Insulin growth factor binding protein-3 enhances dental implant osseointegration against methylglyoxal-induced bone deterioration in a rat model

Abstract

Purpose: The aim of this study was to determine the effect of insulin growth factor binding protein-3 (IGFBP-3) on the inhibition of glucose oxidative stress and promotion of bone formation near the implant site in a rat model of methylglyoxal (MGO)-induced bone loss.

Methods: An in vitro study was performed in MC3T3 E1 cells treated with chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 cDNA followed by MGO. An in vivo study was conducted in a rat model induced by MGO administration after the insertion of a dental implant coated with IGFBP-3.

Results: MGO treatment downregulated molecules involved in osteogenic differentiation and bone formation in MC3T3 E1 cells and influenced the bone mineral density and bone volume of the femur and alveolar bone. In contrast, IGFBP-3 inhibited oxidative stress and inflammation and enhanced osteogenesis in MGO-treated MC3T3 E1 cells. In addition, IGFBP-3 promoted bone formation by reducing inflammatory proteins in MGO-administered rats. The application of Ch-GNPs conjugated with IGFBP-3 as a coating of titanium implants enhanced osteogenesis and the osseointegration of dental implants.

Conclusions: This study demonstrated that IGFBP-3 could be applied as a therapeutic component in dental implants to promote the osseointegration of dental implants in patients with diabetes, which affects MGO levels.

Keywords: Antioxidants; Bone formation; Diabetes mellitus; Inflammation; MC3T3 E1.

Figure 1. MGO impairs the function of bone formation in MC3T3 E1 cells. (A) Alizarin red staining after treatment of MC3T3 E1 cells with MGO for 3, 6, 9, 12, and 15 days. (B) Effects of MGO on ALP activity in MC3T3 E1 cells at 3, 6, 9, 12, and 15 days. MC3T3 E1 cells were treated with OM with 5 mM β-glycerol phosphate, 100 μM ascorbic acid, and 10 nM dexamethasone for the indicated times and ALP was measured. (C) Protein expression of BMP-2, BMP-7, RANKL, and OPG analyzed by western blots after treatment with MGO. (D) Effects of MGO on the expression of inflammatory proteins and anti-oxidant enzymes analyzed by western blots. (E) Expression of IGFBP-3 in MC3T3 E1 cells induced with or without MGO. Each value was reported as the mean±standard deviation of 3 experiments.

MGO: methylglyoxal, ALP: alkaline phosphatase, BMP: bone morphogenetic protein, OM: osteogenic medium, RANKL: receptor activator of nuclear factor-κB ligand, OPG: osteoprotegerin, IGFBP-3: insulin growth factor binding protein-3. ^*P<0.05.

Figure 2. IGFBP-3 increased bone formation in MGO-treated MC3T3 E1 cells. (A) Analysis of osteogenic differentiation in response to Ch-GNPs/IGFBP-3 in MC3T3 E1 cells induced with MGO and measurement of calcium deposition for matrix mineralization by Alizarin red staining. (B) Measurement of ALP activity by Ch-GNPs/IGFBP-3 in MC-3T3 E1 cells induced with MGO. (C) Analysis of protein expression of IGFBP-3 by western blots in MC3T3 E1 cells after treatment with Ch-GNPs/IGFBP-3 and induction with MGO. (D) Expression of osteogenic-related proteins (BMP-2, BMP-7, and OPG) and an osteoclastic protein (RANKL). (E) Expression of inflammatory (TNF-α, IL-6, and RAGE) and anti-oxidant proteins. (F) Measurement of ROS formation by the Muse oxidative stress assay. (G) Graphical representation of ROS formation. Each value was reported as the mean±standard deviation of 3 experiments.

IGFBP-3: insulin growth factor binding protein-3, MGO: methylglyoxal, Ch-GNP: chitosan gold nanoparticle, MGO: methylglyoxal, BMP: bone morphogenetic protein, OPG: osteoprotegerin, RANKL: receptor activator of nuclear factor-κB ligand, TNF: tumor necrosis factor, IL: interleukin, RAGE: receptor for advanced glycation end products, ROS: reactive oxygen species. ^*P<0.05.

Figure 3. MGO impairs femur and alveolar bone formation in MGO-administered rats. (A) μCT (2-dimensional) imaging of rat femur after the administration of MGO (75 mg/kg) for 3 and 6 weeks. Analysis of femoral cortical BMD, trabecular BMD, BV/TV, trabecular number, thickness, and space. (B) Detection of changes in rat alveolar bone by 2-dimensonal μCT after the administration of MGO and analysis of mandible BMD and BV/TV. M1: the mandibular first molar; M: mesial root; B: buccal root; D: distal root; L: lingual root; and T1 and T2: the 2 parallel horizontal planes passing through the alveolar ridge and the apex of the buccal muscle separately. (C) H&E staining of rat mandible with or without the administration of MGO. Data are expressed as mean±SEM at 3 and 6 weeks (n=6).

MGO: methylglyoxal, μCT, micro-computed tomography, BMD: bone mineral density, BV: bone volume, TV: total volume, H&E: hematoxylin and eosin, SEM: standard error of the mean. ^*P<0.05.

Figure 4. IGFBP-3 restores MGO-induced bone deterioration in MGO-administered rats. (A) Two-dimensional μCT analysis of Ch-GNP/LacZ- and Ch-GNs/IGFBP-3-coated implants 3 and 6 weeks after the administration of MGO. (B) Three-dimensional μCT analysis of Ch-GNP/LacZ- and Ch-GNPs/IGFBP-3-coated implants. (C) Analysis of BMD and BV/TV. (D) H&E staining of the mandible around the implant surface 3 and 6 weeks after implantation. Data are expressed as mean ± SEM at 3 and 6 weeks (n=6).

IGFBP-3: insulin growth factor binding protein-3, MGO: methylglyoxal, μCT, micro-computed tomography, Ch-GNP: chitosan gold nanoparticle, BMD: bone mineral density, BV: bone volume, TV: total volume, H&E: hematoxylin and eosin, SEM: standard error of the mean. ^*P<0.05.

Figure 5. Ch-GNP/IGFBP-3-coated implants inhibit inflammatory proteins in MGO-administered rats. (A, B) Analysis of IGFBP-3, TNF-α, IL-6, and RAGE by IHC staining after 3 and 6 weeks. The intensity specific for the brown color correlates with the protein level.

Ch-GNP: chitosan gold nanoparticle, IGFBP-3: insulin growth factor binding protein-3, MGO: methylglyoxal, TNF: tumor necrosis factor, IL: interleukin, RAGE: receptor for advanced glycation end products, IHC: immunohistochemistry. ^*P<0.05.

Figure 6. Ch-GNP/IGFBP-3-coated implants increase osteogenic differentiation in MGO-administered rats. (A, B) IHC staining of BMP-2, BMP-7, OPG, and RANKL for the detection of osteoblast differentiation. The intensity specific for the brown color correlates with the protein level.

Ch-GNP: chitosan gold nanoparticle, IGFBP-3: insulin growth factor binding protein-3, MGO: methylglyoxal, IHC: immunohistochemistry, BMP: bone morphogenetic protein, OPG: osteoprotegerin, RANKL: receptor activator of nuclear factor-κB ligand. ^*P<0.05.