Bilayered chitosan-based scaffolds for osteochondral tissue engineering : influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor

摘要

Osteochondral tissue engineering presents a current research challenge due to the necessity of combining both bone and cartilagetissue engineering principles. In the present study, bilayered chitosan-based scaffolds are developed based on the optimization of bothpolymeric and composite scaffolds. A particle aggregation methodology is proposed in order to achieve an improved integrativebone–cartilage interface needed for this application, since any discontinuity is likely to cause long-term device failure. Cytotoxicitywas evaluated by the MTS assay with the L929 fibroblast cell line for different conditions. Surprisingly, in composite scaffolds usingunsintered hydroxyapatite, cytotoxicity was observed in vitro. This work reports the investigation that was conducted to overcomeand explain this behaviour. It is suggest that the uptake of divalent cations may induce the cytotoxic behaviour. Sintered hydroxyapatitewas consequently used and showed no cytotoxicity when compared to the controls. Microcomputed tomography (micro-CT) was carriedout to accurately quantify porosity, interconnectivity, ceramic content, particle and pore sizes. The results showed that the developedscaffolds are highly interconnected and present the ideal pore size range to be morphometrically suitable for the proposed applications.Dynamical mechanical analysis (DMA) demonstrated that the scaffolds are mechanically stable in the wet state even under dynamic compression.The obtained elastic modulus was, respectively, 4.21 ± 1.04, 7.98 ± 1.77 and 6.26 ± 1.04 MPa at 1 Hz frequency for polymeric,composite and bilayered scaffolds. Bioactivity studies using both a simulated body fluid (SBF) and a simulated synovial fluid (SSF) wereconducted in order to assure that the polymeric component for chondrogenic part would not mineralize, as confirmed by scanning electronmicroscopy (SEM), inductively coupled plasma-optical emission spectroscopy (ICP) and energy-dispersive spectroscopy (EDS) fordifferent immersion periods. The assays were carried out also under dynamic conditions using, for this purpose, a specifically designeddouble-chamber bioreactor, aiming at a future osteochondral application. It was concluded that chitosan-based bilayered scaffolds producedby particle aggregation overcome any risk of delamination of both polymeric and composite parts designed, respectively, for chondrogenicand osteogenic components that are mechanically stable. Moreover, the proposed bilayered scaffolds could serve as alternative,biocompatible and safe biodegradable scaffolds for osteochondral tissue engineering applications.