Abstract
Low-intensity pulsed ultrasound (LIPUS) has positive effects on osteogenic differentiation. However, the effect of LIPUS on osteogenic differentiation of human adipose-derived stem cells (hASCs) is unclear. In the present study, we investigated whether LIPUS could promote the proliferation and osteogenic differentiation of hASCs. hASCs were isolated and osteogenically induced with LIPUS stimulation at 20 and 30 mW cm−2 for 30 min day−1. Cell proliferation and osteogenic differentiation potential of hASCs were respectively analyzed by cell counting kit-8 assay, Alizarin Red S staining, real-time polymerase chain reaction, and Western blotting. The results indicated that LIPUS stimulation did not significantly affect the proliferation of hASCs, but significantly increased their alkaline phosphatase activity on day 6 of culture and markedly promoted the formation of mineralized nodules on day 21 of culture. The mRNA expression levels of runt-related transcription factor, osteopontin, and osteocalcin were significantly up-regulated by LIPUS stimulation. LIPUS stimulation did not affect the expression of heat shock protein (HSP) 27, HSP40, bone morphogenetic protein (BMP)-6 and BMP-9, but significantly up-regulated the protein levels of HSP70, HSP90, BMP-2, and BMP-7 in the hASCs. Further studies found that LIPUS increased the mRNA levels of Smad 1 and Smad 5, elevated the phosphorylation of Smad 1/5, and suppressed the expression of BMP antagonist Noggin. These findings indicated that LIPUS stimulation enhanced osteogenic differentiation of hASCs possibly through the up-regulation of HSP70 and HSP90 expression and activation of BMP signaling pathway. Therefore, LIPUS might have the potential to promote the repair of bone defect.
Highlights
Many studies have confirmed that mesenchymal stem cells (MSCs) have a strong potential to differentiate into different lineages, such as adipocytes, chondrocytes, osteoblasts, and other nonmesoderm types [1,2,3]
The results of flow cytometry showed that these human adipose-derived stem cell (hASC) at passage 3 expressed high levels of cell surface antigens of CD29 (92.61%), CD90 (94.23%), CD44 (90.05%), CD105 (84.25%), and CD29 (86.74%), but with low levels of CD106 (6.43%), CD31 (4.85%), CD34 (3.91%), CD45 (4.26%), and CD14 (4.51%) (Figure 2B), consistent with the mesenchymal stem cells obtained from bone marrow
The results indicated that Alkaline phosphatase (ALP) activity gradually increased through 10 days of culture regardless of Low-intensity pulsed ultrasound (LIPUS) stimulation, and it slightly decreased on day 12 and 14
Summary
Many studies have confirmed that mesenchymal stem cells (MSCs) have a strong potential to differentiate into different lineages, such as adipocytes, chondrocytes, osteoblasts, and other nonmesoderm types [1,2,3]. As a source of MSCs, human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering and regenerative medicine because adipose tissue can be obtained with less c 2018 The Author(s). As a form of mechanical energy, ultrasound stimulation belongs to a biophysical signal that can be conducted into a human body, and results in biochemical events at the cellular level [10]. It has been reported that LIPUS can stimulate fracture healing and has been used to treat bone defects in clinical therapy [14,15], and accelerates bone maturation in distraction osteogenesis cases in animal models [16]
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