The role of hesperetin on osteogenesis of human mesenchymal stem cells and its function in bone regeneration

Abstract

Hesperetin has been suggested to be involved in bone strength. We aimed to investigate the effects of hesperetin on the osteogenic differentiation of human mesenchymal stem cells and its related mechanisms. We showed that hesperetin promoted osteogenic differentiation of human mesenchymal stem cells in vitro. It potentially exerts its effects via the ERK and Smad signaling pathways. Using a rat osteotomy model, we showed that human mesenchymal stem cells combined with a hesperetin/gelatin sponge scaffold resulted in accelerated fracture healing in vivo. Due to the low cost of hesperetin, it could be used as a growth factor for bone tissue engineering or surgical fracture treatment.

Keywords: bone regeneration; gelatin sponge scaffold; hesperetin; mesenchymal stem cells; osteogenesis.

Figure 1. The effect of different concentrations of hesperetin on hMSC expansion

*p < 0.05 vs. the control group. ^#p < 0.05 vs. the lower concentrations (0.1 and 1 μM).

Figure 2. The effects of hesperetin on hMSC migration

(A) Control group without hesperetin in the lower chamber. (B) Hesperetin group with hesperetin (1 μM) in the lower chamber. (C) Hesperetin (1 μM) co-treatment with the p38 inhibitor SB203580 in the lower chamber. Cells were pretreated with SB203580. (D) The number of cells located on the underside of the membrane. *p < 0.05.

Figure 3

The gene expression levels of ALP, COL1A1, OCN, and Runx2 in the control and hesperetin (1 μM) groups on days 3 and 9 (A–D). The hMSCs were stained with alizarin red S on day 14 (E–F). *p < 0.05 vs. the control group.

Figure 4

Immunofluorescence of type I collagen, runt-related transcription factor 2 (RUNX2), phosphorylated ERK (p-ERK), and phosphorylated Smad1/5/8 (p-Smad1/5/8) in hMSCs after 24-h culture in OIM with or without hesperetin (1 μM).

Figure 5

(A) hMSCs were cultured in OIM, OIM + hesperetin, OIM + hesperetin + U0126 or OIM + U0126, after which the expressions of p-ERK and COL1A1 were detected using Western blotting. (B) Protein expression levels were normalized to GAPDH. *, ^#p < 0.05 vs. the control OIM group. (C–E) hMSCs were cultured in OIM, OIM + hesperetin, or OIM + hesperetin + U0126 and stained with alizarin red S (ARS) on day 9. (F) Mineralization was quantified with extraction of ARS-stained cells using 10% cetylpyridinium chloride (CPC). *p < 0.05 vs. OIM, ^#p < 0.05 vs. OIM + hesperetin + U0126.

Figure 6

(A) hMSCs were cultured in OIM, OIM + hesperetin, OIM + hesperetin + LDN-193189 or OIM + LDN-193189, after which the expression of phosphorylated Smad1/5/8 (p-Smad1/5/8) and COL1A1 were detected by Western blotting. (B) Protein expression levels were normalized to GAPDH. *, ^#p < 0.05 vs. the control OIM group. (C–E) hMSCs were cultured in OIM, OIM + hesperetin, or OIM + hesperetin + LDN-193189 and stained with alizarin red S (ARS) on day 9. (f) Mineralization was quantified by the extraction of ARS-stained cells using 10% cetylpyridinium chloride (CPC). *p < 0.05 vs. OIM, ^#p < 0.05 vs. OIM + hesperetin + LDN-193189.

Figure 7

(A) Disc-shaped gelatin sponge scaffolds. (B) The DNA content of MSCs in the gelatin sponge and hesperetin/gelatin sponge scaffolds after 0, 3, 6, and 9 days of culture. Each scaffold was seeded with 100 μL of cells to achieve a density of 1 × 10⁶ cells/scaffold. *p < 0.05 between the hesperetin/gelatin and gelatin sponge scaffolds at the same time point.

Figure 8

(A–D) Radiographic images of osteotomized rat tibias 8 weeks post-operatively. (E–L) Hematoxylin and eosin and Safranin-O staining of osteotomized rat tibias 8 weeks post-operatively. Staining was evaluated at ×50 magnification.

Figure 9. Radiographic scores of fracture sites, *P < 0.05 vs

the control and gelatin groups, ^#P < 0.05 vs. the hesperetin/gelatin group.