Therapy with BMSCs has been considered as a potential therapeutic strategy for many musculoskeletal disorders such as bone defects, osteogenesis imperfecta and osteoporosis [
17]. However, the long-lasting underlying molecular mechanisms of osteogenic differentiation from BMSCs are still elusive. In the present study, we utilized the in vitro osteogenic induction model to study the temporal expression profiles in osteogenic differentiation of BMSCs. The crucial molecule mechanism that cytokines including IL6, LIF and CSF3 activated the phosphorylation of STAT3 to drive the osteogenesis and angiogenesis was identified by overlapping the common EDGs of three inductive periods.
The formation of hematoma at the defect site which induced by inflammation was a typical character of the primary phase of bone fracture healing [
18]. Both macrophages and neutrophils were proved to influence the bone formation [
19,
20]. In the osteogenic induction of BMSCs for 7, 14 and 21 days, inflammatory response and immune response were both contributed as the main biological process (Fig.
3). It was reported that stem cell differentiation to osteoblasts was a dynamic process. Initially, after seven days of induction, the MAPK pathway was activated, but after 14 days of induction, the PPAR pathway was predominant, showing that in the middle of induction, stem cells would further promote osteogenic differentiation by reducing lipogenic differentiation. After longer induction (21 days), the Toll pathway was gradually activated, while the pathways enriched to the first two time points were missing (Fig.
4A,
B). All the above three pathways have been discussed in previous studies [
21‐
23]. The correlation analysis also indicated the distinction between samples from four time-points (Fig.
2D–E). However, osteogenesis is occurred throughout the induction periods, so the co-expression EDGs were overlapped to explore the underlying molecular mechanisms. It demonstrated that the cytokine–cytokine receptor interaction and JAK-STAT signaling pathways were involved in all the osteogenic induction periods (Fig.
4A). The results of the PPI protein interaction analysis indicated the key role of IL6, LIF and CSF3 in the common DEGs (Fig.
4D). These inflammatory cytokines are involved in the Cytokine/JAK/SAT signaling pathway, and their expression levels were up-regulated in the induction of osteogenic induction medium (
Figs. 4I,
5A‐
C). They might medicate the phosphorylation of STAT3 to modulate the osteogenesis of BMSCs. The osteogenic differentiation was inhibited by suppression the phosphorylation of STAT3 (Fig.
6). Increase in IL6 family cytokines activated the co-receptor gp130, as well as the JAK/STAT signaling pathway, resulting in the increased phosphorylation of STAT3 [
24]. IL6 is known as an endogenous regulator in the innate immune system and the transformation from neutrophil to macrophage recruitment after injury [
25]. It participates in the inflammatory phase of bone regeneration in various means. Overexpression of LIF promoted osteogenic differentiation of mouse renal tubular interstitial fibrosis by activating ERK and STAT3 pathway [
26]. Different forms of mutations in colony-stimulating factor 3 receptor (CSF3R) were found to affect STAT3 activation [
27]. JAK/STAT signaling pathway was also reported to participate osteogenic differentiation of MSCs stimulated by leptin and BMP9 [
28].
Bone formation requires a well-coordinated combination of osteogenesis and angiogenesis [
29]. BMSCs differentiated into osteoblasts accompanied with regeneration of neovasculature or neoangiogenesis which were also validated in this study (F
ig.
5D–I). The angiogenesis in the newly formed bone was mediated by various cytokines and signaling pathways. CSF3 had been reported as a central downstream mechanism for the proangiogenic effects of TGF transforming growth factor β1 (TGFβ1) [
8]. As evidenced by microvessel sprouting in aortic ring cultures, LIF overexpression MSCs promoted tube formation of endothelial cells in vitro [
30]. Pro-inflammatory IL6 is one of the cytokines that stimulate angiogenic gene VEGF expression [
31]. Ca
2+/calmodulin-dependent protein kinase II (CaMKII) induced angiogenesis reliance on the regulation of interleukin-6 (IL6)/JAK/STAT3 signaling axis [
32]. Here, we identified the cytokines (CSF3, IL6 and LIF) that contributed to osteogenesis and angiogenesis from the transcriptome perspective (F
ig.
4D–I). These reports supported cytokines-mediated immuno-inflammation via activation of JAK-STAT3 signaling pathway as a promoter of angiogenesis and osteogenesis. Last but not the least, this research seemed to be the first time to reveal the temporal transcriptome of rabbit BMSCs differentiating into osteoblasts, which provides valuable reference for solving the differences between species.