Journal of Periodontology; Copyright 2015
DOI: 10.1902/jop.2015.150083
Osteocytic Sclerostin Expression in Alveolar Bone in Diabetic Rats With Ligature Induced-Periodontitis Ji-Hye Kim*§, Dong-Eun Lee*§, Gye-Hyeong Woo†, Jeong-Heon Cha*‡§, Eun-Jung Bak* and Yun-Jung Yoo*‡ * Department of Oral Biology, Yonsei University College of Dentistry, Seoul, Republic of Korea. † Department of Clinical Science, Semyung University, Jecheon, Republic of Korea. ‡ Department of Applied Life Science, The Graduate School, Yonsei University, Seoul, Republic of Korea. § BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Republic of Korea. Background: Osteocytic sclerostin inhibits bone formation and its expression is stimulated by tumor necrosis factor (TNF)-α. We investigated sclerostin and TNF-α expression in diabetic rats with periodontitis. Methods: Rats were divided into control (C), periodontitis (P), and diabetes + periodontitis (DP) groups. After induction of diabetes by streptozotocin, periodontitis was induced by ligature. At day 0 (control) and at days 3 and 20 after induction of periodontitis, alveolar bone, osteoclasts, osteoid, and TNF-α and sclerostin expression were evaluated. Results: The cementoenamel junction-alveolar bone crest distance of the DP group was longer than that of the P group at day 20 after induction of periodontitis, but the number of osteoclasts was not different. Osteoid area decreased in both the P and DP groups by day 3, but while sustained osteoid suppression was observed in the DP group at day 20, osteoid formation was increased in the P group. The number of sclerostinpositive osteocytes increased in both groups at day 3, but, the increased number of sclerostin-positive osteocytes was maintained only in the DP group through day 20. The number of TNF-α positive cells increased more in the DP group than in the P group. Conclusion: Enhanced alveolar bone loss, suppressed bone formation, and prevalent TNF-α expression were characteristic of the DP group compared to the P group. Suppressed bone formation in the DP was observed simultaneously with increased sclerostin and TNF-α expression. These results suggest that upregulated osteocytic sclerostin expression in periodontitis with diabetes may play a role in suppressed bone formation.
KEYWORDS: Periodontitis, Diabetes Mellitus, Osteogenesis, Sclerostin, Tumor Necrosis Factor-alpha
Bone tissue undergoes remodeling, a process that involves bone resorption by osteoclasts with an equivalent amount of new bone formation by osteoblasts 1, 2. Bone loss occurs by uncoupled remodeling, during which the amount of bone resorption exceeds the amount of new bone formation 1, 2. Various factors regulate bone resorption and formation 3. Sclerostin (SOST), expressed by osteocytes, is an inhibitor of bone formation 4. Sclerostin suppresses osteoblast differentiation by interrupting Wnt signaling through binding to the Wnt coreceptor, low-density lipoprotein receptor-related protein-5/6 (LRP5/6) 4. A role for sclerostin in bone loss has been indicated through the observation of sclerostin deficiency in sclerosteosis patients and the high bone mass phenotype characterizing SOST knockout mice 4-6 . Additionally, increased sclerostin expression and restored bone formation after treatment with anti-sclerostin antibody in postmenopausal women and in animal model suggest that sclerostin inhibition may be a viable approach for developing novel anabolic agents for diseases characterized by bone loss 7-9.
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DOI: 10.1902/jop.2015.150083
Alveolar bone loss is a main characteristic of periodontitis. We previously demonstrated increased osteocytic sclerostin expression during suppressed bone formation in rats with ligature–induced periodontitis, suggesting that osteocytes may be a relevant source of sclerostin during periodontitis-induced alveolar bone loss 10. Another group using ligatureinduced periodontitis model reported that treatment with anti-sclerostin antibody restored alveolar bone mass, providing the rationale for the clinical investigation of sclerostin inhibitors in periodontitis 11. Knowledge about the relationship between periodontitis and diabetes has been increasing over the last decade. Several studies have reported that prevalence and severity of periodontal disease are increased in patients with type 1 and type 2 diabetes, suggesting that enhanced inflammation caused by diabetes may be related to the pathogenesis of periodontitis 12-16. Periodontitis patients exhibit increased levels of interleukin (IL)-1 and tumor necrosis factor (TNF)-α, two pro-inflammatory cytokines, in gingiva and crevicular fluid 17, 18. Increases in TNF-α and IL-1β have been observed in periodontitis patients with type 1 diabetes 18. These inflammatory cytokines are also increased in type 2 diabetic and STZ-induced diabetic rats with periodontitis when compared to non-diabetic rats with periodontitis 18-20. It has also been reported that enhanced TNF-α in type 2 diabetic rats contributes to deficient bone formation by suppressing the expression of bone formation inducing factors 21. Increased sclerostin expression has been observed both in vitro in TNF-α-treated osteocytes and in vivo in the osteocytes of obese mice with bone loss and increased TNF-α expression 22. Together, these results suggest that TNF-α induces osteocytic sclerostin expression and that altered osteocytic sclerostin expression by pro-inflammatory cytokines may play a role in the aggravation of periodontitis-induced alveolar bone loss in diabetes. Although previous studies have demonstrated how diabetes can negatively affect bone formation in periodontitis through enhanced inflammation 21, 23, 24, the potential role of sclerostin in this context remains uninvestigated. Therefore, in this study, we characterized sclerostin and TNF-α expression in diabetic rats with periodontitis.
MATERIALS AND METHODS Animal Models All animal protocols were approved by the Institutional Animal Care and Use Committee of Yonsei University (2012-0020-2). Six-week-old male F344 rats were purchased‖ and adapted for 1 week. Rats were then divided into three groups; control (C), periodontitis (P), and diabetes + periodontitis (DP) (N=6–8/each group/each time point). Diabetes was induced via intravenous administration of streptozotocin¶ (50 mg/kg). One week later, experimental periodontitis was generated by bilateral ligature on the gingival sulcus of the mandibular first molars. C group was sacrificed before ligature (day 0) and P and DP groups were sacrificed at days 3 and 20 after ligature. During the experiments, body weight and fasting glucose levels were measured at each time point. Fasting blood glucose levels were more than 350 mg/dl in diabetic rats and less than 100 mg/dl in control rats. Alveolar Bone Loss The mandibles were removed to perform histomorphometric examination at day 0 and at days 3 and 20 after ligature. Mandibles were fixed in 10% neutral-buffered formalin# overnight and decalcified in 10% EDTA# for 2 months. After embedding in paraffin, sections were cut at a thickness of 4 µm. Sections were then placed on slides and stained with hematoxylin and eosin (H&E). Alveolar bone loss in the distal area of the first molar was examined by
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measuring the cementoenamel junction (CEJ) to alveolar bone crest (ABC) distance using a software program**, using slides at 10× magnification. Osteoid Formation The osteoid area in distal area of first molar was measured to examine new bone formation. Osteoid formation was measured as the area of unmineralized bone matrix (light pink color) between osteoblasts (cuboidal bone-lining cells) and the mineralized bone surface, which was stained a dark pink color 10. The osteoid area, measured in a region of interest (ROI) that extended 0.5 mm from ABC in the distal area of the first molar, was examined under 20× magnification using a software program**. Osteoclast Formation To identify osteoclasts, we performed a tartrate-resistant acid phosphatase (TRAP) assay following the manufacturer's instructions††. The number of osteoclasts, defined as TRAPpositive multinucleated cells, was counted along the alveolar bone surface in a ROI that extended 0.5 mm from ABC in the distal area, using light microscopy at 20× magnification‡‡. The number of osteoclasts was calculated per millimeter length of alveolar bone surface. Immunohistochemistry for Sclerostin, TNF-α, and IL-1β For immunohistochemistry (IHC) staining, bone sections were quenched in 3% H2O2# for 20 min to inhibit endogenous peroxidase, and then incubated with trypsin§§ for antigen retrival. The commercial IHC kits ‖‖,¶¶ were used and all procedures were performed following the manufacturer's instructions. Briefly, after pre-incubating sections with normal horse blocking reagent for 20 min, sections were incubated overnight at 4°C with goat anti-sclerostin antibody## (1:300 dilution), rabbit anti-TNF-α antibody*** (1:400 dilution), and rabbit anti-IL1β antibody††† (1:75 dilution). Sections stained for sclerostin were then further incubated with biotinylated link antibody and peroxidase-labeled streptavidin. Sections stained for TNF-α and IL-1β were incubated with anti-mouse and anti-rabbit IgG conjugated horseradish peroxidase microploymer, respectively. The color was developed with substrate-chromogen, with methyl green as the counterstain. Negative controls were created by omitting the primary antibody. Sclerostin-positive osteocytes were counted in the ROI, which was extended 0.5 mm from ABC in the distal area of alveolar bone, using light microscopy at 40× magnification. Osteocytes were counted per bone area. TNF-α and IL-1β positive cells were counted in a standardized site (0.2 mm × 0.2 mm) located under the gingival epithelium. Statistical Analysis All statistical analyses were performed using a statistical analysis program‡‡‡. One-way analysis of variance (ANOVA) and Student's t-tests were used to determine significant differences. A p-value < 0.05 was considered statistically significant. Data are expressed as the mean ± standard error (SEM).
RESULTS 1. Alveolar Bone Loss in Diabetic Rats With Periodontitis To estimate how diabetes affects alveolar bone loss in periodontitis, we measured the CEJ-ABC distance in the distal area of the first molar (Fig 1A). In the P and DP groups, the increased CEJ-ABC distance was observed at days 3 and 20 post-periodontitis induction (via ligature) compared to the C group (Fig 1B). At day 20, the CEJ-ABC distance in the DP 3
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group was increased compared to that of the P group, and also tend to be increased compared to day 3. 2. Osteoclast Formation in Diabetic Rats With Periodontitis To investigate the effect of diabetes on periodontitis-induced bone resorption, we counted TRAP-positive multinucleated osteoclasts in the distal area (Fig 2A). At days 3 and 20 post-ligature, the number of osteoclasts in the P and DP groups significantly increased compared to the C group (Fig 2B). However, there was no significant difference between the number of osteoclasts detected in the P and DP groups during the entire experiment period. 3. Bone Formation in Diabetic Rats With Periodontitis To investigate the effect of diabetes on bone formation in periodontitis, the osteoid area was measured in the distal area (Fig 3A). At day 3 post-ligature, a reduced osteoid area was observed in both the P and DP groups (Fig 3B). The decreased osteoid area was maintained up to day 20 in the DP group; however, the osteoid area in the P group at day 20 was remarkably increased (approximately 3.3-fold) compared to that of the C group, and was notably higher than that of the DP group. 4. Osteocytic Sclerostin Expression in Diabetic Rats With Periodontitis To investigate the effect of diabetes on sclerostin expression, the number of sclerostin-positive osteocytes was counted (Fig 4A). Sclerostin-positive osteocytes in the P and DP groups markedly increased at day 3 post-ligature compared to the C group (Fig 4B). Interestingly, the number of sclerostin-positive osteocytes in the P group at day 20 decreased to control levels; however, the increased number of sclerostin-positive osteocytes in the DP group was maintained through day 20. 5. TNF-α and IL-1β Expression in Diabetic Rats With Periodontitis To investigate the effect of diabetes on the expression of pro-inflammatory cytokines in periodontitis, we counted the number of TNF-α and IL-1β positive cells that infiltrated the connective tissue beneath the gingival epithelium. The number of TNF-α positive cells increased in both the P and DP groups compared to the C group at days 3 and 20 postligature; additionally, there were more TNF-α positive cells in the DP group than in the P group (Fig. 5A). The quantity of IL-1β positive cells also increased in the P and DP groups than in the C group, with higher levels in the DP group than in the P group at day 20 postligature (Fig. 5B).
DISCUSSION In this study, alveolar bone loss similarly increased at day 3 post-ligature in the P and DP groups. Consistent with our previous study 10, alveolar bone loss in the P group did not further increase at day 20 post-ligature. However, the alveolar bone loss observed in the DP group more increased than that observed in the P group at day 20, and the increase was continual from ligature to day 20. These results indicate that diabetes exacerbates alveolar bone loss and could sustain severe uncoupled bone remodeling in periodontitis. Alveolar bone loss is typically determined using the degree of uncoupled bone remodeling, which is characterized by the imbalance of bone resorption and formation 1, 2. In both the P and DP groups, osteoclast numbers peaked at day 3 post-ligature and then gradually decreased. The difference in osteoclast numbers between these groups was not significant. Osteoclast formation in diabetes with periodontitis is a controversial topic, 4
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according to several reports 21, 23-26. Some groups have reported increased osteoclast formation in type 2 diabetic and STZ-induced diabetic rats with periodontitis 21, 23-25; however, He et al. did not observe increased osteoclast formation in type 2 diabetic mice infected with a periodontopathogen 26. Although the precise role of osteoclast formation in diabetes with periodontitis remains unclear, the association between diminished bone formation and exacerbated alveolar bone loss in type 2 diabetic and STZ-induced diabetic rats with periodontitis has been consistently reported 23, 24. In the current study, we also observed exacerbated alveolar bone loss through diminished bone formation in the DP group at day 20 post-ligature. These results suggest that diabetes more strongly affects bone formation than bone resorption. The pattern of sclerostin expression coincided with that of osteoid formation in both the P and DP groups. At day 3 post-ligature, when osteoid formation markedly decreased in both groups, sclerostin expression increased. At day 20 post-ligature, when osteoid formation in the P group notably increased, sclerostin expression decreased, which we also observed in our previous study 10. Interestingly, increased sclerostin expression in the DP group was sustained until day 20 post-ligature, at which time diminished bone formation was also observed. Additionally, clinical studies have demonstrated increased sclerostin levels in patients with type 2 diabetes and periodontitis 27, 28. In type 2 diabetes patients, high levels of serum sclerostin were observed, and were negatively associated with β-catenin, an essential Wnt pathway molecule involved in osteogenesis 27. In patients with chronic periodontitis and severe alveolar bone loss, gingival tissue and serum sclerostin levels were increased compared to patients without periodontitis 28. Taken together, these findings suggest that persistent and increased sclerostin expression in diabetic rats with periodontitis may contribute to diminished bone formation. In this study, prolonged increased TNF-α and IL-1β expression was observed in the DP group compared to the P group, a result that is consistent with observations in type 1 diabetes patients and type 2 diabetic and STZ-induced diabetic rats 18-20. When inflammation in type 2 diabetic rats with periodontitis was resolved by treatment with a TNF-α inhibitor, bone formation improved, and this group suggested that TNF-α limits bone formation by modulating the expression of several bone formation-stimulating factors, including transforming growth factor β-1, bone morphogenetic protein (BMP)-2, BMP-6, and fibroblast growth factor-2 21. Tanabe et al. reported that alkaline phosphatase activity and calcification in rat osteoblasts decreased upon treatment with IL-1α, which acts similarly to IL-1β 29. In IL-1 receptor antagonist knockout mice infected with Aggregatibacter actinomycetemcomitans, severe alveolar bone loss occurred, suggesting that high levels of IL-1α expression promote the down-regulation of osteoblast-related gene expression 30. Together, these studies indicate that TNF-α and IL-1β are involved in prolonged and decreased bone formation in diabetic rats with periodontitis. Recently, some groups reported that TNF-α is associated with osteocytic sclerostin expression in pathological bone loss caused by estrogen deficiency and obesity. One of these groups observed that treatment with a TNF-α blocker reversed the increased sclerostin expression in estrogen-deficient mice with osteoporosis 31. Additionally, the activity of myocyte enhancer factor 2, a sclerostin transcription factor, was increased by TNF-α, but not by IL-1, in UMR-106 osteosarcoma cells 31. A second group demonstrated that levels of osteocytic sclerostin and serum TNF-α were increased in obese mice with bone loss 22. Treatment with TNF-α increased sclerostin expression in MLO-Y4 osteocytes, suggesting that TNF-α-induced sclerostin expression contributes to bone loss in obesity 22. Considering these studies, we assume that in the diabetic rats with periodontitis, prolonged and prevalent TNF-α expression may play a role in sustained sclerostin expression. To fully elucidate the 5
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role of TNF-α in sclerostin expression in diabetic rats with periodontitis, further studies are needed, including those that can confirm the immunohistochemistry results observed in the current study and those using TNF-α inhibitors.
CONCLUSIONS The results described here show that prolonged and enhanced alveolar bone loss, suppressed bone formation, and increased TNF-α and IL-1β are observed in diabetic rats with periodontitis. Suppressed bone formation with concurrent increased in sclerostin and TNF-α were specifically observed in diabetic rats with periodontitis. These results suggest that up-regulated osteocytic sclerostin expression may play a role in the prolongation of suppressed bone formation in diabetic rats with periodontitis. This study is the first to show increased osteocytic sclerostin expression in periodontitis with concurrent diabetes. The data support the further clinical investigation of relationship between osteocytic sclerostin expression and diminished bone formation in diabetic patients with periodontitis. ACKNOWLEDGEMENT This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A6A3A01053590) (2014R1A2A1A11049412).
CONFLICT OF INTEREST STATEMENT The authors report no conflicts of interest related to this study.
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Correspondence: Yun-Jung Yoo, D.D.S., Ph.D. Department of Oral Biology, Yonsei University College of Dentistry, 134 Sinchon dong, Seodaemun-gu, Seoul 120-752, Republic of Korea, Tel: +82-2-2228-3060, Fax: +82-2-2227-7903, E-mail: [email protected], Eun-Jung Bak, D.V.M., Ph.D. Department of Oral Biology, Yonsei University College of Dentistry, 134 Sinchon dong, Seodaemun-gu, Seoul 120-752, Republic of Korea, Tel: +82-2-22283062, Fax: +82-2-2227-7903, E-mail: [email protected] Submitted February 04, 2015; accepted for publication March 30, 2015. Figure 1. Alveolar bone loss in each group. A) Representative images for the calculation of the CEJ-ABC distance are shown (H&E stain; scale bar = 100 µm [original magnification ×10]). B) Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used (* p < 0.05 compared with day 0. † p < 0.05). Figure 2. Osteoclast numbers in each group. A) Representative images for the calculation of the number of osteoclasts on the alveolar bone surface are shown (TRAP stain; scale bar = 100 µm [original magnification ×20]). B) Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used. (* p < 0.05 compared with day 0); n=number, OCs=osteoclasts, BS=alveolar bone surface. Figure 3. Osteoid area in each group. A) Representative images for the calculation of the osteoid area are shown (H&E stain; scale bar = 100 µm [original magnification ×20]). The osteoid (dotted line) was defined as the unmineralized bone matrix (light pink) between the osteoblasts and the mineralized bone surfaces. B) Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used (* p < 0.05 compared with day 0. † p < 0.05). Figure 4. Osteocytic sclerostin expression in each group. A) Representative images for calculating the number of sclerostin-positive osteocytes are shown (IHC stain; scale bar = 100 µm [original magnification ×40]). White arrowhead indicates representative negative osteocyte and black arrow head indicates representative positive osteocyte. B) Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used (* p < 0.05 compared with day 0. † p < 0.05); n=number. Figure 5. TNF-α and IL-1β expression in each group. The number of TNF-α (A) and IL-1β (B)-positive cells in each group is illustrated. Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used (* p < 0.05 compared with day 0. † p < 0.05); n=number.
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Key findings : Increased sclerostin expression was accompanied with reduced bone formation in diabetic rats with periodontitis. ‖ Oriental Bio, Gyeonggi-do, Korea ¶ Sigma Aldrich, St. Louis, MO, USA # Duksan, Gyeonggi-do, South of Korea ** Image-Pro software, Media Cybernetics, Silver Spring, MD, USA †† Acid Phosphatase, Leukocyte (TRAP), Sigma Aldrich, St. Louis, MO, USA ‡‡ Olympus, Tokyo, Japan §§ Invitrogen, Carlsbad, CA, USA ‖‖ LSAB+ system-HRP, Dako, Carpinteria, CA, USA ¶¶ ImmPRESS™ universal reagent, Vector Laboratories, Inc., Burlingame, CA, USA ## R&D System Inc, Minneapolis, MN, USA *** Abcam, Thousand Oaks, CA, USA ††† Santa Cruz Biotechnology, Santa Cruz, CA, USA ‡‡‡ SPSS 12.0, SPSS, Chicago, IL, USA
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Osteocytic Sclerostin Expression in Alveolar Bone in Diabetic Rats With Ligature Induced-Periodontitis Ji-Hye Kim*§, Dong-Eun Lee*§, Gye-Hyeong Woo†, Jeong-Heon Cha*‡§, Eun-Jung Bak* and Yun-Jung Yoo*‡ * Department of Oral Biology, Yonsei University College of Dentistry, Seoul, Republic of Korea. † Department of Clinical Science, Semyung University, Jecheon, Republic of Korea. ‡ Department of Applied Life Science, The Graduate School, Yonsei University, Seoul, Republic of Korea. § BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, Republic of Korea. Background: Osteocytic sclerostin inhibits bone formation and its expression is stimulated by tumor necrosis factor (TNF)-α. We investigated sclerostin and TNF-α expression in diabetic rats with periodontitis. Methods: Rats were divided into control (C), periodontitis (P), and diabetes + periodontitis (DP) groups. After induction of diabetes by streptozotocin, periodontitis was induced by ligature. At day 0 (control) and at days 3 and 20 after induction of periodontitis, alveolar bone, osteoclasts, osteoid, and TNF-α and sclerostin expression were evaluated. Results: The cementoenamel junction-alveolar bone crest distance of the DP group was longer than that of the P group at day 20 after induction of periodontitis, but the number of osteoclasts was not different. Osteoid area decreased in both the P and DP groups by day 3, but while sustained osteoid suppression was observed in the DP group at day 20, osteoid formation was increased in the P group. The number of sclerostinpositive osteocytes increased in both groups at day 3, but, the increased number of sclerostin-positive osteocytes was maintained only in the DP group through day 20. The number of TNF-α positive cells increased more in the DP group than in the P group. Conclusion: Enhanced alveolar bone loss, suppressed bone formation, and prevalent TNF-α expression were characteristic of the DP group compared to the P group. Suppressed bone formation in the DP was observed simultaneously with increased sclerostin and TNF-α expression. These results suggest that upregulated osteocytic sclerostin expression in periodontitis with diabetes may play a role in suppressed bone formation.
KEYWORDS: Periodontitis, Diabetes Mellitus, Osteogenesis, Sclerostin, Tumor Necrosis Factor-alpha
Bone tissue undergoes remodeling, a process that involves bone resorption by osteoclasts with an equivalent amount of new bone formation by osteoblasts 1, 2. Bone loss occurs by uncoupled remodeling, during which the amount of bone resorption exceeds the amount of new bone formation 1, 2. Various factors regulate bone resorption and formation 3. Sclerostin (SOST), expressed by osteocytes, is an inhibitor of bone formation 4. Sclerostin suppresses osteoblast differentiation by interrupting Wnt signaling through binding to the Wnt coreceptor, low-density lipoprotein receptor-related protein-5/6 (LRP5/6) 4. A role for sclerostin in bone loss has been indicated through the observation of sclerostin deficiency in sclerosteosis patients and the high bone mass phenotype characterizing SOST knockout mice 4-6 . Additionally, increased sclerostin expression and restored bone formation after treatment with anti-sclerostin antibody in postmenopausal women and in animal model suggest that sclerostin inhibition may be a viable approach for developing novel anabolic agents for diseases characterized by bone loss 7-9.
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Alveolar bone loss is a main characteristic of periodontitis. We previously demonstrated increased osteocytic sclerostin expression during suppressed bone formation in rats with ligature–induced periodontitis, suggesting that osteocytes may be a relevant source of sclerostin during periodontitis-induced alveolar bone loss 10. Another group using ligatureinduced periodontitis model reported that treatment with anti-sclerostin antibody restored alveolar bone mass, providing the rationale for the clinical investigation of sclerostin inhibitors in periodontitis 11. Knowledge about the relationship between periodontitis and diabetes has been increasing over the last decade. Several studies have reported that prevalence and severity of periodontal disease are increased in patients with type 1 and type 2 diabetes, suggesting that enhanced inflammation caused by diabetes may be related to the pathogenesis of periodontitis 12-16. Periodontitis patients exhibit increased levels of interleukin (IL)-1 and tumor necrosis factor (TNF)-α, two pro-inflammatory cytokines, in gingiva and crevicular fluid 17, 18. Increases in TNF-α and IL-1β have been observed in periodontitis patients with type 1 diabetes 18. These inflammatory cytokines are also increased in type 2 diabetic and STZ-induced diabetic rats with periodontitis when compared to non-diabetic rats with periodontitis 18-20. It has also been reported that enhanced TNF-α in type 2 diabetic rats contributes to deficient bone formation by suppressing the expression of bone formation inducing factors 21. Increased sclerostin expression has been observed both in vitro in TNF-α-treated osteocytes and in vivo in the osteocytes of obese mice with bone loss and increased TNF-α expression 22. Together, these results suggest that TNF-α induces osteocytic sclerostin expression and that altered osteocytic sclerostin expression by pro-inflammatory cytokines may play a role in the aggravation of periodontitis-induced alveolar bone loss in diabetes. Although previous studies have demonstrated how diabetes can negatively affect bone formation in periodontitis through enhanced inflammation 21, 23, 24, the potential role of sclerostin in this context remains uninvestigated. Therefore, in this study, we characterized sclerostin and TNF-α expression in diabetic rats with periodontitis.
MATERIALS AND METHODS Animal Models All animal protocols were approved by the Institutional Animal Care and Use Committee of Yonsei University (2012-0020-2). Six-week-old male F344 rats were purchased‖ and adapted for 1 week. Rats were then divided into three groups; control (C), periodontitis (P), and diabetes + periodontitis (DP) (N=6–8/each group/each time point). Diabetes was induced via intravenous administration of streptozotocin¶ (50 mg/kg). One week later, experimental periodontitis was generated by bilateral ligature on the gingival sulcus of the mandibular first molars. C group was sacrificed before ligature (day 0) and P and DP groups were sacrificed at days 3 and 20 after ligature. During the experiments, body weight and fasting glucose levels were measured at each time point. Fasting blood glucose levels were more than 350 mg/dl in diabetic rats and less than 100 mg/dl in control rats. Alveolar Bone Loss The mandibles were removed to perform histomorphometric examination at day 0 and at days 3 and 20 after ligature. Mandibles were fixed in 10% neutral-buffered formalin# overnight and decalcified in 10% EDTA# for 2 months. After embedding in paraffin, sections were cut at a thickness of 4 µm. Sections were then placed on slides and stained with hematoxylin and eosin (H&E). Alveolar bone loss in the distal area of the first molar was examined by
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measuring the cementoenamel junction (CEJ) to alveolar bone crest (ABC) distance using a software program**, using slides at 10× magnification. Osteoid Formation The osteoid area in distal area of first molar was measured to examine new bone formation. Osteoid formation was measured as the area of unmineralized bone matrix (light pink color) between osteoblasts (cuboidal bone-lining cells) and the mineralized bone surface, which was stained a dark pink color 10. The osteoid area, measured in a region of interest (ROI) that extended 0.5 mm from ABC in the distal area of the first molar, was examined under 20× magnification using a software program**. Osteoclast Formation To identify osteoclasts, we performed a tartrate-resistant acid phosphatase (TRAP) assay following the manufacturer's instructions††. The number of osteoclasts, defined as TRAPpositive multinucleated cells, was counted along the alveolar bone surface in a ROI that extended 0.5 mm from ABC in the distal area, using light microscopy at 20× magnification‡‡. The number of osteoclasts was calculated per millimeter length of alveolar bone surface. Immunohistochemistry for Sclerostin, TNF-α, and IL-1β For immunohistochemistry (IHC) staining, bone sections were quenched in 3% H2O2# for 20 min to inhibit endogenous peroxidase, and then incubated with trypsin§§ for antigen retrival. The commercial IHC kits ‖‖,¶¶ were used and all procedures were performed following the manufacturer's instructions. Briefly, after pre-incubating sections with normal horse blocking reagent for 20 min, sections were incubated overnight at 4°C with goat anti-sclerostin antibody## (1:300 dilution), rabbit anti-TNF-α antibody*** (1:400 dilution), and rabbit anti-IL1β antibody††† (1:75 dilution). Sections stained for sclerostin were then further incubated with biotinylated link antibody and peroxidase-labeled streptavidin. Sections stained for TNF-α and IL-1β were incubated with anti-mouse and anti-rabbit IgG conjugated horseradish peroxidase microploymer, respectively. The color was developed with substrate-chromogen, with methyl green as the counterstain. Negative controls were created by omitting the primary antibody. Sclerostin-positive osteocytes were counted in the ROI, which was extended 0.5 mm from ABC in the distal area of alveolar bone, using light microscopy at 40× magnification. Osteocytes were counted per bone area. TNF-α and IL-1β positive cells were counted in a standardized site (0.2 mm × 0.2 mm) located under the gingival epithelium. Statistical Analysis All statistical analyses were performed using a statistical analysis program‡‡‡. One-way analysis of variance (ANOVA) and Student's t-tests were used to determine significant differences. A p-value < 0.05 was considered statistically significant. Data are expressed as the mean ± standard error (SEM).
RESULTS 1. Alveolar Bone Loss in Diabetic Rats With Periodontitis To estimate how diabetes affects alveolar bone loss in periodontitis, we measured the CEJ-ABC distance in the distal area of the first molar (Fig 1A). In the P and DP groups, the increased CEJ-ABC distance was observed at days 3 and 20 post-periodontitis induction (via ligature) compared to the C group (Fig 1B). At day 20, the CEJ-ABC distance in the DP 3
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group was increased compared to that of the P group, and also tend to be increased compared to day 3. 2. Osteoclast Formation in Diabetic Rats With Periodontitis To investigate the effect of diabetes on periodontitis-induced bone resorption, we counted TRAP-positive multinucleated osteoclasts in the distal area (Fig 2A). At days 3 and 20 post-ligature, the number of osteoclasts in the P and DP groups significantly increased compared to the C group (Fig 2B). However, there was no significant difference between the number of osteoclasts detected in the P and DP groups during the entire experiment period. 3. Bone Formation in Diabetic Rats With Periodontitis To investigate the effect of diabetes on bone formation in periodontitis, the osteoid area was measured in the distal area (Fig 3A). At day 3 post-ligature, a reduced osteoid area was observed in both the P and DP groups (Fig 3B). The decreased osteoid area was maintained up to day 20 in the DP group; however, the osteoid area in the P group at day 20 was remarkably increased (approximately 3.3-fold) compared to that of the C group, and was notably higher than that of the DP group. 4. Osteocytic Sclerostin Expression in Diabetic Rats With Periodontitis To investigate the effect of diabetes on sclerostin expression, the number of sclerostin-positive osteocytes was counted (Fig 4A). Sclerostin-positive osteocytes in the P and DP groups markedly increased at day 3 post-ligature compared to the C group (Fig 4B). Interestingly, the number of sclerostin-positive osteocytes in the P group at day 20 decreased to control levels; however, the increased number of sclerostin-positive osteocytes in the DP group was maintained through day 20. 5. TNF-α and IL-1β Expression in Diabetic Rats With Periodontitis To investigate the effect of diabetes on the expression of pro-inflammatory cytokines in periodontitis, we counted the number of TNF-α and IL-1β positive cells that infiltrated the connective tissue beneath the gingival epithelium. The number of TNF-α positive cells increased in both the P and DP groups compared to the C group at days 3 and 20 postligature; additionally, there were more TNF-α positive cells in the DP group than in the P group (Fig. 5A). The quantity of IL-1β positive cells also increased in the P and DP groups than in the C group, with higher levels in the DP group than in the P group at day 20 postligature (Fig. 5B).
DISCUSSION In this study, alveolar bone loss similarly increased at day 3 post-ligature in the P and DP groups. Consistent with our previous study 10, alveolar bone loss in the P group did not further increase at day 20 post-ligature. However, the alveolar bone loss observed in the DP group more increased than that observed in the P group at day 20, and the increase was continual from ligature to day 20. These results indicate that diabetes exacerbates alveolar bone loss and could sustain severe uncoupled bone remodeling in periodontitis. Alveolar bone loss is typically determined using the degree of uncoupled bone remodeling, which is characterized by the imbalance of bone resorption and formation 1, 2. In both the P and DP groups, osteoclast numbers peaked at day 3 post-ligature and then gradually decreased. The difference in osteoclast numbers between these groups was not significant. Osteoclast formation in diabetes with periodontitis is a controversial topic, 4
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according to several reports 21, 23-26. Some groups have reported increased osteoclast formation in type 2 diabetic and STZ-induced diabetic rats with periodontitis 21, 23-25; however, He et al. did not observe increased osteoclast formation in type 2 diabetic mice infected with a periodontopathogen 26. Although the precise role of osteoclast formation in diabetes with periodontitis remains unclear, the association between diminished bone formation and exacerbated alveolar bone loss in type 2 diabetic and STZ-induced diabetic rats with periodontitis has been consistently reported 23, 24. In the current study, we also observed exacerbated alveolar bone loss through diminished bone formation in the DP group at day 20 post-ligature. These results suggest that diabetes more strongly affects bone formation than bone resorption. The pattern of sclerostin expression coincided with that of osteoid formation in both the P and DP groups. At day 3 post-ligature, when osteoid formation markedly decreased in both groups, sclerostin expression increased. At day 20 post-ligature, when osteoid formation in the P group notably increased, sclerostin expression decreased, which we also observed in our previous study 10. Interestingly, increased sclerostin expression in the DP group was sustained until day 20 post-ligature, at which time diminished bone formation was also observed. Additionally, clinical studies have demonstrated increased sclerostin levels in patients with type 2 diabetes and periodontitis 27, 28. In type 2 diabetes patients, high levels of serum sclerostin were observed, and were negatively associated with β-catenin, an essential Wnt pathway molecule involved in osteogenesis 27. In patients with chronic periodontitis and severe alveolar bone loss, gingival tissue and serum sclerostin levels were increased compared to patients without periodontitis 28. Taken together, these findings suggest that persistent and increased sclerostin expression in diabetic rats with periodontitis may contribute to diminished bone formation. In this study, prolonged increased TNF-α and IL-1β expression was observed in the DP group compared to the P group, a result that is consistent with observations in type 1 diabetes patients and type 2 diabetic and STZ-induced diabetic rats 18-20. When inflammation in type 2 diabetic rats with periodontitis was resolved by treatment with a TNF-α inhibitor, bone formation improved, and this group suggested that TNF-α limits bone formation by modulating the expression of several bone formation-stimulating factors, including transforming growth factor β-1, bone morphogenetic protein (BMP)-2, BMP-6, and fibroblast growth factor-2 21. Tanabe et al. reported that alkaline phosphatase activity and calcification in rat osteoblasts decreased upon treatment with IL-1α, which acts similarly to IL-1β 29. In IL-1 receptor antagonist knockout mice infected with Aggregatibacter actinomycetemcomitans, severe alveolar bone loss occurred, suggesting that high levels of IL-1α expression promote the down-regulation of osteoblast-related gene expression 30. Together, these studies indicate that TNF-α and IL-1β are involved in prolonged and decreased bone formation in diabetic rats with periodontitis. Recently, some groups reported that TNF-α is associated with osteocytic sclerostin expression in pathological bone loss caused by estrogen deficiency and obesity. One of these groups observed that treatment with a TNF-α blocker reversed the increased sclerostin expression in estrogen-deficient mice with osteoporosis 31. Additionally, the activity of myocyte enhancer factor 2, a sclerostin transcription factor, was increased by TNF-α, but not by IL-1, in UMR-106 osteosarcoma cells 31. A second group demonstrated that levels of osteocytic sclerostin and serum TNF-α were increased in obese mice with bone loss 22. Treatment with TNF-α increased sclerostin expression in MLO-Y4 osteocytes, suggesting that TNF-α-induced sclerostin expression contributes to bone loss in obesity 22. Considering these studies, we assume that in the diabetic rats with periodontitis, prolonged and prevalent TNF-α expression may play a role in sustained sclerostin expression. To fully elucidate the 5
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role of TNF-α in sclerostin expression in diabetic rats with periodontitis, further studies are needed, including those that can confirm the immunohistochemistry results observed in the current study and those using TNF-α inhibitors.
CONCLUSIONS The results described here show that prolonged and enhanced alveolar bone loss, suppressed bone formation, and increased TNF-α and IL-1β are observed in diabetic rats with periodontitis. Suppressed bone formation with concurrent increased in sclerostin and TNF-α were specifically observed in diabetic rats with periodontitis. These results suggest that up-regulated osteocytic sclerostin expression may play a role in the prolongation of suppressed bone formation in diabetic rats with periodontitis. This study is the first to show increased osteocytic sclerostin expression in periodontitis with concurrent diabetes. The data support the further clinical investigation of relationship between osteocytic sclerostin expression and diminished bone formation in diabetic patients with periodontitis. ACKNOWLEDGEMENT This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A6A3A01053590) (2014R1A2A1A11049412).
CONFLICT OF INTEREST STATEMENT The authors report no conflicts of interest related to this study.
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Correspondence: Yun-Jung Yoo, D.D.S., Ph.D. Department of Oral Biology, Yonsei University College of Dentistry, 134 Sinchon dong, Seodaemun-gu, Seoul 120-752, Republic of Korea, Tel: +82-2-2228-3060, Fax: +82-2-2227-7903, E-mail: [email protected], Eun-Jung Bak, D.V.M., Ph.D. Department of Oral Biology, Yonsei University College of Dentistry, 134 Sinchon dong, Seodaemun-gu, Seoul 120-752, Republic of Korea, Tel: +82-2-22283062, Fax: +82-2-2227-7903, E-mail: [email protected] Submitted February 04, 2015; accepted for publication March 30, 2015. Figure 1. Alveolar bone loss in each group. A) Representative images for the calculation of the CEJ-ABC distance are shown (H&E stain; scale bar = 100 µm [original magnification ×10]). B) Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used (* p < 0.05 compared with day 0. † p < 0.05). Figure 2. Osteoclast numbers in each group. A) Representative images for the calculation of the number of osteoclasts on the alveolar bone surface are shown (TRAP stain; scale bar = 100 µm [original magnification ×20]). B) Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used. (* p < 0.05 compared with day 0); n=number, OCs=osteoclasts, BS=alveolar bone surface. Figure 3. Osteoid area in each group. A) Representative images for the calculation of the osteoid area are shown (H&E stain; scale bar = 100 µm [original magnification ×20]). The osteoid (dotted line) was defined as the unmineralized bone matrix (light pink) between the osteoblasts and the mineralized bone surfaces. B) Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used (* p < 0.05 compared with day 0. † p < 0.05). Figure 4. Osteocytic sclerostin expression in each group. A) Representative images for calculating the number of sclerostin-positive osteocytes are shown (IHC stain; scale bar = 100 µm [original magnification ×40]). White arrowhead indicates representative negative osteocyte and black arrow head indicates representative positive osteocyte. B) Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used (* p < 0.05 compared with day 0. † p < 0.05); n=number. Figure 5. TNF-α and IL-1β expression in each group. The number of TNF-α (A) and IL-1β (B)-positive cells in each group is illustrated. Data are presented as mean ± SEM. Student's t-tests and ANOVA followed by Tukey’s post hoc test were used (* p < 0.05 compared with day 0. † p < 0.05); n=number.
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Key findings : Increased sclerostin expression was accompanied with reduced bone formation in diabetic rats with periodontitis. ‖ Oriental Bio, Gyeonggi-do, Korea ¶ Sigma Aldrich, St. Louis, MO, USA # Duksan, Gyeonggi-do, South of Korea ** Image-Pro software, Media Cybernetics, Silver Spring, MD, USA †† Acid Phosphatase, Leukocyte (TRAP), Sigma Aldrich, St. Louis, MO, USA ‡‡ Olympus, Tokyo, Japan §§ Invitrogen, Carlsbad, CA, USA ‖‖ LSAB+ system-HRP, Dako, Carpinteria, CA, USA ¶¶ ImmPRESS™ universal reagent, Vector Laboratories, Inc., Burlingame, CA, USA ## R&D System Inc, Minneapolis, MN, USA *** Abcam, Thousand Oaks, CA, USA ††† Santa Cruz Biotechnology, Santa Cruz, CA, USA ‡‡‡ SPSS 12.0, SPSS, Chicago, IL, USA
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