of encapsulated cells by the culture time. Presented preliminary results show good potential of chitosan-hydroxyapatite/NaHCO as a cell carrier, whose impact on the cell differentiation need to be confirmed by encapsulation of other cell phenotypes.

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Injectable hydrogels have emerged as promising biomaterials for tissue engineering applications. The goal of this study was to evaluate the potential of a pH-responsive chitosan-hydroxyapatite hydrogel to be used as a three-dimensional support for encapsulated mesenchymal stem cells (MSCs) osteogenic differentiation. In vitro enzymatic degradation of the hydrogel, during 28 days of incubation, in simulated physiological condiditons, was characterized by swelling measurements, molecular weight determination and SEM analysis of hydrogel microstructure. Osteogenic differentiation of encapsulated MSCs was confirmed by osteogenic Runx2, collagen type I and osteocalcin immunostaining and alkaline phosphatase quantification. The deposition of late osteogenic markers (calcium phosphates) detected by Alizarin red and von Kossa staining indicated an extracellular matrix mineralization.