The fabrication of halloysite nanotube-based multicomponent hydrogel scaffolds for bone healing

Bone tissue engineering, as an alternative for common available therapeutic approaches, has been developed to focus on reconstructing of the missing tissues and restoring their functionality. In this work, three-dimensional (3D) nanocomposite scaffolds of polycaprolactone-polyethylene glycol-polycaprolactone/gelatin (PCEC/Gel) were prepared by freeze-drying method. Biocompatible nanohydroxyapatite (nHA), iron oxide nanoparticle (Fe₃O₄) and halloysite nanotube (HNT) powders were added to the polymer matrix aiming to combine the osteogenic activity of nHA or Fe₃O₄ with high mechanical strength of HNT. The scanning electron microscope (SEM) methods was utilized to characterize the nanotube morphology of HNT as well as nanoparticles of Fe₃O₄ and nHA. Prepared scaffolds were characterized via Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), and SEM methods. In addition, the physical behavior of scaffolds was evaluated to explore the influence of HNT on the physicochemical properties of composites. Cell viability and attachment were investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay and SEM on human dental pulp-derived mesenchymal stem cells (h-DPSCs) in-vitro. Cell proliferation was observed without any cytotoxicity effect on h-DPSCs for all examined scaffolds. Alizarin red (ARS) and alkaline phosphatase (ALP) staining were carried out to determine the osteoconductivity of scaffolds. The data demonstrated that all PCEC/Gel/HNT hydrogel scaffolds supported osteoblast differentiation of hDPSCs with moderate effects on cell proliferation. Moreover, PCEC/Gel/HNT/nHA with proper mechanical strength showed better biological activity compared to PCEC/Gel/HNT/Fe₃O₄ and PCEC/Gel/HNT scaffolds. Therefore, this study suggested that with proper fillers content, PCEC/Gel/HNT nanocomposite hydrogels alone or in a complex with nHA, Fe₃O₄ could be a suitable candidate for hard tissue regeneration.