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

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.