Polarized M2 macrophages induced by mechanical stretching modulate bone regeneration of the craniofacial suture for midfacial hypoplasia treatment

The underlying mechanism of the trans-sutural distraction osteogenesis (TSDO) technique as an effective treatment that improves the symptoms of midfacial hypoplasia syndromes is not clearly understood. Increasing findings in the orthopedics field indicate that macrophages are mechanically sensitive and their phenotypes can respond to mechanical cues. However, how macrophages respond to mechanical stretching and consequently influence osteoblast differentiation of suture-derived stem cells (SuSCs) remains unclear, particularly during the TSDO process. In the present study, we established a TSDO rat model to determine whether and how macrophages were polarized in response to stretching and consequently affected bone regeneration of the suture frontal edge. Notably, after performing immunofluorescence, RNA-sequencing, and micro-computed tomography, it was demonstrated that macrophages are first recruited by various chemokines factors and polarized to the M2 phenotype upon optimal stretching. The latter in turn regulates SuSC activity and facilitates bone regeneration in sutures. Moreover, when the activated M2 macrophages were suppressed by pharmacological manipulation, new bone microarchitecture could rarely be detected under mechanical stretching and the expansion of the sutures was clear. Additionally, macrophages achieved M2 polarization in response to the optimal mechanical stretching (10%, 0.5 Hz) and strongly facilitated SuSC osteogenic differentiation and human umbilical vein endothelial cell angiogenesis using an indirect co-culture system in vitro. Collectively, this study revealed the mechanical stimulation-immune response-bone regeneration axis and clarified at least in part how sutures achieve bone regeneration in response to mechanical force.