Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo

Abstract

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin's influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.

Keywords: bone; bone morphogenetic protein; collagen; critical-sized defect; extracellular matrix; hesperidin; osteogenesis; regeneration.

Figure 1

MC3T3-E1 cell proliferation (A) and viability (B) upon treatment with hesperidin (H) at concentrations of 1, 5 (H5) and 100 (H100) μM show no significant differences (n = 3/group, p > 0.05).

Figure 2

Effect of hesperidin (H) on gene expression of osteogenic markers of MC3T3-E1 pre-osteoblast cells at 7 (A) and 14 (B) days as analyzed by qRT-PCR. Different superscript letters (a, b, c) indicate statistical difference among groups (p < 0.05). H, on day 7, at a dose of 1 µM (H1) showed consistent elevation of osteogenic markers compared to control. On day 14, both lower concentrations of H (1 and 5 µM) showed significantly more elevated Runx2, Osx and Bsp (n = 3/group).

Figure 3

Picrosirius red (PSR) staining of MC3T3-E1 cell-produced collagenous matrices after culture for 21 days with and without hesperidin (H) in the medium (1, 100 or 500 μM). Three independent experiments produced similar results and one representative polarized image of each condition is shown. The collagen matrices in H-treated samples maintained collagen at similar levels except for H500. H500 had reduced collagen accumulation and a significant decrease in red fibrils compared to other groups of treatment (red-color signal indicates presence of mature collagen) (** indicates difference compared to osteogenic medium alone at p < 0.05; *** indicates difference compared to osteogenic and other H groups at p < 0.05). Yellow fibrils (immature) increased in H500 (* indicates statistical difference compared to control). No green signal was detected in cultures (green depicts the least mature fibril, typical of early collagen deposits). All cells had similar viability as determined by CellTiter-Blue Cell Viability Assay (n = 3, 10X magnification).

Figure 4

Average FT-IR-derived parameters for control and treated samples demonstrate matrix composition and quality changes. Total protein was affected by hesperidin (H)1 (1 µM) and H5 (5 µM) doses (A) and collagen matrix amount was reduced significantly with H5 and H100 (100 µM) doses (C) (* represents statistical difference p < 0.05). Standard deviation or heterogeneity plots indicate the variation in matrix composition and quality within the sample. Parameters from H-treated samples were less heterogeneous (B,D,F,H–J), i.e., a more homogenous distribution of tissue components. Mineral-to-matrix ratio was significantly increased with higher doses of H (G).There were no changes in average mineral content among samples (E) (n = 3/group).

Figure 5

In vivo study of bone regeneration modulation by local delivery of bone morphogenetic protein 2 (BMP2) and hesperidin (H) in a rat mandible critical-sized defect (n = 4/group). Microcomputed tomography (µCT) 3D images (top row) show increased bone formation with use of a large dose of BMP2 (optimal dose of 1 µg of BMP2, optBMP) compared to a small dose (suboptimal dose of 100 ng of BMP2, subBMP). Different superscript letters on the top-right chart (BV, bone volume) indicate statistical difference among groups (p < 0.05). When subBMP was delivered with three different doses of H, a significantly greater amount of newly formed bone (NFB) was found for subBMP+ H1 (1 µM) and H100 (100 µM) compared to subBMP alone (3D and 2D µCT panels and BV graph). The 2D µCT view is a coronal cross-section of the 5-mm mandible defect (rat mandible depicted on bottom left with red arrow indicating where the 2D cross-section was taken). The 2D view provides additional understanding of the bone fill within the defect as compared to the 3D view, which is a median plane perspective that does not show the ectopic nature of optBMP. The NFB for H-treated samples was found to be within the constraints of the defect (µCT 2D panels B–D) as opposed to optBMP (A). BV for H100 alone was not statistically different from control subBMP (top-right bar graph). Bottom-right chart shows the effect of H on collagen organization of the NFB during BMP-induced bone formation. Polarized light images (20×, bar 200 µm) of PSR-stained NFB (bottom row) highlight differences in the organization/maturation of the bone matrix as there was an increase in more mature collagen (red fibrils) in H-treated samples with lower doses of H (B and C) (* in bar graph indicates statistical difference in red fibril percentage compared to optBMP, p < 0.05). SubBMP and H100 PSR alone were not quantified due to the limited amount of NFB. (n = 4 images/animal).