Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications : scaffold design and its performance when seeded with goat bone marrow stromal cells

摘要

Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used toregenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffoldarchitecture that can support the formation de novo of either bone and cartilage tissue, or both, as in the case of osteochondral defects.The later has been attracting a great deal of attention since it is considered a difficult goal to achieve. This work consisted on developingnovel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show thepotential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds werecharacterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanningelectron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. Invitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This wasdone by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffoldsand HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mousefibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culturestudies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goatmarrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEMwhile the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation ofGBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate andosteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenicmedium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemicaland biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use inTE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects.