The treatment of bone defects caused by diseases, trauma, or tumor has always been a great clinical challenge. Implantation of bone biomaterials into bone defect areas is an effective method for bone injury repair. In this study, we used three-dimensional (3D) printing technology to prepare nano-hydroxyapatite (nHA)/sodium alginate (SA)/gelatin (Gel) hydrogel scaffolds loaded with different ratios (0, 0.13, 0.26, 0.39, 0.53, and 0.79‰) of emodin (EM) (EM/nHA/SA/Gel). Scanning electron microscopy showed that the scaffolds had a smooth surface without fracture and nHA was evenly distributed on the surface. The cell proliferation and migration results showed that the 0.39‰ EM group, in particular, could significantly promote the proliferation and migration of mouse embryonic osteoblast precursor (MC3T3-E1) cells and significantly increase the mRNA expression of osteogenic differentiation-related genes (bone morphogenetic protein/BMP-2, BMP-9, osteocalcin). In addition, the 0.39‰ EM group exhibited the best effect on osteogenic differentiation-related proteins (alkaline phosphatase, Runx 2, OSX). The expression of M2 polarization-related genes (arginase-1, CD206) also significantly increased after the treatment with the 0.39‰ EM group. Micro-CT showed that in the rat skull defect model, the EM/nHA/SA/Gel scaffold group significantly promoted bone regeneration after being implanted into the skull for 30 days. Our results indicate that the EM/nHA/SA/Gel hydrogel scaffolds can not only directly promote the proliferation and differentiation of osteoblasts but also indirectly promote osteogenic differentiation by supporting M2 polarization of macrophages. EM/nHA/SA/Gel hydrogel scaffolds are potential bone tissue engineering materials for bone regeneration.

Current studies have found that low‐dose irradiation (IR) can promote bone regeneration. However, mechanism studies of IR‐triggered bone regeneration mainly focus on the effects of osteoblasts, neglecting the role of the surrounding immune microenvironment. Here in this study, in vitro proliferation experiments showed that low‐dose IR ≤2 Gy could promote the proliferation of bone marrow mesenchymal stem cells (BMSCs), and qRT‐PCR assay showed that low‐dose IR ≤2 Gy could exert the M2 polarization of Raw264.7 cells, while IR >2 Gy inhibited BMSC proliferation and triggered M1 polarization in Raw264.7 cells. The ALP and mineralized nodules staining showed that low‐dose IR ≤2 Gy not only promoted osteoblast mineralization through IR‐triggered osteoblast proliferation but also through M2 polarization of Raw264.7 cells, while high‐dose IR >2 Gy had the opposite effect. The co‐incubation of BMSC with low‐dose IR irradiated Raw264.7 cell supernatants increased the mRNA expression of BMP‐2 and Osx. The rat cranial defects model revealed that low‐dose IR ≤2 Gy gradually promoted bone regeneration, while high‐dose IR >2 Gy inhibited bone regeneration. Detection of macrophage polarity in peripheral blood samples showed that low‐dose IR ≤2 Gy increased the expression of CD206 and CD163, but decreased the expression of CD86 and CD80 in macrophages, which indicated M2 polarization of macrophages in vivo, while high‐dose IR had the opposite effect. Our finding innovatively revealed that low‐dose IR ≤2 Gy promotes bone regeneration not only by directly promoting the proliferation of osteoblasts but also by triggering M2 polarization of macrophages, which provided a new perspective for immune mechanism study in the treatment of bone defects with low‐dose IR.