J Periodont Res 2014 All rights reserved
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12165
Diabetic characteristics and alveolar bone loss in streptozotocin- and streptozotocin-nicotinamidetreated rats with periodontitis
J-H. Kim1,2,3, D-E. Lee1,2,3, S-H. Choi4, J-H. Cha1,2,3, E-J. Bak5*, Y-J. Yoo1,2* 1 Department of Applied Life Science, The Graduate School, Yonsei University, Seoul, South Korea, 2Department of Oral Biology, Yonsei University College of Dentistry, Seoul, South Korea, 3BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, South Korea, 4Department of Periodontology, Research Institute for Periodontal Regeneration, Yonsei University College of Dentistry, Seoul, South Korea and 5Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul, South Korea
Kim J-H, Lee D-E, Choi S-H, Cha J-H, Bak E-J, Yoo Y-J. Diabetic characteristics and alveolar bone loss in streptozotocin- and streptozotocinnicotinamide-treated rats with periodontitis. J Periodont Res 2014; doi: 10.1111/ jre.12165. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: Experimental models showing variable diabetic status are necessary to understand the relationship between diabetes and periodontitis. The streptozotocin (STZ)-induced diabetes model allows control of diabetic status by nicotinamide (NA), which protects against STZ-induced b-cell necrosis. Therefore, we compared diabetic characteristics and alveolar bone loss in STZand STZ-NA-treated rats with periodontitis. Material and Methods: STZ-treated rats were generated by intravenous (IV) administration of STZ (50 mg/kg). STZ-NA-treated rats were induced by intraperitoneal administration of NA (270 mg/kg) 15 min before IV administration of STZ (65 mg/kg). Periodontitis was induced by ligature around the left mandibular first molar 1 wk after injection. Blood glucose level, glucose tolerance and serum insulin levels were determined at day 0 and/or 20 after ligature. At day 20, tibia bone loss was assessed using micro-computed tomography and hematoxylin and eosin staining. Alveolar bone loss was histologically measured as the distance of the cementoenamel junction to the alveolar bone crest in distal and the percentage of periodontal ligament area in the first molar furcation, respectively. The number of inflammatory cells, receptor activator of nuclear factor kappa-B ligand (RANKL)-positive cells and the area of osteoid were determined. Results: In STZ-treated rats, obvious hyperglycemia over 300 mg/dL and severe body weight loss were observed. The insulin level was approximately 14% compared to that of control rats. STZ-NA-treated rats were impaired in glucose tolerance compared to control rats; however, body weight and insulin levels were not significantly different. Tibia bone loss was increased in STZ-treated rats, but significant change was not observed in STZ-NA-treated rats compared to control rats. In ligatured teeth, alveolar bone loss was increased in both STZand STZ-NA-treated rats compared to control rats. Alveolar bone loss, the number of inflammatory cells and RANKL-positive cells in STZ-treated rats were greater than in STZ-NA-treated rats. The area of osteoid decreased in STZ-treated rats compared to control, but not STZ-NA-treated rats.
Yun-Jung Yoo, DDS, PhD, Department of Oral Biology, Yonsei University College of Dentistry, 134 Sinchon dong, Seodaemun-gu, Seoul 120752, South Korea Tel: +82 2 2228 3060 Fax: +82 2 2227 7903 e-mail: [email protected] and Eun-Jung Bak, DVM, PhD, Oral Cancer Research Institute, Yonsei University College of Dentistry, 134 Sinchon dong, Seodaemun-gu, Seoul 120-752, South Korea Tel: +82 2 2228 3062 Fax: +82 2 2227 7903 e-mail: [email protected] *These authors contributed equally to this work. Key words: bone loss; nicotinamide;
periodontitis; streptozotocin Accepted for publication December 13, 2013
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Conclusion: These results indicate that STZ- and STZ-NA-treated rats exhibit diabetic characteristics similar to type 1 diabetes mellitus and a pre-diabetic state, respectively. In addition, alveolar bone loss in response to periodontitis and tibia loss depend on diabetic status. Diabetic status-dependent bone remodeling imbalance and inflammation could affect the alveolar bone loss in the two models. Both STZ- and STZ-NA-treated rats may be useful to investigate differences in periodontitis sensitivity associated with diabetic status and to develop therapeutic agents for periodontitis in patients with diabetes.
Diabetes is a chronic disease characterized by hyperglycemia that leads to long-term damage to several organs, including the heart, eye, kidneys, nerves, vascular system and bone. Hyperglycemia is the result of the absolute or relative lack of insulin production or resistance to insulin (1,2). Type 1 diabetes mellitus (T1DM) is present in 5–10% of patients with diabetes and is the result of b-cell destruction, which usually leads to a total loss of insulin secretion. Type 2 diabetes mellitus (T2DM) presents in 90–95% of patients with diabetes and is characterized by abnormal insulin production as well as insulin resistance, which alters the utilization of endogenously produced insulin by target cells. Periodontitis is an inflammatory disease caused by bacteria, and alveolar bone loss surrounding the roots of teeth is one of its characteristics. Alveolar bone loss is induced by an imbalance of bone resorption and bone formation caused by bacteria and a subsequent immune reaction (3,4). Several researchers have investigated the association of diabetes with periodontitis through epidemiological surveys and in vivo studies (5–9). A relationship between diabetes and the increased incidence of periodontitis has been proposed, but the exact mechanism for diabetes-related periodontitis is still unclear. In addition, it is not clear whether there is any difference in periodontitis pathogenesis depending on diabetic status. Various animal models that mimic characteristics of patients with diabetes have been generated by genetic, chemical, dietary or surgical means, or a combination of these techniques (10,11). Chemicals such as streptozo-
tocin (STZ) and nicotinamide (NA) have been demonstrated to induce experimental diabetes in rats (12). STZ has a toxic effect on pancreatic b cells, decreases insulin biosynthesis and secretion, and consequently induces hyperglycemia (13). On the other hand, a suitable dose of NA can exert partial protection against STZ’s cytotoxic effect on pancreatic b-cells (12,14). STZ-NA-induced diabetic rats have been used for diabetes drug screening (15,16). A genetic or STZ model was employed in a periodontitis study, but the STZ-NA diabetic model has never been used. Patients with diabetes show variable diabetic status, including levels of hyperglycemia and insulinopenia and the presence of osteoporosis. To understand better the mechanism that links diabetes and periodontitis and to develop new drugs and preventive strategies, experimental models showing variable diabetic status are necessary. STZ-induced diabetic model is cheaper and easier to develop compared with other diabetic models, in addition, NA can be used to control diabetic characteristics. To investigate the suitability of these animal models in the study of periodontitis pathogenesis-related diabetic status, we investigated diabetic characteristics and levels of alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis.
Material and methods Animal models
Six-wk-old male F344 rats were purchased from Oriental Bio (Gyeonggi-do, Korea) and acclimated for 1 wk. The
animals were housed in cages and were fed with a standard laboratory diet and tap water. The animals were maintained in a temperaturecontrolled room (22 2°C) on a 12 h light–dark cycle. Animals were randomly divided into a control group and two groups treated with STZ or STZ-NA (five to six animals per group). After a 15 h fast, the STZ group was given intravenous STZ (50 mg/kg; Sigma-Aldrich, St Louis, MO, USA) dissolved in 0.1 M citrate buffer (Sigma-Aldrich), the STZ-NA group was given intraperitoneal NA (270 mg/kg; Sigma-Aldrich) 15 min before STZ administration (65 mg/ kg), and the control group was injected with citrate buffer alone. At 7 d after injection, all rats were anesthetized with 1 : 2 mixtures of Zoletil 50 (Virbac, Carros, France) and Rompun (Bayer, Ansan, Korea) to induce periodontitis. Dental floss was placed around the cervix of the left mandibular first molars and knotted mesially. The contralateral right side was used as the non-ligatured control. During the experimental period, the location of dental floss, gross appearance, and food and water intake were observed daily. All animal procedure protocols were approved by the Institutional Animal Care and Use Committee of Yonsei University (2010-0209). Evaluation of diabetic characteristics
Body weight was measured every day during the experimental period under fasting and non-fasting conditions. Fasting and non-fasting blood glucose levels were measured from tail veins
Diabetic characteristics and alveolar bone loss at days 0 and 20 after inducing periodontitis. For the oral glucose tolerance test, rats were fasted for 15 h and were given glucose (2 g/kg body weight) orally using gavage. Blood glucose levels were measured with the Accu-check active system (Roche, Mannheim, Germany) in accordance with the manufacturer’s instructions 0, 30, 60, 90 and 120 min after glucose administration. Serum insulin was determined using an enzymelinked immunosorbent assay kit (Shibayagi, Gunma, Japan). Evaluation of tibia bone loss
Tibia trabecular bone loss was assessed by micro-computed tomography (micro-CT) (Skyscan, Antwerp, Belgium) and histomorphometric analysis. Tibias were scanned using micro-CT and three-dimensional images were reconstructed. The region of interest (ROI) was determined, which extended 2.5 mm at 1.5 mm below the growth plate toward the diaphysis and excluded the outer cortical shell. Trabecular bone loss was evaluated by the trabecular bone volume fraction (BVF), which was obtained by dividing the trabecular bone volume by the total ROI volume. For histomorphometric analysis, tibias were fixed in 10% neutralbuffered formalin and decalcified in 5% nitric acid for 1 wk. Three serial sections of 4 lm thickness were cut and stained with hematoxylin and eosin (H&E). The ROI for trabecular bone volume extended 1 mm at 1.5 mm below the growth plate toward the diaphysis and excluded the outer cortical shell. Trabecular BVF was calculated by data from three
serial sections using Image-Pro software (Media Cybernetics, Silver Spring, MD, USA). Evaluation of alveolar bone loss, osteoid formation and inflammatory cells infiltration
For histopathological examination, mandibles were removed after being killed and fixed in 10% neutralbuffered formalin overnight and decalcified in 5% nitric acid for 1 wk. The paraffin-embedded sections were cut at a thickness of 4 lm. Sections were selected based on clear appearance of the dental pulp of the mesial and distal roots of the first molars and were H&E stained. Alveolar bone loss in the distal and furcation areas of the first molar was examined under 100 9 magnification. Alveolar bone loss in the distal area of the first molar was examined by measuring the distance from cementoenamel junction (CEJ) to the alveolar bone crest (ABC) using Image-Pro software (Media Cybernetics). Alveolar bone loss in the furcation area was estimated by the percentage of periodontal ligament (PDL) area in the ROI. The height of the ROI was 0.8 mm from the apical furcation. To examine bone formation, the area of osteoid was measured in the distal area of the first molar under 200 9 magnification. The new osteoid was considered as unmineralized bone matrix between osteoblasts and the mineralized bone surface. For evaluation of inflammatory cell infiltration, the number of inflammatory cells including polymorphonuclear leukocytes and mononuclear cells was counted within a standardized site (0.1 mm 9 0.1 mm)
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located at the junctional epithelium and subepithelial connective tissue of the distal area of the ligated first molar. A single examiner, blind to the treatment assignment, performed the analyses using a light microscope under 500 9 magnification. Evaluation of receptor activator of nuclear factor kappa-B ligandpositive cells
The sections of mandible were mounted onto glass slides coated with silane (Muto Pure Chemicals, Tokyo, Japan), deparaffinized in xylene and rehydrated. For immunohistochemical staining, the LSAB+ system-HRP kit (Dako, Carpinteria, CA, USA) was used and all procedures followed the manufacturer’s instructions. Briefly, after endogenous peroxidase activity was blocked, sections were incubated overnight at 4°C with goat antireceptor activator of nuclear factor kappa-B ligand (anti-RANKL) antibody (1 : 500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with biotinylated link antibody and peroxidase-labeled streptavidin. The color was developed with substrate-chromogen and methyl green was used as a counterstain. RANKL-positive cells were counted in a standardized site (0.1 mm 9 0.1 mm) located in the distal area of the ligated first molar under 400 9 magnification. Statistical analyses
All statistical analyses were performed using SPSS 12.0 (SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) and Student’s
Table 1. Body weight, and fasting and non-fasting blood glucose levels in the control, STZ- and STZ-NA-treated rats with periodontitis Day 0
Day 20
Diabetes characteristics
Control
STZ-NA
Body weight (g) Fasting blood glucose (mg/dL) Non-fasting blood glucose (mg/dL)
258 13 99 5 131 8
241 10 109 9 222 105
STZ 157 8* 355 23* –
NA, nicotinamide; STZ, streptozotocin. Data are expressed as the mean SEM. *Significant difference at p < 0.05, when compared with control rats.
Control
STZ-NA
285 10 97 11 125 7
264 18 105 7 236 121
STZ 156 7* 517 28* –
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t-tests were used to determine significant differences. p < 0.05 was considered statistically significant. Data are expressed as the mean standard error (SEM).
Results Blood glucose levels and gross changes in streptozotocin- and streptozotocin-nicotinamide-treated rats with periodontitis
To estimate the induction and maintenance of diabetic characteristics, we measured body weight and fasting or non-fasting glucose levels (Table 1). In STZ-treated rats, severe body weight loss was observed compared with control rats. Moreover, their fasting blood glucose level was significantly increased by 355 23 mg/dL and 517 28 mg/dL on days 0 and 20 after ligature compared to
99 5 mg/dL and 97 11 mg/dL in control rats, respectively. In addition, classic signs of T1DM, such as polyuria, polydipsia and polyphagia were observed during the experimental period (data not shown). STZNA-treated rats showed a normal increase of body weight similar to control rats. STZ-NA-treated rats exhibited a mild increase in nonfasting blood glucose levels. During the 2 h GTT, STZ-NA-treated rats had larger areas under the curve at days 0 and 20 compared to control rats (Fig. 1A and B). Serum insulin levels in streptozotocin- and streptozotocinnicotinamide-treated rats with periodontitis
To estimate insulin secretion in STZ and STZ-NA-treated rats, we measured serum insulin levels after 15 h
of fasting at day 20 (Fig. 2). The serum insulin level of STZ-treated rats was approximately 14% that of control rats. Although serum insulin levels in STZ-NA-treated rats were 70% that of control rats, the difference was not statistically significant. Tibia bone loss in streptozotocinand streptozotocin-nicotinamidetreated rats with periodontitis
To observe skeletal bone loss, trabecular BVF of the tibia was analyzed using micro-CT and H&E staining at day 20 (Fig. 3). For micro-CT (Fig. 3A), tibia BVF in STZ-treated rats was about 28% compared to that of control rats. However, tibia BVF in STZ-NA-treated rats was not significantly different from that of control rats. These results were similar to the histomorphometric analysis (Fig. 3B). Only STZ-treated
A
B
Fig. 1. Glucose tolerance test in STZ-NA-treated rats with periodontitis. At days 0 (A) and 20 (B) after ligature, oral glucose tolerance tests were performed in STZ-NA-treated rats. The animals were orally administered with glucose (2 g/kg) after 15 h of fasting, and blood glucose levels were measured at 0, 30, 60, 90 and 120 min. AUC at days 0 and 20 were compared, and data are presented as mean SEM. *Significant difference at p < 0.05 compared with control rats. AUC, area under the curve; STZ-NA, streptozotocinnicotinamide.
Diabetic characteristics and alveolar bone loss
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A
Fig. 2. Serum insulin levels in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, serum insulin levels were estimated in STZ- and STZ-NA-treated rats by enzyme-linked immunosorbent assay. Data are represented as mean SEM. *Significant difference at p < 0.05 compared with control rats. NA, nicotinamide; STZ, streptozotocin.
rats showed a significantly reduced tibia BVF compared to control rats. Alveolar bone loss in streptozotocin- and streptozotocinnicotinamide-treated rats with periodontitis
To estimate alveolar bone loss of non-ligatured and ligatured teeth in control and treated rats, the distance of CEJ-ABC and percentage of the PDL area were measured in distal and furcation areas at day 20, respectively (Fig. 4). In non-ligatured teeth, there was no difference in either measure between treated and control rats (Fig. 4A and 4B). However, marked alveolar bone loss was present in ligatured teeth in both treated and control rats. The CEJ-ABC distance of ligatured teeth was slightly increased in STZ-NA-treated rats compared to control rats, but the difference was not significant; however, we observed a significant difference in STZtreated rats compared to control rats (Fig. 4A). The percentage of PDL area of ligatured teeth was increased in STZ-NA-treated rats as well as STZ-treated rats compared to control rats. (Fig. 4B). Inflammation of gingival tissue in streptozotocin- and streptozotocinnicotinamide-treated rats with periodontitis
To estimate the degree of inflammation, the infiltration of inflammatory
B
Fig. 3. Tibia bone loss in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, tibias were analyzed using micro-computed tomography (A) and hematoxylin and eosin staining (B). Trabecular bone loss was evaluated by trabecular BVF, which is obtained by dividing the trabecular bone volume by the total region of interest volume. Data are presented as mean SEM. *Significant difference at p < 0.05 compared with control rats (bar = 500 lm). BVF, bone volume fraction; NA, nicotinamide; STZ, streptozotocin.
cells, such as polymorphonuclear leukocytes and mononuclear cells, were counted in gingiva (Fig. 5). The number of inflammatory cells infiltrating the junctional epithelium and connective tissue were increased in ligatured teeth of the three groups com-
pared to non-ligatured teeth (Fig. 5A and 5B). In non-ligatured teeth, there was no difference in either the location between treated and control rats. In junctional epithelium of ligatured teeth, STZ-treated rats showed a marked increase of infiltration com-
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A
cells were counted by immunohistochemistry (Fig. 6A). RANKL-positive cells were located more in ligatured teeth than non-ligatured teeth. The number of RANKL-positive cells was markedly increased in STZ-treated rats as well as in STZ-NA-treated rats compared to control rats. The number of RANKL-positive cells was higher in STZ-treated rats than in STZ-NA-treated rats. To estimate coupled bone formation after bone resorption, the area of osteoid was measured (Fig. 6B). In non-ligatured teeth, there was no difference in the area between treated and control rats. However, a significant increase was evident in ligatured teeth of control and STZ-NA-treated rats, but not in STZ-treated rat compared with each non-ligatured teeth. The area of osteoid in ligatured teeth of control and STZ-NA-treated rats was 11.8fold and 9.4-fold larger than STZ-treated rats.
B
Discussion
Fig. 4. Alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, alveolar bone levels of non-ligatured and ligatured first molars were examined by light microscopy (100 9 magnification). Alveolar bone loss in distal and furcation areas of teeth was estimated by the distance of CEJ-ABC (A) and the percentage of PDL area to region of interest area (B), respectively. The height of the region of interest was 0.8 mm from the apical furcation. Data are presented as mean SEM. *Significant difference at p < 0.05 compared with non-ligatured teeth. #Significant difference at p < 0.05 (bar = 100 lm). ABC, alveolar bone crest; CEJ, cementoenamel junction; NA, nicotinamide; PDL, periodontal ligament; STZ, streptozotocin.
pared to control rats, but STZ-NAtreated rats were not significant (Fig. 5A). In connective tissue of ligatured teeth, infiltration of inflammatory cells was increased in both STZ-NA- and STZ-treated rats compared to control rats (Fig. 5B).
RANKL expression and osteoid formation in streptozotocin- and streptozotocin-nicotinamide-treated rats with periodontitis
To identify expression of osteoclast differentiation factor, RANKL-positive
In this study, we compared diabetic characteristics and alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis. We observed blood glucose levels and glucose tolerance, as well as insulin levels. STZ has a selective toxic effect on pancreatic b-cells and causes hyperglycemia due to absolute insulin deficiency. STZ is known to cause alkylation of DNA strands and a consequent increase in the activity of poly-ADP-ribose polymerase. This leads to the depletion of NAD, which results in reduced insulin synthesis in b-cells and cell necrosis (13). This cytotoxicity is prevented by NA, a component of NAD, which inhibits poly-ADP-ribose polymerase and prevents NAD depletion in b-cells (12,14). Therefore, the administration of NA lessens the severity of diabetic status by STZ (14). In this study, STZ-treated rats showed characteristics that resembled T1DM, including obvious hyperglycemia, body weight loss and markedly decreased serum insulin levels. While STZ-NA-treated rats did not show significant differences in fasting or non-fasting glucose levels or serum insulin level compared to control rats,
Diabetic characteristics and alveolar bone loss
A
B
Fig. 5. Inflammation in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, infiltration of inflammatory cells, such as polymorphonuclear leukocytes and mononuclear cells, were examined in a standardized site (0.1 mm 9 0.1 mm) located in junctional epithelium (A) and subepithelial connective tissue (B) of the distal area of the non-ligatured and ligatured first molar (500 9 magnification). Data are represented as mean SEM. *Significant difference at p < 0.05 compared with non-ligatured teeth. # Significant difference at p < 0.05. NA, nicotinamide; STZ, streptozotocin.
they showed slightly increases in nonfasting glucose levels. In the oral glucose tolerance test, STZ-NA-treated rats showed impaired glucose tolerance compared with control rats. These results indicated that STZ- and STZ-NA-treated rats have characteristics similar to the T1DM and prediabetic state, respectively. Bone loss is one of the complications in diabetes. It has been proposed that the incidence of bone loss is different between T1DM and T2DM (17–20). The majority of T2DM studies have determined that bone mineral density is normal or increased. Conversely, 50% of T1DM patients have lower bone mass than expected, and approximately 20% meet the criteria for osteoporosis.
T1DM studies have reported that late bone turnover is associated with increased bone resorption and decreased bone formation. These indicate that the prevalence of bone loss is influenced by diabetic status. To investigate skeletal bone loss in both treated rats, we measured tibia BVF using micro-CT and H&E staining. We observed a significant decrease of tibia BVF in STZ-treated rat, but not STZNA-treated rats in both analyses. In STZ-NA-treated rats, the absence of an effect on tibia BVF could be due to the short time of induction period which STZ-NA-treated rats were in the pre-diabetic state. For exact evaluation on bone loss in pre-diabetic state, long-term study of the chronic state will be needed. Taken together,
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these results suggest that STZ-treated rats are useful for studying T1DM accompanied by osteoporosis. To compare the effect of diabetic status on alveolar bone loss in response to periodontitis, we investigated alveolar bone loss of ligatured teeth by measuring CEJ-ABC distances in distal areas and the percentage of PDL areas in furcation. There were no differences among the groups for non-ligatured teeth. However, alveolar bone loss in ligatured teeth was increased in treated rats compared to control rats. Interestingly, STZtreated rats showed that alveolar bone loss occurred more than in STZ-NAtreated rats. These results indicate that STZ- and STZ-NA-treated rats show severe and mild alveolar bone loss in response to periodontitis, respectively. Alveolar bone loss in response to periodontitis is caused by an imbalance of bone resorption and bone formation (3,4). As the different degree of alveolar bone loss dependent on diabetic status, we estimated the RANKL expression and the area of osteoid associated with bone resorption and bone formation (21,22). In ligatured teeth, the number of RANKL-positive cells was increased in treated rats and was higher in STZ-treated rats than in STZ-NAtreated rats. The area of osteoid, an indicator of newly formed bones, was significantly increased in ligatured teeth of control and STZ-NA-treated rats, but not in STZ-treated rats compared to each non-ligatured teeth. These results suggest that the degree of RANKL expression and osteoid formation in response to periodontitis affects alveolar bone loss in the two models. The alveolar bone loss in STZ-treated rats might be caused by increased bone resorption and depressed coupled bone formation. The diminished alveolar bone loss in STZ-NA-treated rats may be due to the maintenance of normal bone formation via a protective effect of NA against STZ-induced b-cell damage. Inflammation is one factor related to bone loss (23). Ligation raised the inflammatory cells infiltration in both treated and control rats. While STZ-treated rats showed higher
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Kim et al. may be related to the enhancement of bone resorption and the depression of bone formation in STZ-treated rats. In conclusion, this study is the first to compare diabetic characteristics and alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis. Our results indicate that STZ- and STZ-NA-treated rats exhibit pathologies similar to T1DM and pre-diabetic state, respectively, and suggest that the level of bone loss in alveolar bone and tibia depend on diabetic status. Diabetic status-dependent bone remodeling imbalance and inflammation could contribute to the difference in the degree of alveolar bone loss in the two models. Evaluations using different diabetic models would provide valuable comparative information on the relationship between diabetes and periodontitis. STZ- and STZ-NAtreated rats may be useful to study variations in periodontitis pathogenesis depending on diabetic status and for developing therapeutic agents.
A
B
Acknowledgements Fig. 6. RANKL expression and area of osteoid in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, the number of RANKL-positive cells was counted in a standardized site (0.1 mm 9 0.1 mm) located in the distal area of the non-ligatured and ligatured first molar (A; 400 9 magnification). The area of osteoid, unmineralized bone matrix between osteoblasts and the mineralized bone surface, was measured in distal area of the first molar under 200 9 magnification (B). Data are presented as mean SEM. *Significant difference at p < 0.05 compared with non-ligatured teeth. # Significant difference at p < 0.05. NA, nicotinamide; RANKL, receptor activator of nuclear factor kappa-B ligand; STZ, streptozotocin.
inflammatory cells infiltration in both junctional epithelium and connective tissue compared to control, STZ-NAtreated rats displayed it in connective tissues. These results indicate that the severity of inflammation is influenced by diabetic status. Various cells including osteoblasts, fibroblasts, lymphocytes and neutrophils produce RANKL (24,25). In addition, cytokines, such as interleukin (IL)-1, IL-6 and tumor necrosis factor, produced by inflammatory cells affect bone level by stimulating osteoclast differentiation and suppressing osteoblast differentiation (23). Thus, increased inflammation in STZ-treated rats may be one factor that leads to a net alveolar bone loss. A previous study on
diabetic complications reported that accumulation of irreversible advanced glycation end products (AGEs) as a result of hyperglycemia may contribute to enhancing inflammation (7,26). AGE deposition was elevated in gingival tissue of diabetic patients with periodontitis (27). These suggest that AGE deposition in gingival tissue may be related to enhancement of inflammation in STZ-treated rats. As well as enhancing the inflammation, AGEs increase RANKL expression and decreased collagen type I, osteocalcin and alkaline phosphatase expression in osteoblast (28,29). In addition, insulin promotes osteoblast differentiation (30). Therefore, AGE accumulation and insulin deficiency
The authors thank Jang Gi Cho, Department of Oral Biology, Yonsei University College of Dentistry, for assistance with the micro-CT analysis and graphics. This study was supported by a faculty research grant of Yonsei University College of Dentistry for 2012 (6-2012-0062).
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15. Pierre W, Gildas AJ, Ulrich MC, Modeste WN, Benoit NT, Albert K. Hypoglycemic and hypolipidemic effects of Bersama engleriana leaves in nicotinamide/streptozotocin-induced type 2 diabetic rats. BMC Complement Altern Med 2012;12:264. 16. Tahara A, Matsuyama-Yokono A, Nakano R, Someya Y, Shibasaki M. Hypoglycaemic effects of antidiabetic drugs in streptozotocin-nicotinamideinduced mildly diabetic and streptozotocin-induced severely diabetic rats. Basic Clin Pharmacol Toxicol 2008;103:560–568. 17. Barrett-Connor E, Holbrook TL. Sex differences in osteoporosis in older adults with non-insulin-dependent diabetes mellitus. JAMA 1992;268:3333–3337. 18. Hamann C, Kirschner S, Gunther KP, Hofbauer LC. Bone, sweet bone– osteoporotic fractures in diabetes mellitus. Nat Rev Endocrinol 2012;8:297–305. 19. van Daele PL, Stolk RP, Burger H et al. Bone density in non-insulin-dependent diabetes mellitus. The Rotterdam study. Ann Intern Med 1995;122:409–414. 20. Sealand R, Razavi C, Adler RA. Diabetes mellitus and osteoporosis. Curr Diab Rep 2013;13:411–418. 21. Pacios S, Kang J, Galicia J et al. Diabetes aggravates periodontitis by limiting repair through enhanced inflammation. FASEB J 2012;26:1423–1430. 22. Tobon-Arroyave SI, Isaza-Guzman DM, Restrepo-Cadavid EM, Zapata-Molina SM, Martinez-Pabon MC. Association of salivary levels of the bone remodelling regulators sRANKL and OPG with periodontal clinical status. J Clin Periodontol 2012;39:1132–1140.
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23. Graves DT, Li J, Cochran DL. Inflammation and uncoupling as mechanisms of periodontal bone loss. J Dent Res 2011; 90:143–153. 24. Kajiya M, Giro G, Taubman MA, Han X, Mayer MP, Kawai T. Role of periodontal pathogenic bacteria in RANKLmediated bone destruction in periodontal disease. J Oral Microbiol 2010;2: 5532. 25. Mori G, D’Amelio P, Faccio R, Brunetti G. The interplay between the bone and the immune system. Clin Dev Immunol 2013;2013:720504. 26. Roszer T. Inflammation as death or life signal in diabetic fracture healing. Inflamm Res 2011;60:3–10. 27. Zizzi A, Tirabassi G, Aspriello SD, Piemontese M, Rubini C, Lucarini G. Gingival advanced glycation endproducts in diabetes mellitus-associated chronic periodontitis: an immunohistochemical study. J Periodontal Res 2013; 48:293–301. 28. Franke S, Siggelkow H, Wolf G, Hein G. Advanced glycation endproducts influence the mRNA expression of RAGE, RANKL and various osteoblastic genes in human osteoblasts. Arch Physiol Biochem 2007;113:154–161. 29. Franke S, Ruster C, Pester J, Hofmann G, Oelzner P, Wolf G. Advanced glycation end products affect growth and function of osteoblasts. Clin Exp Rheumatol 2011;29:650–660. 30. Fulzele K, Riddle RC, DiGirolamo DJ et al. Insulin receptor signaling in osteoblasts regulates postnatal bone acquisition and body composition. Cell 2010; 142:309–319.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12165
Diabetic characteristics and alveolar bone loss in streptozotocin- and streptozotocin-nicotinamidetreated rats with periodontitis
J-H. Kim1,2,3, D-E. Lee1,2,3, S-H. Choi4, J-H. Cha1,2,3, E-J. Bak5*, Y-J. Yoo1,2* 1 Department of Applied Life Science, The Graduate School, Yonsei University, Seoul, South Korea, 2Department of Oral Biology, Yonsei University College of Dentistry, Seoul, South Korea, 3BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, South Korea, 4Department of Periodontology, Research Institute for Periodontal Regeneration, Yonsei University College of Dentistry, Seoul, South Korea and 5Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul, South Korea
Kim J-H, Lee D-E, Choi S-H, Cha J-H, Bak E-J, Yoo Y-J. Diabetic characteristics and alveolar bone loss in streptozotocin- and streptozotocinnicotinamide-treated rats with periodontitis. J Periodont Res 2014; doi: 10.1111/ jre.12165. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: Experimental models showing variable diabetic status are necessary to understand the relationship between diabetes and periodontitis. The streptozotocin (STZ)-induced diabetes model allows control of diabetic status by nicotinamide (NA), which protects against STZ-induced b-cell necrosis. Therefore, we compared diabetic characteristics and alveolar bone loss in STZand STZ-NA-treated rats with periodontitis. Material and Methods: STZ-treated rats were generated by intravenous (IV) administration of STZ (50 mg/kg). STZ-NA-treated rats were induced by intraperitoneal administration of NA (270 mg/kg) 15 min before IV administration of STZ (65 mg/kg). Periodontitis was induced by ligature around the left mandibular first molar 1 wk after injection. Blood glucose level, glucose tolerance and serum insulin levels were determined at day 0 and/or 20 after ligature. At day 20, tibia bone loss was assessed using micro-computed tomography and hematoxylin and eosin staining. Alveolar bone loss was histologically measured as the distance of the cementoenamel junction to the alveolar bone crest in distal and the percentage of periodontal ligament area in the first molar furcation, respectively. The number of inflammatory cells, receptor activator of nuclear factor kappa-B ligand (RANKL)-positive cells and the area of osteoid were determined. Results: In STZ-treated rats, obvious hyperglycemia over 300 mg/dL and severe body weight loss were observed. The insulin level was approximately 14% compared to that of control rats. STZ-NA-treated rats were impaired in glucose tolerance compared to control rats; however, body weight and insulin levels were not significantly different. Tibia bone loss was increased in STZ-treated rats, but significant change was not observed in STZ-NA-treated rats compared to control rats. In ligatured teeth, alveolar bone loss was increased in both STZand STZ-NA-treated rats compared to control rats. Alveolar bone loss, the number of inflammatory cells and RANKL-positive cells in STZ-treated rats were greater than in STZ-NA-treated rats. The area of osteoid decreased in STZ-treated rats compared to control, but not STZ-NA-treated rats.
Yun-Jung Yoo, DDS, PhD, Department of Oral Biology, Yonsei University College of Dentistry, 134 Sinchon dong, Seodaemun-gu, Seoul 120752, South Korea Tel: +82 2 2228 3060 Fax: +82 2 2227 7903 e-mail: [email protected] and Eun-Jung Bak, DVM, PhD, Oral Cancer Research Institute, Yonsei University College of Dentistry, 134 Sinchon dong, Seodaemun-gu, Seoul 120-752, South Korea Tel: +82 2 2228 3062 Fax: +82 2 2227 7903 e-mail: [email protected] *These authors contributed equally to this work. Key words: bone loss; nicotinamide;
periodontitis; streptozotocin Accepted for publication December 13, 2013
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Conclusion: These results indicate that STZ- and STZ-NA-treated rats exhibit diabetic characteristics similar to type 1 diabetes mellitus and a pre-diabetic state, respectively. In addition, alveolar bone loss in response to periodontitis and tibia loss depend on diabetic status. Diabetic status-dependent bone remodeling imbalance and inflammation could affect the alveolar bone loss in the two models. Both STZ- and STZ-NA-treated rats may be useful to investigate differences in periodontitis sensitivity associated with diabetic status and to develop therapeutic agents for periodontitis in patients with diabetes.
Diabetes is a chronic disease characterized by hyperglycemia that leads to long-term damage to several organs, including the heart, eye, kidneys, nerves, vascular system and bone. Hyperglycemia is the result of the absolute or relative lack of insulin production or resistance to insulin (1,2). Type 1 diabetes mellitus (T1DM) is present in 5–10% of patients with diabetes and is the result of b-cell destruction, which usually leads to a total loss of insulin secretion. Type 2 diabetes mellitus (T2DM) presents in 90–95% of patients with diabetes and is characterized by abnormal insulin production as well as insulin resistance, which alters the utilization of endogenously produced insulin by target cells. Periodontitis is an inflammatory disease caused by bacteria, and alveolar bone loss surrounding the roots of teeth is one of its characteristics. Alveolar bone loss is induced by an imbalance of bone resorption and bone formation caused by bacteria and a subsequent immune reaction (3,4). Several researchers have investigated the association of diabetes with periodontitis through epidemiological surveys and in vivo studies (5–9). A relationship between diabetes and the increased incidence of periodontitis has been proposed, but the exact mechanism for diabetes-related periodontitis is still unclear. In addition, it is not clear whether there is any difference in periodontitis pathogenesis depending on diabetic status. Various animal models that mimic characteristics of patients with diabetes have been generated by genetic, chemical, dietary or surgical means, or a combination of these techniques (10,11). Chemicals such as streptozo-
tocin (STZ) and nicotinamide (NA) have been demonstrated to induce experimental diabetes in rats (12). STZ has a toxic effect on pancreatic b cells, decreases insulin biosynthesis and secretion, and consequently induces hyperglycemia (13). On the other hand, a suitable dose of NA can exert partial protection against STZ’s cytotoxic effect on pancreatic b-cells (12,14). STZ-NA-induced diabetic rats have been used for diabetes drug screening (15,16). A genetic or STZ model was employed in a periodontitis study, but the STZ-NA diabetic model has never been used. Patients with diabetes show variable diabetic status, including levels of hyperglycemia and insulinopenia and the presence of osteoporosis. To understand better the mechanism that links diabetes and periodontitis and to develop new drugs and preventive strategies, experimental models showing variable diabetic status are necessary. STZ-induced diabetic model is cheaper and easier to develop compared with other diabetic models, in addition, NA can be used to control diabetic characteristics. To investigate the suitability of these animal models in the study of periodontitis pathogenesis-related diabetic status, we investigated diabetic characteristics and levels of alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis.
Material and methods Animal models
Six-wk-old male F344 rats were purchased from Oriental Bio (Gyeonggi-do, Korea) and acclimated for 1 wk. The
animals were housed in cages and were fed with a standard laboratory diet and tap water. The animals were maintained in a temperaturecontrolled room (22 2°C) on a 12 h light–dark cycle. Animals were randomly divided into a control group and two groups treated with STZ or STZ-NA (five to six animals per group). After a 15 h fast, the STZ group was given intravenous STZ (50 mg/kg; Sigma-Aldrich, St Louis, MO, USA) dissolved in 0.1 M citrate buffer (Sigma-Aldrich), the STZ-NA group was given intraperitoneal NA (270 mg/kg; Sigma-Aldrich) 15 min before STZ administration (65 mg/ kg), and the control group was injected with citrate buffer alone. At 7 d after injection, all rats were anesthetized with 1 : 2 mixtures of Zoletil 50 (Virbac, Carros, France) and Rompun (Bayer, Ansan, Korea) to induce periodontitis. Dental floss was placed around the cervix of the left mandibular first molars and knotted mesially. The contralateral right side was used as the non-ligatured control. During the experimental period, the location of dental floss, gross appearance, and food and water intake were observed daily. All animal procedure protocols were approved by the Institutional Animal Care and Use Committee of Yonsei University (2010-0209). Evaluation of diabetic characteristics
Body weight was measured every day during the experimental period under fasting and non-fasting conditions. Fasting and non-fasting blood glucose levels were measured from tail veins
Diabetic characteristics and alveolar bone loss at days 0 and 20 after inducing periodontitis. For the oral glucose tolerance test, rats were fasted for 15 h and were given glucose (2 g/kg body weight) orally using gavage. Blood glucose levels were measured with the Accu-check active system (Roche, Mannheim, Germany) in accordance with the manufacturer’s instructions 0, 30, 60, 90 and 120 min after glucose administration. Serum insulin was determined using an enzymelinked immunosorbent assay kit (Shibayagi, Gunma, Japan). Evaluation of tibia bone loss
Tibia trabecular bone loss was assessed by micro-computed tomography (micro-CT) (Skyscan, Antwerp, Belgium) and histomorphometric analysis. Tibias were scanned using micro-CT and three-dimensional images were reconstructed. The region of interest (ROI) was determined, which extended 2.5 mm at 1.5 mm below the growth plate toward the diaphysis and excluded the outer cortical shell. Trabecular bone loss was evaluated by the trabecular bone volume fraction (BVF), which was obtained by dividing the trabecular bone volume by the total ROI volume. For histomorphometric analysis, tibias were fixed in 10% neutralbuffered formalin and decalcified in 5% nitric acid for 1 wk. Three serial sections of 4 lm thickness were cut and stained with hematoxylin and eosin (H&E). The ROI for trabecular bone volume extended 1 mm at 1.5 mm below the growth plate toward the diaphysis and excluded the outer cortical shell. Trabecular BVF was calculated by data from three
serial sections using Image-Pro software (Media Cybernetics, Silver Spring, MD, USA). Evaluation of alveolar bone loss, osteoid formation and inflammatory cells infiltration
For histopathological examination, mandibles were removed after being killed and fixed in 10% neutralbuffered formalin overnight and decalcified in 5% nitric acid for 1 wk. The paraffin-embedded sections were cut at a thickness of 4 lm. Sections were selected based on clear appearance of the dental pulp of the mesial and distal roots of the first molars and were H&E stained. Alveolar bone loss in the distal and furcation areas of the first molar was examined under 100 9 magnification. Alveolar bone loss in the distal area of the first molar was examined by measuring the distance from cementoenamel junction (CEJ) to the alveolar bone crest (ABC) using Image-Pro software (Media Cybernetics). Alveolar bone loss in the furcation area was estimated by the percentage of periodontal ligament (PDL) area in the ROI. The height of the ROI was 0.8 mm from the apical furcation. To examine bone formation, the area of osteoid was measured in the distal area of the first molar under 200 9 magnification. The new osteoid was considered as unmineralized bone matrix between osteoblasts and the mineralized bone surface. For evaluation of inflammatory cell infiltration, the number of inflammatory cells including polymorphonuclear leukocytes and mononuclear cells was counted within a standardized site (0.1 mm 9 0.1 mm)
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located at the junctional epithelium and subepithelial connective tissue of the distal area of the ligated first molar. A single examiner, blind to the treatment assignment, performed the analyses using a light microscope under 500 9 magnification. Evaluation of receptor activator of nuclear factor kappa-B ligandpositive cells
The sections of mandible were mounted onto glass slides coated with silane (Muto Pure Chemicals, Tokyo, Japan), deparaffinized in xylene and rehydrated. For immunohistochemical staining, the LSAB+ system-HRP kit (Dako, Carpinteria, CA, USA) was used and all procedures followed the manufacturer’s instructions. Briefly, after endogenous peroxidase activity was blocked, sections were incubated overnight at 4°C with goat antireceptor activator of nuclear factor kappa-B ligand (anti-RANKL) antibody (1 : 500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with biotinylated link antibody and peroxidase-labeled streptavidin. The color was developed with substrate-chromogen and methyl green was used as a counterstain. RANKL-positive cells were counted in a standardized site (0.1 mm 9 0.1 mm) located in the distal area of the ligated first molar under 400 9 magnification. Statistical analyses
All statistical analyses were performed using SPSS 12.0 (SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) and Student’s
Table 1. Body weight, and fasting and non-fasting blood glucose levels in the control, STZ- and STZ-NA-treated rats with periodontitis Day 0
Day 20
Diabetes characteristics
Control
STZ-NA
Body weight (g) Fasting blood glucose (mg/dL) Non-fasting blood glucose (mg/dL)
258 13 99 5 131 8
241 10 109 9 222 105
STZ 157 8* 355 23* –
NA, nicotinamide; STZ, streptozotocin. Data are expressed as the mean SEM. *Significant difference at p < 0.05, when compared with control rats.
Control
STZ-NA
285 10 97 11 125 7
264 18 105 7 236 121
STZ 156 7* 517 28* –
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t-tests were used to determine significant differences. p < 0.05 was considered statistically significant. Data are expressed as the mean standard error (SEM).
Results Blood glucose levels and gross changes in streptozotocin- and streptozotocin-nicotinamide-treated rats with periodontitis
To estimate the induction and maintenance of diabetic characteristics, we measured body weight and fasting or non-fasting glucose levels (Table 1). In STZ-treated rats, severe body weight loss was observed compared with control rats. Moreover, their fasting blood glucose level was significantly increased by 355 23 mg/dL and 517 28 mg/dL on days 0 and 20 after ligature compared to
99 5 mg/dL and 97 11 mg/dL in control rats, respectively. In addition, classic signs of T1DM, such as polyuria, polydipsia and polyphagia were observed during the experimental period (data not shown). STZNA-treated rats showed a normal increase of body weight similar to control rats. STZ-NA-treated rats exhibited a mild increase in nonfasting blood glucose levels. During the 2 h GTT, STZ-NA-treated rats had larger areas under the curve at days 0 and 20 compared to control rats (Fig. 1A and B). Serum insulin levels in streptozotocin- and streptozotocinnicotinamide-treated rats with periodontitis
To estimate insulin secretion in STZ and STZ-NA-treated rats, we measured serum insulin levels after 15 h
of fasting at day 20 (Fig. 2). The serum insulin level of STZ-treated rats was approximately 14% that of control rats. Although serum insulin levels in STZ-NA-treated rats were 70% that of control rats, the difference was not statistically significant. Tibia bone loss in streptozotocinand streptozotocin-nicotinamidetreated rats with periodontitis
To observe skeletal bone loss, trabecular BVF of the tibia was analyzed using micro-CT and H&E staining at day 20 (Fig. 3). For micro-CT (Fig. 3A), tibia BVF in STZ-treated rats was about 28% compared to that of control rats. However, tibia BVF in STZ-NA-treated rats was not significantly different from that of control rats. These results were similar to the histomorphometric analysis (Fig. 3B). Only STZ-treated
A
B
Fig. 1. Glucose tolerance test in STZ-NA-treated rats with periodontitis. At days 0 (A) and 20 (B) after ligature, oral glucose tolerance tests were performed in STZ-NA-treated rats. The animals were orally administered with glucose (2 g/kg) after 15 h of fasting, and blood glucose levels were measured at 0, 30, 60, 90 and 120 min. AUC at days 0 and 20 were compared, and data are presented as mean SEM. *Significant difference at p < 0.05 compared with control rats. AUC, area under the curve; STZ-NA, streptozotocinnicotinamide.
Diabetic characteristics and alveolar bone loss
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A
Fig. 2. Serum insulin levels in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, serum insulin levels were estimated in STZ- and STZ-NA-treated rats by enzyme-linked immunosorbent assay. Data are represented as mean SEM. *Significant difference at p < 0.05 compared with control rats. NA, nicotinamide; STZ, streptozotocin.
rats showed a significantly reduced tibia BVF compared to control rats. Alveolar bone loss in streptozotocin- and streptozotocinnicotinamide-treated rats with periodontitis
To estimate alveolar bone loss of non-ligatured and ligatured teeth in control and treated rats, the distance of CEJ-ABC and percentage of the PDL area were measured in distal and furcation areas at day 20, respectively (Fig. 4). In non-ligatured teeth, there was no difference in either measure between treated and control rats (Fig. 4A and 4B). However, marked alveolar bone loss was present in ligatured teeth in both treated and control rats. The CEJ-ABC distance of ligatured teeth was slightly increased in STZ-NA-treated rats compared to control rats, but the difference was not significant; however, we observed a significant difference in STZtreated rats compared to control rats (Fig. 4A). The percentage of PDL area of ligatured teeth was increased in STZ-NA-treated rats as well as STZ-treated rats compared to control rats. (Fig. 4B). Inflammation of gingival tissue in streptozotocin- and streptozotocinnicotinamide-treated rats with periodontitis
To estimate the degree of inflammation, the infiltration of inflammatory
B
Fig. 3. Tibia bone loss in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, tibias were analyzed using micro-computed tomography (A) and hematoxylin and eosin staining (B). Trabecular bone loss was evaluated by trabecular BVF, which is obtained by dividing the trabecular bone volume by the total region of interest volume. Data are presented as mean SEM. *Significant difference at p < 0.05 compared with control rats (bar = 500 lm). BVF, bone volume fraction; NA, nicotinamide; STZ, streptozotocin.
cells, such as polymorphonuclear leukocytes and mononuclear cells, were counted in gingiva (Fig. 5). The number of inflammatory cells infiltrating the junctional epithelium and connective tissue were increased in ligatured teeth of the three groups com-
pared to non-ligatured teeth (Fig. 5A and 5B). In non-ligatured teeth, there was no difference in either the location between treated and control rats. In junctional epithelium of ligatured teeth, STZ-treated rats showed a marked increase of infiltration com-
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Kim et al.
A
cells were counted by immunohistochemistry (Fig. 6A). RANKL-positive cells were located more in ligatured teeth than non-ligatured teeth. The number of RANKL-positive cells was markedly increased in STZ-treated rats as well as in STZ-NA-treated rats compared to control rats. The number of RANKL-positive cells was higher in STZ-treated rats than in STZ-NA-treated rats. To estimate coupled bone formation after bone resorption, the area of osteoid was measured (Fig. 6B). In non-ligatured teeth, there was no difference in the area between treated and control rats. However, a significant increase was evident in ligatured teeth of control and STZ-NA-treated rats, but not in STZ-treated rat compared with each non-ligatured teeth. The area of osteoid in ligatured teeth of control and STZ-NA-treated rats was 11.8fold and 9.4-fold larger than STZ-treated rats.
B
Discussion
Fig. 4. Alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, alveolar bone levels of non-ligatured and ligatured first molars were examined by light microscopy (100 9 magnification). Alveolar bone loss in distal and furcation areas of teeth was estimated by the distance of CEJ-ABC (A) and the percentage of PDL area to region of interest area (B), respectively. The height of the region of interest was 0.8 mm from the apical furcation. Data are presented as mean SEM. *Significant difference at p < 0.05 compared with non-ligatured teeth. #Significant difference at p < 0.05 (bar = 100 lm). ABC, alveolar bone crest; CEJ, cementoenamel junction; NA, nicotinamide; PDL, periodontal ligament; STZ, streptozotocin.
pared to control rats, but STZ-NAtreated rats were not significant (Fig. 5A). In connective tissue of ligatured teeth, infiltration of inflammatory cells was increased in both STZ-NA- and STZ-treated rats compared to control rats (Fig. 5B).
RANKL expression and osteoid formation in streptozotocin- and streptozotocin-nicotinamide-treated rats with periodontitis
To identify expression of osteoclast differentiation factor, RANKL-positive
In this study, we compared diabetic characteristics and alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis. We observed blood glucose levels and glucose tolerance, as well as insulin levels. STZ has a selective toxic effect on pancreatic b-cells and causes hyperglycemia due to absolute insulin deficiency. STZ is known to cause alkylation of DNA strands and a consequent increase in the activity of poly-ADP-ribose polymerase. This leads to the depletion of NAD, which results in reduced insulin synthesis in b-cells and cell necrosis (13). This cytotoxicity is prevented by NA, a component of NAD, which inhibits poly-ADP-ribose polymerase and prevents NAD depletion in b-cells (12,14). Therefore, the administration of NA lessens the severity of diabetic status by STZ (14). In this study, STZ-treated rats showed characteristics that resembled T1DM, including obvious hyperglycemia, body weight loss and markedly decreased serum insulin levels. While STZ-NA-treated rats did not show significant differences in fasting or non-fasting glucose levels or serum insulin level compared to control rats,
Diabetic characteristics and alveolar bone loss
A
B
Fig. 5. Inflammation in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, infiltration of inflammatory cells, such as polymorphonuclear leukocytes and mononuclear cells, were examined in a standardized site (0.1 mm 9 0.1 mm) located in junctional epithelium (A) and subepithelial connective tissue (B) of the distal area of the non-ligatured and ligatured first molar (500 9 magnification). Data are represented as mean SEM. *Significant difference at p < 0.05 compared with non-ligatured teeth. # Significant difference at p < 0.05. NA, nicotinamide; STZ, streptozotocin.
they showed slightly increases in nonfasting glucose levels. In the oral glucose tolerance test, STZ-NA-treated rats showed impaired glucose tolerance compared with control rats. These results indicated that STZ- and STZ-NA-treated rats have characteristics similar to the T1DM and prediabetic state, respectively. Bone loss is one of the complications in diabetes. It has been proposed that the incidence of bone loss is different between T1DM and T2DM (17–20). The majority of T2DM studies have determined that bone mineral density is normal or increased. Conversely, 50% of T1DM patients have lower bone mass than expected, and approximately 20% meet the criteria for osteoporosis.
T1DM studies have reported that late bone turnover is associated with increased bone resorption and decreased bone formation. These indicate that the prevalence of bone loss is influenced by diabetic status. To investigate skeletal bone loss in both treated rats, we measured tibia BVF using micro-CT and H&E staining. We observed a significant decrease of tibia BVF in STZ-treated rat, but not STZNA-treated rats in both analyses. In STZ-NA-treated rats, the absence of an effect on tibia BVF could be due to the short time of induction period which STZ-NA-treated rats were in the pre-diabetic state. For exact evaluation on bone loss in pre-diabetic state, long-term study of the chronic state will be needed. Taken together,
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these results suggest that STZ-treated rats are useful for studying T1DM accompanied by osteoporosis. To compare the effect of diabetic status on alveolar bone loss in response to periodontitis, we investigated alveolar bone loss of ligatured teeth by measuring CEJ-ABC distances in distal areas and the percentage of PDL areas in furcation. There were no differences among the groups for non-ligatured teeth. However, alveolar bone loss in ligatured teeth was increased in treated rats compared to control rats. Interestingly, STZtreated rats showed that alveolar bone loss occurred more than in STZ-NAtreated rats. These results indicate that STZ- and STZ-NA-treated rats show severe and mild alveolar bone loss in response to periodontitis, respectively. Alveolar bone loss in response to periodontitis is caused by an imbalance of bone resorption and bone formation (3,4). As the different degree of alveolar bone loss dependent on diabetic status, we estimated the RANKL expression and the area of osteoid associated with bone resorption and bone formation (21,22). In ligatured teeth, the number of RANKL-positive cells was increased in treated rats and was higher in STZ-treated rats than in STZ-NAtreated rats. The area of osteoid, an indicator of newly formed bones, was significantly increased in ligatured teeth of control and STZ-NA-treated rats, but not in STZ-treated rats compared to each non-ligatured teeth. These results suggest that the degree of RANKL expression and osteoid formation in response to periodontitis affects alveolar bone loss in the two models. The alveolar bone loss in STZ-treated rats might be caused by increased bone resorption and depressed coupled bone formation. The diminished alveolar bone loss in STZ-NA-treated rats may be due to the maintenance of normal bone formation via a protective effect of NA against STZ-induced b-cell damage. Inflammation is one factor related to bone loss (23). Ligation raised the inflammatory cells infiltration in both treated and control rats. While STZ-treated rats showed higher
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Kim et al. may be related to the enhancement of bone resorption and the depression of bone formation in STZ-treated rats. In conclusion, this study is the first to compare diabetic characteristics and alveolar bone loss in STZ- and STZ-NA-treated rats with periodontitis. Our results indicate that STZ- and STZ-NA-treated rats exhibit pathologies similar to T1DM and pre-diabetic state, respectively, and suggest that the level of bone loss in alveolar bone and tibia depend on diabetic status. Diabetic status-dependent bone remodeling imbalance and inflammation could contribute to the difference in the degree of alveolar bone loss in the two models. Evaluations using different diabetic models would provide valuable comparative information on the relationship between diabetes and periodontitis. STZ- and STZ-NAtreated rats may be useful to study variations in periodontitis pathogenesis depending on diabetic status and for developing therapeutic agents.
A
B
Acknowledgements Fig. 6. RANKL expression and area of osteoid in STZ- and STZ-NA-treated rats with periodontitis. At day 20 after ligature, the number of RANKL-positive cells was counted in a standardized site (0.1 mm 9 0.1 mm) located in the distal area of the non-ligatured and ligatured first molar (A; 400 9 magnification). The area of osteoid, unmineralized bone matrix between osteoblasts and the mineralized bone surface, was measured in distal area of the first molar under 200 9 magnification (B). Data are presented as mean SEM. *Significant difference at p < 0.05 compared with non-ligatured teeth. # Significant difference at p < 0.05. NA, nicotinamide; RANKL, receptor activator of nuclear factor kappa-B ligand; STZ, streptozotocin.
inflammatory cells infiltration in both junctional epithelium and connective tissue compared to control, STZ-NAtreated rats displayed it in connective tissues. These results indicate that the severity of inflammation is influenced by diabetic status. Various cells including osteoblasts, fibroblasts, lymphocytes and neutrophils produce RANKL (24,25). In addition, cytokines, such as interleukin (IL)-1, IL-6 and tumor necrosis factor, produced by inflammatory cells affect bone level by stimulating osteoclast differentiation and suppressing osteoblast differentiation (23). Thus, increased inflammation in STZ-treated rats may be one factor that leads to a net alveolar bone loss. A previous study on
diabetic complications reported that accumulation of irreversible advanced glycation end products (AGEs) as a result of hyperglycemia may contribute to enhancing inflammation (7,26). AGE deposition was elevated in gingival tissue of diabetic patients with periodontitis (27). These suggest that AGE deposition in gingival tissue may be related to enhancement of inflammation in STZ-treated rats. As well as enhancing the inflammation, AGEs increase RANKL expression and decreased collagen type I, osteocalcin and alkaline phosphatase expression in osteoblast (28,29). In addition, insulin promotes osteoblast differentiation (30). Therefore, AGE accumulation and insulin deficiency
The authors thank Jang Gi Cho, Department of Oral Biology, Yonsei University College of Dentistry, for assistance with the micro-CT analysis and graphics. This study was supported by a faculty research grant of Yonsei University College of Dentistry for 2012 (6-2012-0062).
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