Biological Behavior of Chitosan-Fibroin-Hydroxyapatite Scaffolds with STRO+1A, MC3T3-E1 and SaOS2 Cells

Hybrid composites with chitosan (CHI), silk fibroin (SF) and hydroxyapatite (HA) are biocompatible and attractive for bone engineering applications. The objective of this work was to evaluate in vitro cells behavior in contact with CHI-SF-HA scaffolds. The scaffolds were produced from a chitosan solution (2%wt) with SF (1%wt) and HA powders (1%wt) in acetic acid. This solution was molded, frozen and the scaffolds were freeze-dried, crosslinked, and then, submitted to in vitro tests with STRO+1A, MC3T3-E1 and SaOS2 cells under static conditions for 7, 14 and 21 days. The scaffolds were characterized through X-ray diffraction (XRD), infrared spectroscopy (FTIR) and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Cell viability and activity was assessed by MTT reduction and alkaline phosphate (ALP) activity detection. XRD patterns showed characteristic peaks at 8.8o, 20.3o and 24.6o (corresponding to SF) and peaks at 31.8o, 32.2o and 32.9o (corresponding to HA). The FTIR presented characteristic bands of the amide groups (SF) and of the phosphate and carbonate groups (HA). The EDS showed the presence of the C, O, N, P and Ca elements. SEM analyses showed the scaffold morphology, and indicated cell growth across the surface of the sample. The STRO+1A and MC3T3-E1 cells presented the best cell adhesion. MTT assay showed an increase of the cell number with time and the 21 days analyses showed the best proliferation for SaOS2 cells (p<0.01), STRO+1A cells (p<0.04) and MC3T3-E1 cells, respectively. The ALP activity was higher at 21 days for all cells types and the SaOS2 cells presented the best results in all analyses (p<0.01). Molecular studies are under progress to evaluate more deeply the biocompatibility of these scaffolds. Moreover, dynamic studies in bioreactor are under progress to improve cell colonization inside scaffolds.