Normal development of craniofacial sutures is crucial for cranial and facial growth in all three dimensions. These sutures provide a unique niche for suture stem cells (SuSCs), which are indispensable for homeostasis, damage repair, as well as stress balance. Expansion appliances are now routinely used to treat underdevelopment of the skull and maxilla, stimulating the craniofacial sutures through distraction osteogenesis. However, various treatment challenges exist due to a lack of full understanding of the mechanism through which mechanical forces stimulate suture and bone remodeling. To address this issue, we first identified crucial steps in the cycle of suture and bone remodeling based on the established standard suture expansion model. Observed spatiotemporal morphological changes revealed that the remodeling cycle is approximately 3 to 4 weeks, with collagen restoration proceeding more rapidly. Next, we traced the fate of the Gli1+ SuSCs lineage upon application of tensile force in three dimensions. SuSCs were rapidly activated and greatly contributed to bone remodeling within 1 month. Furthermore, we confirmed the presence of Wnt activity within Gli1+ SuSCs based on the high co‐expression ratio of Gli1+ cells and Axin2+ cells, which also indicated the homogeneity and heterogeneity of two cell groups. Because Wnt signaling in the sutures is highly upregulated upon tensile force loading, conditional knockout of β‐catenin largely restricted the activation of Gli1+ SuSCs and suppressed bone remodeling under physiological and expansion conditions. Thus, we concluded that Gli1+ SuSCs play essential roles in suture and bone remodeling stimulated by mechanical force and that Wnt signaling is crucial to this process. © 2022 American Society for Bone and Mineral Research (ASBMR).

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Background Metabolism shifts from glycolysis to mitochondrial oxidative phosphorylation are vital during the differentiation of stem cells. Mitochondria have a direct function in differentiation. However, the metabolic shift and the effect of mitochondria in regulating the osteogenic differentiation of human dental pulp stem cells (hDPSCs) remain unclear. Methods Human dental pulp stem cells were collected from five healthy donors. Osteogenic differentiation was induced by osteogenic induction medium. The activities of alkaline phosphatase, hexokinase, pyruvate kinase, and lactate dehydrogenase were analyzed by enzymatic activity kits. The extracellular acidification rate and the mitochondrial oxygen consumption rate were measured. The mRNA levels of COL-1, ALP, TFAM, and NRF1 were analyzed. The protein levels of p-AMPK and AMPK were detected by western blotting. Results Glycolysis decreased after a slight increase, while mitochondrial oxidative phosphorylation continued to increase when cells grew in osteogenic induction medium. Therefore, the metabolism of differentiating cells switched to mitochondrial respiration. Next, inhibiting mitochondrial respiration with carbonyl cyanide-chlorophenylhydrazone, a mitochondrial uncoupler inhibited hDPSCs differentiation with less ALP activity and decreased ALP and COL-1 mRNA expression. Furthermore, mitochondrial uncoupling led to AMPK activation. 5-Aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, simulated the effect of mitochondrial uncoupling by inhibiting osteogenic differentiation, mitochondrial biogenesis, and mitochondrial morphology. Mitochondrial uncoupling and activation of AMPK depressed mitochondrial oxidative phosphorylation and inhibited differentiation, suggesting that they may serve as regulators to halt osteogenic differentiation from impaired mitochondrial oxidative phosphorylation.