Comparison of EDC/NHS and Pentasodium Triphosphate (TPP) as Cross-Linking Agents for Nanohydroxyapatite, Silk Fibroin, and Chitosan 3D Scaffolds

The present study compares two scaffold cross-linking strategies, EDC/NHS and pentasodium triphosphate (TPP), applied to nanohydroxyapatite/silk fibroin/chitosan (nHA/SF/CTS) scaffolds. The effects of the cross-linking strategy on scaffold morphology, ATR-FTIR profile, swelling, in vitro degradation, and compressive properties were evaluated. In addition, the biological response of selected 20:40:40 and 15:70:15 (w/w) scaffold variants was assessed using SaOS-2 cells. The IR spectra of TPP- and EDC/NHS-cross-linked materials were qualitatively similar, and no distinct bands attributable to residual EDC or NHS were observed after washing. SEM observations indicated that the TPP-cross-linked scaffolds tended to have smaller and more spherical pores, whereas the EDC/NHS-cross-linked materials showed a more elongated pore morphology. These qualitative morphological differences were accompanied by differences in swelling, degradation, and low-strain mechanical response. The 20:40:40 formulation cross-linked with EDC/NHS showed the most favorable low-strain response, whereas PBS swelling markedly reduced the early compressive response of all scaffolds. Biological assessment showed that the 20:40:40 scaffolds supported viable SaOS-2 populations and maintained osteogenic activity, although cell colonization remained lower than on the control surfaces. The 15:70:15 variant performed less favorably, particularly after TPP cross-linking. These findings indicate that both cross-linking strategies can be used to obtain nHA/SF/CTS scaffolds, but their effects depend strongly on scaffold composition and on the property being considered.