Fisetin: An Integrated Approach to Identify a Strategy Promoting Osteogenesis

Abstract

Flavonoids may modulate the bone formation process. Among flavonoids, fisetin is known to counteract tumor growth, osteoarthritis, and rheumatoid arthritis. In addition, fisetin prevents inflammation-induced bone loss. In order to evaluate its favorable use in osteogenesis, we assayed fisetin supplementation in both in vitro and in vivo models and gathered information on nanoparticle-mediated delivery of fisetin in vitro and in a microfluidic system. Real-time RT-PCR, Western blotting, and nanoparticle synthesis were performed to evaluate the effects of fisetin in vitro, in the zebrafish model, and in ex vivo samples. Our results demonstrated that fisetin at 2.5 µM concentration promotes bone formation in vitro and mineralization in the zebrafish model. In addition, we found that fisetin stimulates osteoblast maturation in cell cultures obtained from cleidocranial dysplasia patients. Remarkably, PLGA nanoparticles increased fisetin stability and, consequently, its stimulating effects on RUNX2 and its downstream gene SP7 expression. Therefore, our findings demonstrated the positive effects of fisetin on osteogenesis and suggest that patients affected by skeletal diseases, both of genetic and metabolic origins, may actually benefit from fisetin supplementation.

Keywords: PLGA; differentiation; fisetin; mesenchymal stem cells; osteogenesis.

FIGURE 1

Fisetin supplementation did not affect MSC viability in a statistically significant manner. The cell viability was evaluated by performing the XTT test in hMSCs cultured for 7 days in the presence of fisetin ranging from 0 to 5 µM.

FIGURE 2

Effects of fisetin supplementation during osteogenic differentiation. The upregulation of osteogenic genes was much more evident when fisetin was supplemented at a concentration of 2.5 µM (A). Expression of RUNX2 in treated cells decreased after 7 days of differentiation (B). ROS levels were modulated by fisetin supplementation at the late differentiation phase (14 days of differentiation) (C). Analyses were performed in hMSCs cultured in the presence of the osteogenic differentiation medium. *p < 0.05; **p < 0.01; ***p < 0.005.

FIGURE 3

Effects of fisetin supplementation on the late phase of osteogenic differentiation. Fisetin increased the expression levels of osteogenic genes (A) and the mineral deposition (B) after 21 days of differentiation (mineralization phase) (a, control; b, 0.625 µM; c, 1.25 µM; d, 2.5 µM) *p < 0.05; **p < 0.001. Magnification ×4.

FIGURE 4

Fisetin, PEDF and AsA were able to upregulate osteogenic genes expression in MSCs after 7 (A) and 14 (B) days of supplementation. *p < 0.05; **p < 0.01; ***p < 0.005.

FIGURE 5

Fisetin effects on in vivo model. Fisetin supplementation increased osteogenic genes expression in zebrafish larvae after 7 (9 dpf) days of treatment (A). The fluorescence density produced by calcein staining showed an increased vertebrae area in larvae after 7 days of fisetin (B). Osteogenic genes were upregulated also in adult zebrafish supplemented with fisetin for 14 days (C). Fisetin supplementation for 14 days increased bone mineralization, evaluated by Alizarin red staining (ARS) in adult zebrafish (D). *p<0.05; **p<0.01; ***p<0.005.

FIGURE 6

RUNX2 expression was not affected by fisetin in CCD patients. However, fisetin supplementation strongly upregulated the expression of SPARC in control as well as in osteogenic differentiating cells of both RUNX2 mutant patients (normal control vs treated normal control, untreated patients' cells vs treated patients' cells respectively)) (D). p<0.05; **p<0.005. ND, Normal Donor; T-ND, Treated Normal Donor; P1, Patient 1; T-P1, Treated Patient 1; P2, Patient 2; T-P2, Treated Patient 2.

FIGURE 7

The graphics show absorption peaks of fisetin at 360 nm in culture medium at different times (in x axis): with the passage of time the molecule degrades and the peak is lowered, until it disappears later 6h. The image on the left (A) shows the entire duration of the run (from 0 to 8h time lapse in culture medium), while the one on the right (B) only around the retention time (from 4.5 to 7h). As control we used the colture medium (Medium) without fisetin supplementation. The degradation kinetics of fisetin is shown in graphic C.

FIGURE 8

Release test performed at different temperatures (4°C (red line) and 37°C (purple line) in PBS and in citric acid pH 5 (37°C (blue line) in a final volume of 1 ml. b. In-vitro release study in 100 ml of physiological solution that fits with a second grade polynomial function curve (red dots).

FIGURE 9

PLGA (Fis) are visible after 4 h of treatment in cultures and fisetin (in green, FITC) diffuses in intercellular spaces (A). However, after 6 h, fisetin is completely absorbed, and only cellular nuclei [blue, 4′,6–diamidino-2-fenilindolo diidrocloruro (DAPI) stained nuclei] are visible (B). After 7 days of osteogenic differentiation, PLGA (Fis) increased the expression of the osteogenic transcription factors RUNX2 and SP7 (C). COL1A1 chain levels increased in cells treated with PLGA (Fis) compared to control (D). *p < 0.05. Magnification × 40.