Effect of Flavonoid Supplementation on Alveolar Bone Healing-A Randomized Pilot Trial

Abstract

We investigated the effects of two common dietary supplements on bone healing in dental extraction sockets in humans. In this randomized pilot trial, male subjects took Grape Seed Extract [GSE] or Grapefruit Extract [GFE] starting two weeks prior to dental extraction and maintained this regimen for sixty days after surgery. Extraction sockets were filled with a collagen plug. After 24 h, a socket sample was collected and processed for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and an 84-gene wound healing assay. Sixty days after tooth extraction, a core of newly formed bone was obtained prior to dental implant placement and processed for histology. qRT-PCR revealed that GFE led to a significant decrease in platelet-derived growth factor and interleukin (IL)1-β compared to GSE, and a significant decrease in IL-6 and CXCL2 compared to control. GSE led to a significant increase in coagulation factor Von Willebrand and inflammatory marker IL1-β compared to GFE. WISP1 and CXCL5 were upregulated in both groups. Overall, GFE showed a downregulation of inflammation and GSE led to a decrease in collagen density and increased osteoclasts. This pilot trial highlights the need for further investigation on the mechanism of action of such supplements on bone healing and oral health.

Keywords: collagen; dietary supplements; flavonoid; osteoclast; phytochemicals; tooth socket.

Figure 1

CONSORT flow diagram of enrollment, randomization, and treatment. None of the allocated patients withdrew from the study.

Figure 2

Clinical figures representative of alveolar sockets and sample collection in the study. (A) Extraction socket. (B) Collagen sponge in socket. Sponges were collected after 24 h for RNA extraction and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis. (C,D) Central core removed via trephine bur 60 days post-extraction for histological analyses.

Figure 3

qRT-PCR results. No differences found for transforming growth factor TGF-β1 (A) and vascular endothelial growth factor (VEGFA) (B). Grapefruit extract (GFE) led to a significant decrease in the growth factor PDGF (C), and inflammatory marker IL1-β (D) compared to grape seed extract (GSE), and a significant decrease in inflammatory markers IL-6 (E) and CXCL2 (F) compared to control. GSE led to a significant increase in the coagulation von Willebrand factor (VWF) (G), and inflammatory marker IL1-β (D) compared to GFE. (H) Wound healing PCR array highlighted results. Statistically significant genes indicated by * (p < 0.05). Standard text indicates upregulated and bold text indicates downregulated. Bars are SDs.

Figure 4

Representative H&E samples at 40× magnification. (A) Control group, (B) GSE treatment, and (C) GFE treatment. Scale bar indicates 1 mm. Histomorphometric analysis: Whole tissue masks (D) and bone area masks (E) were generated using Adobe^® Photoshop^® software. Bone area and tissue area were calculated using ImageJ software. (F) Average percent bone area. GSE treatment led to significantly reduced bone area compared to control and GFE groups. Statistically significant results indicated by * (p < 0.05). Bars are SDs.

Figure 5

Representative picrosirius red (PSR) samples at 40× magnification (top row), and 200× magnification (middle row). Scale bars indicate 300 μm. Qualitative analysis revealed control (A,D) had less evidence of new woven bone than GSE samples (B,E). GFE samples (C,F) also exhibited a clear pattern of woven bone compared to control group, but not to the extent of GSE treated samples. Control and GFE samples appeared to have more mature lamellar bone in both loose and densely packed structures. Quantitatively, GSE treatment (middle column) led to significantly decreased yellow and green signals, and significantly increased red signal compared to the control group (first column) (G). Statistically significant results indicated by * (p < 0.05). Bars are SDs.

Figure 6

Osteoclast quantification. Sample images of control (A), GSE (B), and GFE (C) shown at 200× magnification. Positive tartrate-resistant acid phosphatase (TRAP) stained cells (red) with at least three nuclei were counted as an osteoclast for the entire sample and normalized to total bone area (D). GSE treatment led to a significant increase in osteoclast density compared to control and GFE groups (D) Statistically significant results indicated by * (p < 0.05). Bars are SDs.