Spontaneous Alveolar Bone Loss Development by Alloxan-induced Diabetes Mellitus in Rats Diabetes Mellitus and Alveolar Bone Loss Daniela Martins de Souza1*, Ana Carolina Medeiros2, Juliana Oliveira Santos2, MĂĄrcia Costa Marques Lima2, Alan de Aquino Silva3, Claudemir de Carvalho4 Periodontist, Titular Professor of the Dentistry Course. Faculty of Pindamonhangaba-Christian Life University Foundation, Pindamonhangaba, Sao Paulo, Brazil 1*
Dentistry graduate student. Faculty of Pindamonhangaba-Christian Life University Foundation, Pindamonhangaba, Sao Paulo, Brazil 2
Dentistry undergraduate student, Faculty of Pindamonhangaba-Christian Life University Foundation, Pindamonhangaba, Sao Paulo, Brazil 3
Titular Professor of Dentistry Course, Faculty of Pindamonhangaba-Christian Life University Foundation, Pindamonhangaba, Sao Paulo, Brazil 4
*
danimart.voy@gmail.com
Abstract Diabetes mellitus is associated with the occurrence and severity of alveolar bone loss. This study evaluated the effect of alloxaninduced diabetes mellitus on the percentage of remaining periodontal bone support (PBS) and periodontal bone loss (PBL) in adult rats. Twenty female rats were injected with alloxan diluted at 0.2% in a 0.05 M citrate buffer, pH 4.5 (150 mg/kg, i.p.) and eight of them became diabetic. The glycaemic indexes were investigated weekly. After 42 days, the rats were euthanized and left mandibles were radiographed to measure the PBS. The same left mandibles were defleshed and stained and PBL was evaluated morphometrically by measuring the distance between the cementoenamel junction and alveolar bone crest. The comparison between the diabetic and control groups showed that the induced diabetes provoked statistically lower percentage of PBS and higher periodontal bone destruction (p<0.05). In conclusion, the induction of diabetes mellitus in adult rats may cause alveolar bone loss and reduce the remaining periodontal bone support. Keywords Alveolar Bone Loss; Periodontal Bone Support; Periodontal Disease; Diabetes Mellitus; Induced Diabetes; Alloxan
Introduction The supporting structures of the teeth include the periodontal ligament, alveolar bone, and gingival tissues [1]. Periodontal disease is an infectious pathology with a limited number of specific bacteria required, but not sufficient for its onset, and host factors are major determinants for the disease occurrence and progression [2]. Many factors influence the periodontal disease progression, including social and behavioural factors, systemic, genetic, and dental factors [1]. Studies suggest that diabetes is a risk factor for periodontal disease and the prevalence, incidence, and severity of periodontal disease are higher among individuals with diabetes than in healthy individuals [3, 4]. Type II diabetic individuals were 11 times more at risk for loosing alveolar bone during a two-year period than a non-diabetic person. Furthermore, adults with diabetes are 2.9 times more at risk to have periodontitis than nondiabetic adults. On the other hand, individuals with controlled diabetes do not have a significantly higher risk of developing periodontitis [2].
International Journal of Advance in Medical Science, Vol. 3, No. 2â&#x20AC;&#x201D;November 2015 2327-7238/15/02 037-8 Š 2015 DEStech Publications, Inc. doi:10.12783/ams.2015.0302.01
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Daniela Martins de Souza, Ana Carolina Medeiros, Juliana Oliveira Santos, Márcia Costa Marques Lima, Alan
de Aquino Silva, Claudemir de Carvalho
The presence of diabetes influences the periodontal tissues by altering neutrophil function and collagen synthesis, inducing vascular abnormalities, and through genetic predisposition [5]. The use of rat models in the evaluation of periodontal pathogenesis has been applied because of the similarity of the structure of the periodontal tissue between rats and humans [6] and the influence of risk/indicator factors, such as oestrogen deficiency [7], alcohol consumption [8,9] and diabetes induction [10] on disease progression. There is substantial information on potential mechanistic pathways that support a close association between diabetes and periodontitis, but there is a real need for longitudinal clinical studies using larger patient groups together with animal model and cell/tissue in vitro studies [4]. Considering that more studies are necessary to test the hypothesis that diabetes can increase periodontal bone loss, this study aimed to investigate the effect of alloxan-induced diabetes mellitus on alveolar bone level in adult rats. Materials and Methods Animals A total of 30 female Wistar rats (200-275g) were obtained from the animal house of the School of Pindamonhangaba. The animals were acclimatized for 10 days before the onset of the experiment and were housed in a temperaturecontrolled (23 ± 1ºC) animal facility under a 12-hour (7pm to 7am) light/dark cycle, with animal laboratory chow and water. The experimental protocols were carried out following ethical principles for laboratory animal study and approved by the Institutional Animal Research Committee (Protocol CEEA 024/13). Diabetes Induction Twenty rats that were pre-investigated for normal glycaemic condition were injected, after a 24-hour fast, with a single intraperitoneal dose of freshly prepared alloxan [alloxan monohydrate [2,4,5,6 (1H, 3H) Pirimidintotrone, 150 mg/kg body weight, i.p.] diluted at 0.2% in a 0.05 M citrate buffer, pH 4.5 (Sigma Aldrich, St. Louis, MO USA) to induce diabetes. Six hours after alloxan injection, 10% glucose solution was offered as a unique hydric source for 24 hours to avoid fatal hypoglycaemia as a consequence of massive insulin liberation that occurs after ß cells destruction. Glucose concentrations (expressed as mg/dL) were measured at the seventh day after alloxan injection with a commercial Accu-Check Active® glucometer (Roche Diagnostics GmbH, 68298 Mannheim, Germany) with blood obtained by puncture of the tail vein. Only eight rats with blood glucose concentrations of 200 mg/dL and body weight loss were considered severely diabetic. Necropsy After 42 days, the rats were euthanized by administration of ketamine (80 mg/kg) and xylazine (8 mg/kg). Their mandibles were carefully removed and fixed in 10% neutral (pH 7.4) formalin for 48h. Radiographic Analysis The left mandible was positioned so that buccal and lingual cusps of the first and second molars were superimposed and splinted on the sensor by a plastic slab. Digital radiographs were obtained using an Intra-oral Xray System (Kodak 2.200 radiology Inc.-Marietta/USA) that uses X-Scan Duo-Micro image capture system through phosphor plates. Sensors were exposed at 65 Kv and 7 mA for 0.1 s and the source-to-sensor distance was always 30cm. To obtain sufficient reproducibility of the image alignment, two criteria had to be fulfilled: the teeth should not overlap inter-proximally with each other and the tip of the buccal cusp of the first and second molars should be superimposed on the corresponding lingual cusp tip. Periodontal bone support (PBS) was measured on radiograph images through Image Tool software v.3.0 (UTHSCSA, San Antonio, TX, USA). All measurements were made at the distal surface of the mandibular first molars by one trained examiner, who did not have knowledge of the group to which the specimen was assigned.
Spontaneous Alveolar Bone Loss Development by Alloxan-induced Diabetes Mellitus in Rats
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Three points were taken as references: the apex of the distal root (A), the tip of the distal cusp (C) and the bottom of the deepest bony defect distal to the tooth (B). Afterwards, the apex-cusp tip (AC) and apex-deepest bone defect (AB) distances were measured in mm. Periodontal bone support was determined by the following formula: PBS=AB/AC x100 [11] (Fig.1).
FIGURE 1. PHOTOGRAPH OF ALVEOLAR BONE LOSS EVALUATED MORPHOMETRICALLY BY MEASURING THE LINEAR DISTANCE BETWEEN THE CEMENTOENAMEL JUNCTION AND ALVEOLAR BONE CREST AT THREE LINGUAL SITES ON THE FIRST MANDIBULAR MOLAR.
Morphometric Analysis The same left mandibles were defleshed and stained with aqueous methylene blue solution (1.0%) in order to differentiate bone from teeth. Periodontal bone loss (PBL) was evaluated morphometrically by measuring the distance between the cementoenamel junction (CEJ) and alveolar bone crest (ABC). The specimens were photographed in Microscope Axiostar (Carl Zeiss, Oberkochen, Germany) (50X magnification) and PBL was measured on images by AxioVision Rel.4.6 Software (Carl Zeiss, Oberkochen, Germany). The method of measuring distance on digitized images described by Crawford et al. [12] was used to perform linear measurements from the CEJ to the ABC, on half of each root following the axis at seven lingual sites in each mandible. Three measurements were obtained for the first molar, two for the second molar, and two for the third molar. This method can be observed from the lingual aspect of the left mandible (Fig.2). The mean of these measurements per tooth was used to obtain the mean alveolar bone loss in each group. All measurements were made by the same trained examiner, who did not know the origin of the specimens.
FIGURE 2. PHOTOGRAPH OF LINGUAL ASPECT OF MANDIBLE (FIRST, SECOND, AND THIRD MOLARS) (X50). THE LINES INDICATE THE SEVEN DISTANCES MEASURED ON THE TEETH.
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Daniela Martins de Souza, Ana Carolina Medeiros, Juliana Oliveira Santos, MĂĄrcia Costa Marques Lima, Alan
de Aquino Silva, Claudemir de Carvalho
Reproducibility Before the measurement of animal groups, one examiner performed double measurements on 25% of the specimens with a one-week interval. There were no differences between mean values for the radiographic method (p=0.4479) and the morphometric method (p=0.3976). Additionally, Pearsonâ&#x20AC;&#x2122;s correlation coefficient was determined for the two sets of measurements indicating a very high correlation in both the radiographic (99%: r=0.9977, p = 0.000) and morphometric methods (99%: r=0.9986, p = 0.000). Statistical Analysis During the experimental period, the glycaemic indexes were evaluated as the mean and standard deviation for each group at different periods (pre-induction, diabetes confirmation, and six-week following-up). The paired t test (p<0.05) was used for comparisons of blood glucose before alloxan injection (pre-induction) and seven days after to confirm diabetes. The one-way analysis of variance (ANOVA) and Tukey test for subsequent multiple comparisons (p<0.05) were used to determine significant differences in glucose concentrations between the test and control groups at one week, subsequent weeks between one and six weeks, and at six weeks. The independent t test (p<0.05) was used for comparisons of glycaemic indexes between test and control groups at all periods. For radiographic (%) and morphometric (mm) analyses, the data were expressed as mean and standard deviation. The independent t test (p<0.05) was used to determine significant differences in periodontal bone support between treated (diabetic) and control (non-diabetic) groups. Results Glycaemic Analysis There were no significant differences (p>0.05) in glucose concentrations between test and control groups prior to induction of diabetes (Table 1). TABLE 1. MEAN AND STANDARD DEVIATION OF GLUCOSE CONCENTRATIONS (MG/DL) FOR TEST AND CONTROL GROUPS AT THE PRE-INDUCTION AND DIABETES-CONFIRMATION PERIODS.
Pre-induction diabetes 103.4 (9.3)* 103.0 (5.2) 0.4611
Diabetes Control p**
Diabetes confirmation 290.3 (132.9)** 104.9 (4.6) 0.0028
p* 0.0029 0.1213
* paired t-test (p<0.05) in rows and ** independent t test (p<0.05) in columns.
Glucose concentrations were significantly different (p=0.0029) between the pre-induction and diabetes confirmation periods in the test group and not different (p=0.3424) between the pre-induction and diabetes confirmation periods in the control group (Table 1). At the confirmation and six-week follow-up periods, glucose concentrations were significantly different (p=0.0028) between the diabetic and non-diabetic groups (p<0.05) (Tables 1 and 2). During the six-week period, no significant differences among blood glucose values (p=0.7830) were observed in the control group. However, when diabetes was induced, analysis revealed greater glycaemic values (p<0.05; p=0.0000) in the alloxan-treated rats at the third, fourth, fifth and sixth weeks than at the first and second weeks (Table 2). TABLE 2. MEAN AND STANDARD DEVIATION OF GLUCOSE CONCENTRATIONS (MG/DL) FOR TEST AND CONTROL GROUPS AT SIX-WEEK FOLLOWING-UP PERIOD.
Weeks
1st
2nd
3rd
4th
5th
6th
Diabetes
332.6 Aa (108.3)
393.3 Aa (92.2)
512.5 Ba (70.1)
497.8 Ba (71)
Control
102.5 Ab (7.6)
103.1 Ab (4.4)
101.4 Ab (3.2)
101.3 Ab (2.5)
528.1 Ba (73.1) 101.0 Ab (3.0)
574.3 Ba (38.3) 103.5 Ab (2.7)
Uppercase letters should be considered in rows (ANOVA, p < 0.05). Lowercase letters should be considered in columns (independent t test, p < 0.05). Means followed by different letters differ statistically.
Spontaneous Alveolar Bone Loss Development by Alloxan-induced Diabetes Mellitus in Rats
41
Radiographic Analysis Data analysis indicated significant differences (p=0.0201) in the percentage of PBS for diabetic rats compared with the control group. When diabetes mellitus was induced, the analysis revealed less periodontal bone support in the diabetic rats (57.59 ± 2.24%) than in the control group (65.70 ± 8.86%) (Table 3). TABLE 3. MEAN AND STANDARD DEVIATION OF PERIODONTAL BONE SUPPORT (PBS; %) AND PERIODONTAL BONE LOSS (PBL; MM) FOR TEST AND CONTROL GROUPS.
Diabetes Control p*
PBS (%)
PBL (mm)
57.59 (2.24)* 65.70 (8.86) 0. 0201
1.30 (0.27)* 1.09 (0.09) 0,0441
* Independent t test (p<0.05) in columns
Morphometric Analysis Analysis revealed greater morphometric periodontal bone destruction (p=0.0441) in the diabetic group (1.30 ± 0.27 mm) compared with the control group (1.09 ± 0.09 mm) (Table 3). Discussion The present study was designed to evaluate the effect of alloxan-induced diabetes on the level of alveolar bone in adult rats. Our study demonstrated that this animal model was capable of evaluating the association between diabetes and alveolar bone level, showing spontaneous alveolar bone loss following induction of experimental diabetes in rats. Several methods have been applied to assess periodontal bone loss in rats: morphometric analysis in defleshed jaws [9,13,14], histometric evaluation at the furcation region [15,16], and radiographic analysis [8,17]. However, morphometric methods of assessing alveolar bone level are not capable of detecting intra-osseous defects. Therefore, an additional radiographic method was proposed by Klausen et al. [11] and both were used in this study. Rats and mice are commonly used as animal models to study diabetes. Claudino [18] induced diabetes in rats by intravenous administration of alloxan into the caudal vein; He et al. [19] used C57BL Db/db mice that developed diabetes at six to eight weeks of age to study decreased osteoclastgenesis in bacteria-stimulated bone loss. Type II diabetic model Goto-Kakizaki (GK) and normal glycaemic Wistar rats were used by Placios [20] to study diabetesaggravating periodontitis. Jiang [10] and Tesseromatis [21] induced diabetes in rats by injection of freshly prepared streptozotocin to study the dynamic changes in the levels of TNF-α, IL-1β and LPS in the gingival crevicular fluid and to study morphological changes of gingiva, respectively. Claudino et al. [18] evaluated adult male Wistar rats injected with a single dose of 42 mg alloxan/kg of body weight into the caudal vein. Only rats having two successive determinations of blood glucose concentrations (7 and 14 days after alloxan injection) greater than 250 mg/dL were considered diabetic and were included in the experiment. The glucose concentrations during the experiment were typically 400–450 mg/dL in diabetic rats, while serum glucose concentrations of control rats ranged from 90 to 130 mg/dL. Similarly, the present study also used alloxan, but glucose concentrations ranged from 333 to 574 mg/dL in diabetic rats, while serum glucose concentrations of control rats were 101-105 mg/dL. Kodama et al. [17] observed induced or enhanced periodontal disease and dental caries in diabetic and nondiabetic rats. The examination showed that dental caries progressed from acute to sub-acute inflammation due to bacterial infections and necrosis in the pulp when the caries penetrated the dentin. In the most advanced stage of dental caries, inflammatory changes caused root abscess and subsequent apical lesion. The results suggest that diabetic conditions enhance dental caries and that periodontal apical lesions were secondary to dental caries. The authors observed that diabetes did not induce or enhance periodontal disease, but enhanced the incidence and severity of caries in rats. In contrast, the present study showed reduction of alveolar bone following the induction of diabetes in rats.
42
Daniela Martins de Souza, Ana Carolina Medeiros, Juliana Oliveira Santos, Mรกrcia Costa Marques Lima, Alan
de Aquino Silva, Claudemir de Carvalho
Salvi et al. [22] reported an enhanced expression of cytokines in vitro that are capable of stimulating bone resorption in diabetes mellitus patients. These include pro-inflammatory mediators such as IL-1, TNF, IL-6, and prostaglandin E2. It has been proposed that the higher level of cytokine expression associated with diabetes is, in part, due to the formation of advanced glycation end products. Lalla [13] affirmed that blockade of advanced glycation end products reduces the over expression of these cytokines and inhibits alveolar bone loss stimulated by Porphyromonas gingivalis (Pg) in diabetic mice. Because of the association between diabetes and higher levels of cytokines, particularly in response to bacterial products, it has been suggested that the increased risk and greater severity of periodontal disease in diabetics may be due to enhanced inflammation and bone resorption. According to Sugiyama et al. [14], evaluation of alveolar bone by the morphometric method showed that periodontal tissue resorption was enhanced in non-diabetic and diabetic groups orally challenged with Pg, with this effect being significantly greater in the diabetic group. Diabetes alone did not induce or enhance alveolar bone loss in rats. In contrast, the present study showed reduction of alveolar bone in diabetic rats. Jiang [10] reported resorption values of alveolar bone in the upper second molar of four groups of rats at different time points. The rats with periodontal disease (PD) and diabetes mellitus (DM) showed more alveolar bone loss than those with PD alone, DM alone, and control rats at the same time point. Animals with PD only had greater resorption values than those with DM alone (p < 0.05) and control rats (p < 0.05). Additionally, DM rats had higherresorption values compared with the control rats (p < 0.05). Similarly, our study showed spontaneous increase of alveolar bone loss in diabetic compared with non-diabetic rats. He et al. [19] examined pathologic remodelling initiated by an inflammatory stimulus (microorganism inoculation) in an animal model of type II diabetes. Under these conditions, they observed the inhibition of osteoclast genesis and resorption in the diabetic animals. Both parameters were decreased in the diabetic group. Diabetes also suppressed reparative bone formation measured histologically by the expression of osteocalcin. The impact of diabetes on new bone formation coincided with that of diabetes on apoptosis of bone-lining cells. Thus, diabetes may cause a loss of bone because the suppression of bone formation is greater than the suppression of bone resorption. Liu et al. [23] observed that diabetes increased the intensity and duration of the inflammatory infiltrate (p < 0.05). Thus, diabetes caused a more persistent inflammatory response, greater loss of attachment, greater alveolar bone resorption, and reduction of new bone formation. Conclusion In conclusion, alloxan-induced diabetes mellitus may cause alveolar bone loss and reduce periodontal bone support in adult rats. Further studies must be carried out to improve knowledge on the interaction between diabetes and alveolar bone loss in rats, which may serve as a basis for the development of more effective strategies for prevention and treatment of periodontitis in diabetic patients. ACKNOWLEDGMENT
The authors thank Ronald Lima for providing the Intra-oral X-ray System (Kodak 2.200) and for his technical support. They also thank the Dentistry Department of the University of Taubate-UNITAU for providing the Microscope Axiostar (Carl Zeiss, Oberkochen, Germany). REFERENCES
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[10] Jiang, Z. L., Cui, Y. Q., Gao; R., Li, Y., Fu, Z. C., Zhang, B. et al., A Study of TNF-α, IL-1β and LPS levels in the gingival crevicular fluid of a rat model of diabetes mellitus and periodontitis, Dis Markers, Vol. 34, Nº 5, 2013, pp. 295-304. [11] Klausen, B., Evans, R. T., Sfintescu, C., Two complementary methods of assessing periodontal bone level, Scand J Dent Res, Vol. 97, 1989, pp. 494-499. [12] Crawford, J. M., Taubman, M. A., Smith, D. J., The natural history of periodontal bone loss in germfree and gnotobiotic rats infected with periodontopathic microorganisms, J Periodontol Res, Vol. 13, Nº 4, 1978, pp. 316-325. [13] Lalla, E., Lamster, I., Feit, M., Huang, L., Spessot, A., Qu, W., et al., Blockade of RAGE suppresses periodontitis associated bone loss in diabetic mice, J Clin Invest, Vol. 105, 2000, pp. 1117-1124. [14] Sugiyama, S., Takahashi, S., Tokutomi, F., Yoshida, A., Kobayashi, K., Yoshino, F., et al., Gingival vascular functions are altered in type 2 diabetes mellitus model and/or periodontitis model, J Clin. Biochem. Nutr,, Vol. 51, Nº 12, 2012, pp. 108113. [15] Vasconcelos, D. F. P., Silva, M. A. D., Marques, M. R., Gibilini, C., Vasconcelos, A. C. C. G., Barros, S.P., Effects of simultaneous nicotine and alcohol used in periodontitis progression in rats: A histomorphometric study. J Clin Exp Dent, Vol. 5, Nº 2, pp. 95-99. [16] Souza, D. M., Ricardo, L. H., Rocha, R. F., Effects of alcohol intake in periodontitis progression in female rats: A histometric study. Braz J Oral Sci, Vol. 13, Nº 3, 2014, pp. 229-234. [17] Kodama, Y., Matsuura, M., Sano, T., Nakahara, Y., Ozaki, K., Narama I., et al., Diabetes enhances dental caries and apical periodontitis in caries-susceptible WBN\KobSlc rats. Comparative Medicine, Vol. 61, Nº 1, 2011, pp. 53-59. [18] Claudino, M., Gennaro, G., Cestari, T. M., Spadella, C. T., Garlet, G. P., Assis, G. F., Spontaneous periodontitis development in diabetic rats involves an unrestricted expression of inflammatory cytokines and tissue destructive factors in the absence of major changes in commensal oral microbiota. Experimental Diabetes Research, 2012, Article ID 356841, 10 pages. [19] He, H., Liu, R., Desta, T., Leone, C., Gerstenfeld, L. C., Graves, D. T., Diabetes causes decreased osteoclastogenesis, reduced bone formation and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss, Endocrinology, Vol. 145, Nº 1, 2004, pp. 447-452. [20] Pacios, S., Kang, J., Galicia, J., Gluck, K., Patel, H., Ovaydi-Mandel, M., et al., Diabetes aggravates periodontitis by limiting repair through enhanced inflammation, The FASEB Journal, Vol. 26, 2012, pp. 1423-1430. [21] Tesseromatis, C., Kotsiou, A., Parara, H., Vairaktaris, E., Tsamouri, M., Morphological changes of gingive in streptozotocin diabetic rats. International Journal of Dentistry, Vol. 2009, 2009, pp. 1-4. [22] Salvi, G., Yalda, B., Collins, J., Jones, B. H., Smith, F. W., Arnold, R. R., et al., Inflammatory mediator response as a potential risk marker for periodontal diseases in insulin-dependent diabetes mellitus patients. J Periodontol, Vol. 68, 1997, pp. 127-135.
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Daniela Martins de Souza, Ana Carolina Medeiros, Juliana Oliveira Santos, Márcia Costa Marques Lima, Alan
de Aquino Silva, Claudemir de Carvalho [23] Liu R., Bal H.S., Desta T., Krothapalli N., Alyassi M., Luan Q., et al., Diabetes enhances periodontal bone loss through enhanced resorption and diminished bone formation. Journal Dent Res. 2006, Vol. 859, Nº 6, pp. 510-514. D. M. Souza received the Bachelor and M.Sc degrees in Dentistry from Federal University of Rio Grande do Sul, Rio Grande do Sul Satate, Brazil, in 1995 and 2001 respectively. She received the Ph.D. degree in Oral Biopathology from Paulista State University Júlio de Mesquita Filho, São Paulo Satate, Brazil in 2006. Dr. D. M. Souza research interests are dentistry, focusing on Risk factors for periodontal disease and Oral Radiology, acting on the following subjects: subtraction radiography and intra oral radiographic techniques; ethanol consumption, diabetes mellitus, obesity and alveolar bone loss in rats.