Fig 2: Schematic representation of bone healing process focused on bone formation and part of bone remodeling phases. Runt-related transcription factor (RUNX2) is early expressed during bone formation, keeping the osteoblasts in immature state; successively, the simultaneous down-regulation of RUNX2 and the up-regulation of osterix (OSX) allows osteoblasts’ maturation. The activating transcription factor 4 (ATF4) regulates osteoblast differentiation, working in cooperation with OSX, promoting bone matrix protein synthesis such as osteocalcin (OCN), which has the ability to bind calcium to modulate the calcium metabolism by mediating its association with hydroxyapatite (HA) [9]; osteopontin (OPN) regulates osteoclast genesis and osteoclast activity, contributing to bone formation and resorption [10,11]; and osteonectin (ONN) is a regulator of the calcium release by binding collagen and HA crystals, thereby influencing the mineralization of collagen during bone formation [12]. Collagen a-1 type I (COL1A1), which can promote osteogenic differentiation and mineralization, and alkaline phosphatase (ALP), which contributes to mineralization [13], at the beginning of phase 3 both are weakly expressed, and then matured osteoblasts increase col1a1 and alp expression. Bone sialoprotein (ibsp) is vital in the regulation of osteoblast differentiation and initiation of matrix mineralization in bone tissue, whereas dentin matrix acidic phosphoprotein 1 (DMP1) and sclerostin (SOST) are specific osteocyte markers [14,15]. Created in BioRender.com.

Fig 3: Immunofluorescence images of RUNX2 and gene expression at 14 days culture time. All the data reported are expressed as the mean ± S.E.M. of six independent experiments. Before the statistical analysis was performed, the raw data were tested for normality with Kolmogorov–Smirnov normality test and further analyzed with unpaired t-test on GraphPad Prism 8.4.2. The results were evaluated as statistically significant (a) when the p-value < 0.05. Scale bar: 50 µm.

Fig 4: Gene expression results of hASCs cultured both in GM and OM with or w/o the PEMF treatment for 7 and 14 days. The results shown in panels (A–C) refer to treated and untreated (PEMFs+ and w/o) hASCs kept in GM, whereas the panels (D–F) show the results of PEMFs+ and w/o hASCs committed towards the osteogenic differentiation (OM). The gene expression results in the bar plots (A,D) are reported as the mean ± S.E.M. of fold changes (FC). The results in (B,E) are the identity ratios of treated and untreated hASCs expressed in the plots as the mean ± S.E.M. of FC ratios. Finally, in (C,F) are reported “part of whole” plots, which highlight the proportion of bone-specific genes (expressed as FC) of treated and untreated hASCs. The bold letters a, b and c in the plots indicate the statistically significant results (p < 0.05). In (A,D), the data were analyzed with an unpaired t-test: w/o vs. PEMFs+. In (B,E), the identity ratios were tested with a One-Way ANOVA followed by Tukey’s test for multiple comparison: a—runx2/col1a1 vs. col1a1/alp; b—runx2/col1a1 vs. col1a1/alp; c—runx2/alp vs. col1a1/alp. All the results reported are the mean ± S.E.M. of six independent experiments, tested for normality and further analyzed with GraphPad Prism 8.4.2.