Experimental animals

Twenty-four male Wistar rats weighing 100 to 130 g were used. They were divided into 3 groups of 8 animals each:

• I.

  Experimental orthodontics (ORT)

• II.

  Experimental diabetes and orthodontics (DBT + ORT)

• III.

  Experimental diabetes treated with insulin and experimental orthodontics (DBT + INS + ORT)

The animals were kept in groups of 4 in each cage. They were given regular rat chow and water ad libitum . The animals were kept under 12-hour light-dark cycles (light 7:00 am to 7:00 pm ), at room temperature (18–22°C) and relative humidity (40%–60%). All procedures for treatment and maintenance of the animals were conducted in keeping with internationally accepted guidelines (“Guidelines for the care and use of laboratory animals”, NIH Publication, 1985) and were approved by the Ethics Committee of the School of Dentistry of the University of Buenos Aires.

Experimental diabetes induction

Diabetes was induced on day 1 of the experiment in groups II and III by administering a single intraperitoneal injection of streptozotocin (STZ) 60 mg/kg body weight (Sigma-Aldrich, Inc., St. Louis, Mo), dissolved in freshly prepared citrate buffer pH 4.0. STZ is a cytotoxic agent that is relatively selective against pancreatic β cells in animals, causing an irreversible diabetic state. Therefore, diabetes induced by this method is similar to type 1 diabetes, which is insulin-dependent or insulin-requiring diabetes. The animals in the nondiabetic group (group I) received an equivalent volume of citrate buffer. The orthodontic device was applied 6 weeks after diabetes was induced.

Sampling and determination of blood glucose

Twenty-four hours after administration of STZ in the diabetic groups and administration of the vehicle in the control group, blood samples were obtained from all animals for quantitative determination of blood glucose using the glucose oxidase method with Accu-Chek Sensor Comfort test strips (Roche Products Ltd, Santiago, Chile) in the Accu-Chek Sensor (Roche Diagnostics GmbH, Mannheim, Germany). Only animals with blood glucose levels higher than 250 mg/dL were included in the different groups of experimental diabetes, and only those with blood glucose levels less than 120 mg/dL were included in group I.

Determinations were repeated once a week in non–insulin treated groups, to ensure that glycemic levels were as expected, and every 48 hours in insulin-treated rats. All the animals included in the study met the inclusion criteria corresponding to their particular group.

Insulin treatment

Insulin therapy was initiated in the insulin-treated diabetic group the day after STZ injection by daily administration of human NPH insulin subcutaneously (Humulin N, Eli Lilly y Compañía de México S.A. de C.V., Mexico D.F., Mexico) according to the requirements of each animal.

Installation of the orthodontic appliance and sample collection

Orthodontic forces were applied to all animals the first day of the seventh week of the experiment. The orthodontic appliance was specifically designed for the rat and meets the basic standards applied in the clinical setting. It consists of 2 stainless steel molar bands with tubes welded to their palatal aspect. These bands are cemented to the upper first molars of rats with glass ionomer cement. A 0.014-in circular cross-section stainless steel wire was shaped into a helical spring to exert a force magnitude of 120 ± 15 g toward the vestibular plate ( Fig 1 ).

Photograph showing the orthodontic device before and after being placed in the rat.

The animals were anesthetized with a xylazine and ketamine solution (Kensol, Laboratorios König SA, Buenos Aires, Argentina; Ketamine 50, Holliday-Scott SA, Buenos Aires, Argentina) in order to place the device. Forty-eight hours after placement of the orthodontic device and after measuring blood glucose levels, all the animals were killed (day 44 of the experiment) by an intraperitoneal overdose of sodium thiopental (150 mg/kg) (Pentovet, Richmond Laboratorios, Buenos Aires, Argentina) and acepromazine maleate (3 mg/kg) (Acedan, Holliday-Scott SA, Buenos Aires, Argentina).

Immediately after death, body weight was recorded and the maxillae were excised and fixed in formalin buffer (pH 7.0) for 48 hours. After this time, the samples were carefully dissected and decalcified by immersion in a solution of 10% EDTA (ethylenediaminetetraacetic acid), pH 7, for 30 days. The samples were then processed using routine histologic techniques. Buccopalatal and longitudinally oriented sections of the mesial root of the upper first molar were obtained under stereoscopic microscope; the blocks were then reoriented under stereoscopic microscope, changing the angulation of the platen of the microtome to obtain sections of the distal roots, which were also buccopalatal and longitudinally oriented. All the sections were oriented in an apicocoronal direction, parallel to the long axis of the corresponding root. All the sections were stained with hematoxylin and eosin.

Digital photographs were taken of the histologic sections with a digital camera Canon Powershot A640 10.0 megapixel 4x optical zoom lens (Canon Inc., Tokyo, Japan) mounted on an optical microscope Carl Zeiss Axioskop 2 (Carl Zeiss Mikroskopie, Jena, Germany) with a 5x objective. The images obtained were used for the histomorphometric study using the Image-Pro Plus 4.0 image analysis program (Media Cybernetics, Inc, Bethesda, Md) ( Fig 2 ).

Histologic hematoxylin and eosin (H & E) sections used to perform the histomorphometric studies of the mesial root (A) and the distal roots (B) of the upper first molar of the rat. Bone activity was assessed on the entire surface of the periodontal cortex of the mesial root (A). Bone volume was assessed in the interradicular bone of the distal roots (B).

Body weight record

Body weight of each animal was recorded periodically with a digital laboratory balance Ohaus CS 2000 (Ohaus Corp, Pine Brook, NJ) to draw a curve of weight gain.

Histologic and histomorphometric studies

The following histomorphometric parameters were assessed on the dental alveolus according to stereologic principles and using current nomenclature as stated by Parfitt et al :

• •

  Bone activity

  • On digital images of sections of the mesial root, static bone activity was evaluated by determining the percentage of the following parameters on the surface of the periodontal cortical of alveolar bone in the studied area:

    • Areas covered by osteoblasts (Obs/BS)

    • Total erosive surfaces (ES/BS)

    • Erosive surfaces covered with osteoclasts (ES(Oc+)/BS)

    • Erosive surfaces not covered with osteoclasts (ES(Oc−)/BS)

    • Resting surfaces (rest)

    • Surfaces facing hyalinized areas (nec): percentage of the surface of the periodontal cortex of alveolar bone in the studied area presenting surfaces facing necrotic periodontal ligament (hyalinized areas). These areas were defined as areas of necrosis.

• •

  Number of osteoclasts

  • Total number of cells that are morphologically recognizable as osteoclasts found lining the periodontal cortex of alveolar bone measured at the mesial root.

• •

  Bone volume of the interradicular bone (BV/TV)

  • Fraction of bone tissue in total volume. Total volume was taken as bone tissue plus bone marrow evaluated at the level of the distal roots.

Statistical analysis

The values are expressed as mean ± standard deviation. The statistical analysis of the results was performed using 1-way analysis of variance (ANOVA). Multiple comparisons were made using the Bonferroni test. The level of statistical significance was set at a value of P <0.05.