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

Abstract

Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used to regenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffold architecture 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 developing novel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show the potential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds were characterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanning electron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. In vitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This was done by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffolds and HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mouse fibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culture studies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goat marrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEM while the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation of GBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate and osteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenic medium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemical and biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use in TE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects.

A simple rocker‐induced mechanical stimulus upregulates mineralization by human osteoprogenitor cells in fibrous scaffolds

“…Cell lysates were freeze‐thawed three times, vortexed and centrifuged. Cell lysate and PicoGreen ® solution were mixed and the fluorescence intensity measured spectrofluorometrically with an excitation of 480 nm and emission of 520 nm (Oliveira et al, 2006). The total DNA was calculated from the fluorescence emission to ng/ml using a standard curve.…”

Engineering osteochondral constructs through spatial regulation of endochondral ossification

“…Tissue engineering applications aim to replace or regenerate damaged tissues through the combinations of cells, three-dimensional scaffolds and signalling molecules [4][5]. A number of strategies have been implemented to engineer osteochondral constructs including bi-phasic scaffolding [6][7][8][9][10][11][12], bioreactor technologies [13][14][15][16], and growth factor/gene delivery [17][18][19][20]. Engineered anatomically accurate osteochondral grafts have also been suggested as a potential approach to joint condyle repair [21][22].…”

Chitosan–hydroxyapatite composite biomaterials made by a one step co-precipitation method: preparation, characterization and in vivo tests

“…This mineral can be regarded, with some limitations, as a crystallochemical analogue of the main mineral constituent of human and animal skeletal tissues [6]. A wide range of biomaterials for different clinical applications can be created on the basis of two components: nanocrystalline apatite and chitosan [3][4][5][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Chitin is the second (after cellulose) most abundant natural polysaccharide.…”