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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.

Biocompatible hydrogels are promising approaches for bone repair and engineering. A novel therapeutic nanocomposite hydrogel was designed based on triblock copolymer PCL-PEG-PCL and natural gelatin (PCEC/GEL) and reinforced with halloysite nanotube (HNT). Gentamicin (GM) loaded HNT was immobilized in polymeric hydrogel matrix to fabricate scaffolds using the freeze-drying method. Scaffolds were characterized via FT-IR, XRD, and SEM methods. The swelling ratio, density, porosity, degradation, and mechanical behavior were evaluated to investigate the effects of HNT on the physicochemical properties of the composite. Cell viability and cell attachment were investigated by MTT assay and SEM. Cell proliferation was observed without any cytotoxicity effect on h-DPSCs. Alizarin red staining (ARS) and real-time QRT-PCR assay were carried out to monitor the osteoconductivity of scaffolds on human dental pulp-derived mesenchymal stem cells (h-DPSCs) which were seeded drop wise onto the top of scaffolds. The quantification of the mRNA expression of osteogenic marker genes, BMP2, SPARK, BGLAP and RUNEX2 over a period of 21 days of cell seeding, demonstrated that cell-encapsulating PCEC/GEL/HNT-GM hydrogel scaffolds supported osteoblast differentiation of hDPSCs into osteogenic cells through the up-regulation of related genes along with moderate effects on cell viability. Moreover, the antibiotics loading reduced bacterial growth while maintaining the osteogenic properties of the scaffold. Therefore, the bactericidal PCEC/gelatin/HNT-GM hydrogel nanocomposite, with enhanced durability, maintenance the functionality of seeded cells in vitro that can be a remarkable dual-functional candidate for hard tissue reconstruction and customized bone implants fabrication via the direct incorporation of bactericidal drug to prevent infection.