Int J Stem Cells.  2023 Nov;16(4):406-414. 10.15283/ijsc23027.

Low-Intensity Pulsed Ultrasound Promotes BMP9 Induced Osteoblastic Differentiation in Rat Dedifferentiated Fat Cells

• Setoguchi F
• Sena K
• Noguchi K

• KMID: 2548004
• DOI: http://doi.org/10.15283/ijsc23027

Abstract

Dedifferentiated fat cells (DFATs) isolated from mature adipocytes have a multilineage differentiation capacity similar to mesenchymal stem cells and are considered as promising source of cells for tissue engineering. Bone morphogenetic protein 9 (BMP9) and low-intensity pulsed ultrasound (LIPUS) have been reported to stimulate bone formation both in vitro and in vivo. However, the combined effect of BMP9 and LIPUS on osteoblastic differentiation of DFATs has not been studied. After preparing DFATs from mature adipose tissue from rats, DFATs were treated with different doses of BMP9 and/or LIPUS. The effects on osteoblastic differentiation were assessed by changes in alkaline phosphatase (ALP) activity, mineralization/calcium deposition, and expression of bone related genes; Runx2, osterix, osteopontin. No significant differences for ALP activity, mineralization deposition, as well as expression for bone related genes were observed by LIPUS treatment alone while treatment with BMP9 induced osteoblastic differentiation of DFATs in a dose dependent manner. Further, co-treatment with BMP9 and LIPUS significantly increased osteoblastic differentiation of DFATs compared to those treated with BMP9 alone. In addition, upregulation for BMP9-receptor genes was observed by LIPUS treatment. Indomethacin, an inhibitor of prostaglandin synthesis, significantly inhibited the synergistic effect of BMP9 and LIPUS co-stimulation on osteoblastic differentiation of DFATs. LIPUS promotes BMP9 induced osteoblastic differentiation of DFATs in vitro and prostaglandins may be involved in this mechanism.

Keyword

Dedifferentiated fat cells; Bone morphogenetic protein 9; Low-intensity pulsed ultrasound; Prostaglandins

Figure

• 

  Fig. 1 ALP activity of DFATs stimulated with BMP9 and/or LIPUS. DFATs were seeded at a density of 2×104 cells/cm2 in CM. After 24 h of incubation, cells were cultured with BMP9 (0∼100 ng/ml) for 6 days in ODM. LIPUS treated cells were exposed to LIPUS for 20 minutes every day. Enhanced ALP activity by LIPUS treatment was observed in DFATs with BMP9 concentrations at 10 ng/ml or higher. Data represent means±SD (n=4). *p<0.05 vs. LIPUS (-) 100 ng/ml. #p<0.05 vs. LIPUS (-) 10 ng/ml.

• 

  Fig. 2 Mineralization of DFATs treated with BMP9 and/or LIPUS. (A) Alizarin red S staining. (B) Quantification of Alizarin red S staining. The cells were cultured with BMP9 (0∼100 ng/ml) for 21 days in ODM. LIPUS treated cells were exposed to LIPUS for 20 minutes every day. Intense Alizarin red S staining was observed in DFATs treated with BMP9 concentrations at 100 ng/ml and higher amount of stain was noted by co-treatment with LIPUS. Values are shown as mean±SD (n=4). *p<0.05 vs. LIPUS (-) 100 ng/ml.

• 

  Fig. 3 mRNA expression of bone-related genes in DFATs treated with BMP9 and/or LIPUS. (A∼C) The effects of BMP9 and/or LIPUS treatment on the expression of bone-related genes (Runx2, Osx, Opn) were analyzed by quantitative real-time PCR. The cells were cultured with BMP9 (0∼100 ng/ml) for 2 days (A, B) and 6 days (C) in ODM. LIPUS treated cells were exposed to LIPUS for 20 minutes every day. Values are shown as mean±SD (n=3). **p<0.01 vs. LIPUS (-) 100 ng/ml.

• 

  Fig. 4 Expression of BMP9-related receptor genes in DFATs treated with BMP9 and/or LIPUS. (A∼D) The expression of (A) ALK-1, (B) ALK-2, (C) BMP receptor II and (D) Endoglin in DFATs analyzed by real-time PCR. The cells were cultured with BMP9 (100 ng/ml) for 2 days in ODM. LIPUS treated cells were exposed to LIPUS for 20 minutes every day. Values are shown as mean±SD (n=4). *p<0.05, **p<0.01.

• 

  Fig. 5 Effects of indomethacin on ALP activity and mRNA levels of bone-related genes in DFATs treated with BMP9 and/or LIPUS. (A) The effect of indomethacin on ALP activity. (B∼D) The effect of indomethacin on expression of bone-related genes; (B) Runx2, (C) Osx, (D) Opn. DFATs were cultured with BMP9 (100 ng/ml) and indomethacin (1 μM) for 2 days (A∼C) and 6 days (D) in ODM. LIPUS treated cells were exposed to LIPUS for 20 minutes every day. Values are shown as mean±SD (n=4). **p<0.01.

• 

  Fig. 6 Release of PGE2 from DFATs treated with BMP9 and/or LIPUS and effect of indomethacin. DFATs were stimulated with BMP9 and/or LIPUS in ODM. Some samples were treated with indomethacin. The cells were cultured with BMP9 (100 ng/ml) and indomethacin (1 μM) for 24 h in ODM. LIPUS treated cells were exposed to LIPUS for 20 minutes every day. Values are shown as mean±SD (n=4). **p<0.01.

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