Critical role of cellular communication network factor 1 in early osteogenesis of mesenchymal stem cells primed by low‐intensity pulsed ultrasound and bone morphogenetic protein 2

Abstract There is a known reciprocal inhibitory relationship between adipogenesis and osteogenesis in mesenchymal stem cells (MSCs). Our previous study showed that low‐intensity pulsed ultrasound (LIPUS) did not promote osteogenesis of a murine MSC line C3H10T1/2, although LIPUS treatment suppressed adipogenesis. Therefore, we investigated the effect of a combination of an osteoinductive factor such as bone morphogenic protein‐2 (BMP‐2) and LIPUS treatment on osteogenesis. This combination significantly upregulated the gene expressions of BMP receptors and RUNX2, which is a key early osteogenic transcription factor. Interestingly, cellular communication network factor 1 (CCN1) was significantly increased at the mRNA and protein levels. Additionally, Runx2 expression was increased by a recombinant CCN1 protein and decreased in CRISPR‐Cas9‐generated Ccn1 knockout (KO) cells treated with the combination of LIPUS treatment and BMP‐2. Moreover, Runx2 expression was recovered through the transfection of a Ccn1 expression plasmid into Ccn1 KO cells. These findings indicate that treatment with the combination of LIPUS treatment and BMP‐2 promoted CCN1 production, and the CCN1 regulates the Runx2 expression in C3H10T1/2 cells, thus suggesting that CCN1 induced by mechanical stimulation and an osteoinductive factor plays a pivotal role in osteogenesis of MSCs.