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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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Being a major component of bone tissue, hydroxyapatite is the most investigated calcium phosphate in the design and development of bone implants. The high brittleness and poor load-bearing properties have led researchers to manipulate hydroxyapatite performance by applying polymer or metal materials. The present study focuses on biomimetic approach of the hydroxyapatite synthesis from the cuttlefish bone in order to preserve highly porous structure. The low stiffness of hydroxyapatite scaffold was altered by thin polycaprolactone/poly(lactic acid) coating, resulting in remarkably 18-fold increase of Young's modulus. The mechanical test revealed that poly(lactic acid) increases the stiffness of composite scaffolds which depends on the polycaprolactone/poly(lactic acid) volume ratio. The composite scaffolds are bioactive supporting the deposition of new calcium phosphates when incubated in simulated physiological medium for 21 days. Moreover, the culture of human embryonic kidney cells indicated non-cytotoxicity of the composite scaffolds with emphasis on the cell proliferation during three days of culture. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018.

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.

Highly porous chitosan/hydroxyapatite composite structures with different weight ratios (100/0; 90/10; 80/20; 70/30; 60/40; 50/50; 40/60) have been prepared by precipitation method and freeze-gelation technique using calcite, urea phosphate and chitosan as starting materials. The composition of prepared composite scaffolds was characterized by X-ray diffraction analysis and Fourier transformed infrared spectroscopy, while morphology of scaffolds was imaged by scanning electron microscopy. Mercury intrusion porosimetry measurements of prepared scaffolds have shown different porosity and microstructure regarding to the HA content, along with SEM observations of scaffolds after being immersed in physiological medium. The results of swelling capacity and compressive strength measured in Dulbecco's phosphate buffer saline (DPBS) have shown higher values for composite scaffolds with lower in situ HA content. Viability, proliferation and differentiation of MC3T3-E1 cells seeded on different scaffolds have been evaluated by live dead assay and confocal scan microscopy. Our results suggest that the increase of HA content enhance osteoblast differentiation confirming osteogenic properties of highly porous CS/HA scaffolds for tissue engineering applications in bone repair.