In Vitro Osteogenic Response to Copper‐Doped Eggshell‐Derived Hydroxyapatite with Macrophage Supplements

The high bioactivity and biocompatibility of hydroxyapatite (HAP) make it a useful bone graft material for bone tissue engineering. However, the development superior osteoconductive and osteoinductive materials for bone regeneration remains a challenge. To overcome these constraints, Cu-doped hydroxyapatite (HAP(Cu)) from waste eggshells has been produced for bone tissue engineering. The materials produced were characterized using Fourier transform infrared spectroscopy, x-ray diffraction, and photoelectron spectroscopy. The scanning microscopy images revealed that the developed HAP was a rod-like crystalline structure with a typical 80-150 nm diameter. Energy-dispersive x-ray spectroscopy showed that the generated HAP was mostly composed of calcium, oxygen, and phosphorus. The Ca/P molar ratios in eggshell-derived and copper-doped HAP were 1.61 and 1.67, respectively, similar to the commercially available HAP ratio (1.67). The WST-8 assay was used to assess the biocompatibility of HAPs with hBMSCs. HAP(Cu) in the media significantly altered the cytotoxicity of biocompatible HAP(Cu). The osteogenic potential of HAP(Cu) was demonstrated by greater mineralization than that of pure HAP or the control. HAP(Cu) showed higher osteogenic gene expression than pure HAP and the control, indicating its stronger osteogenic potential. Furthermore, we assessed the effects of sample-treated macrophage-derived conditioned medium (CM) on hBMSCs' osteogenesis. CM-treated HAP(Cu) demonstrated a significantly higher osteogenic potential vis-à-vis pure HAP(Cu). These findings revealed that HAP(Cu) with CM significantly improved osteogenesis in hBMSCs and can be explored as a bone graft in bone tissue engineering.