Introduction
Given the overlap between the age of onset of type 1 diabetes mellitus (DM) and the typical timing of rapid maxillary expansion, this study aimed to evaluate the short-term effects of hyperglycemia and insulin treatment on maxillary new bone formation using radiologic and histologic analyses in an experimental rat model.
Methods
Wistar rats with type 1 DM induced with streptozotocin underwent a 5-day maxillary expansion and 12-day retention procedure (groups: control, DM, expansion, DM + expansion, and DM + expansion + insulin). Bone microstructure was assessed in the maxillae of the sacrificed rats using micro-computed tomography imaging. Then, histopathologic examination was performed to assess new bone and vascular formation and osteoblast density.
Results
In all expansion groups, new bone formation, osteoblast number, and vascularity were histologically increased compared with the nonexpansion groups ( P < 0.01). The diabetic expansion (DM + expansion and DM + expansion + insulin) groups have increased bone mineral density, bone volume/trabecular volume, and trabecular thickness. The diabetic expansion (DM + expansion and DM + expansion + insulin) groups have decreased bone surface/volume ratio; trabecular separation was detected compared with the control, DM, and expansion groups, but no difference was detected in tissue volume and trabecular number.
Conclusions
Although metabolic status does not appear to significantly affect early bone remodeling activity associated with maxillary expansion in type 1 DM, timely intervention remains critical because of reduced bone remodeling capacity.
Highlights
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This is the first study examining diabetic effects on bone during maxillary expansion.
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Rapid expansion increased bone formation despite early-stage diabetes.
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Histology showed higher osteoblast density and vascularity in expanded bones.
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Diabetes altered bone microstructure, but did not prevent osteogenesis.
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Insulin treatment had no significant short-term effect on bone remodeling.
Transverse deficiency of the maxilla is one of the most common skeletal problems in the craniofacial region. The narrow maxilla is corrected by gradually expanding it over a period of 1-4 weeks using the rapid expansion technique. , The goal of this method is to stimulate the formation of new bone in the suture area through the application of mechanical forces. Rapid maxillary expansion can be affected by the administration of hormones, chemical agents, drugs, vitamins and proteins, various diseases, and metabolic conditions.
It is well established that the rate of bone formation and resorption is closely associated with metabolic status. Type 1 diabetes mellitus (DM), which starts with insufficient insulin production as a result of autoimmune destruction of beta cells and leads to hyperglycemia, adversely affects bone metabolism by decreasing bone formation and increasing bone resorption. These changes lead to deterioration in bone quality, turnover, microarchitecture, mineralization, and increased microdamage. Experimental studies have demonstrated that diabetes induces osteoblast apoptosis and enhances osteoclast activity. It is also known that diabetes-induced inflammation disrupts the differentiation of mesenchymal stem cells, which are responsible for new bone formation, into osteoblasts by activating nuclear factor-kappa B and tumor necrosis factor–α, thereby inhibiting the Osx promoter. However, the interplay between the effects of DM on bone and the influence of mechanical forces remains unclear.
Type 1 DM is most commonly diagnosed in children aged 5-7 years and during adolescence, which coincides with the typical age range for orthodontic patients. However, the impact of type 1 DM on the success of maxillary expansion treatment remains uncertain. Given the potential overlap between the typical onset of type 1 DM and the timing of orthodontic interventions, this study aimed to elucidate the short-term impact of rapid maxillary expansion on new bone formation in controlled and uncontrolled diabetic states, using radiologic and histologic evaluation.
Material and methods
Ethics committee approval for the study was granted by the experimental animals local ethics committee of Bolu Abant İzzet Baysal University (decision number: 2023/02), and the experiment was conducted in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines. Animals were procured from the experimental animal research center of Bolu Abant İzzet Baysal University, and they were cared for with ad libitum access to water and pellet feed, under controlled conditions of 19 ° C ± 2 ° C, 55%-60% relative humidity, and a 12-hour light and dark cycle. The study included the following groups (n = 8): control, expansion, DM, DM + expansion, and DM + expansion + insulin. Wistar Albino male rats, weighing 200-250 g and 4 weeks of age, were included in the study. Rats with normoglycemic status before DM induction were included in the DM groups.
Streptozotocin (STZ, S0130-1G; Sigma-Aldrich, Mo), dissolved in 0.1 M sodium citrate buffer (pH 4.5), was administered intraperitoneally to the diabetic groups at a dose of 65 mg/kg after confirming normoglycemia and fasting the animals for 12 hours. Blood glucose levels were monitored on the first, third, and seventh days postinjection (Accu-Check Instant; Sweden) to confirm the development of the model. Blood glucose levels ≥200 mg/dL after STZ were included in the DM groups. Insulin treatment with NPH (Humulin 100 IU/ml, Ind) commenced on the eighth day, with 1 U of insulin administered subcutaneously at 1:00 pm and 4 U at 7:00 pm. Blood glucose and body weight were regularly monitored on a weekly basis ( Fig 1 ). Of the animals initially included, rats that did not meet the hyperglycemia criteria (blood glucose levels <200 mg/dL), developed hypoglycemia (<60 mg/dL), exhibited excessive weight loss (>20% of baseline), or experienced mechanical complications during expansion were excluded from the study. All remaining animals completed the experimental protocol and were included in the analyses.
Experimental flowchart.
Under anesthesia (xylazine + ketamine combination at doses of 0.5 mL/kg and 1 mL/kg intramuscularly, respectively), grooves were created in the 2 maxillary incisors of the rats using a small round bur at the level of the gingival papilla, ensuring the pulps of the teeth were not exposed (ie, no perforation). To induce expansion of the premaxilla region using mechanical forces, helical springs made of 0.014-in round stainless-steel wire were placed between the incisors and secured with 0.009-in ligature wire. The applied force from the helical springs was calibrated to 30 g using an orthodontic force gauge (Morelli Orthodontics; Sorocaba, São Paulo, Brazil). The helical springs were removed under anesthesia 5 days after their application, and 0.017 × 0.025-in stainless-steel wire was tied in place for a 12-day retention phase. Upon completion of the retention phase, the animals were euthanized with high-dose anesthesia, and maxillary bone tissues were harvested for radiologic and histomorphologic analysis.
Micro-computed tomography (micro-CT) scanning and analysis were performed using a high-resolution micro-CT device (SkyScan 1172; Bruker Micro-CT, Kontich, Belgium). After scanning, analysis was performed in a 2.40 × 0.90 × 0.60 mm area at the premaxillary suture in a region of interest defined with reference to the palatal surfaces of the incisors ( Fig 2 ). These dimensions were selected based on a previous study that analyzed maxillary expansion and midpalatal suture vremodeling in rats using a similar micro-CT protocol. Samples were evaluated for bone mineral density (BMD) (g/cm 3), bone volume (BV) (mm 3), tissue volume (TV) (mm 3), bone volume fraction (BV/TV) (%), bone surface/volume ratio (BS/BV) (1/mm), trabecular number (Tb.N) (1/mm), trabecular thickness (Tb.Th) (mm), and trabecular separation (Tb.Sp) (mm) at the suture.
Region of interest at the premaxillary suture measured by micro-CT.
The maxillary tissue samples were fixed in 10% buffered neutral formalin solution and then subjected to decalcification in 4% formic acid solution. The acid solution was changed weekly. A routine tissue follow-up protocol was applied to the decalcified samples, which were examined at regular intervals until they reached a sufficient level for sectioning. The tissues washed in running water were passed through 70%, 80%, 96%, 100% alcohol series, respectively. Afterward, the tissues that were cleared with xylene series were blocked using paraffin at 58 ° C. Sections obtained from tissues embedded in paraffin blocks with half sampling and 4 μm thickness according to the systematic random sampling rule by microtome were placed on polylysine slides. According to the systematic random sampling rule, the total number of sections taken was 30 for each animal. The sections obtained were stained with hematoxylin-eosin. New bone formation, new vessel formation, and osteoblast density changes were evaluated under a Nikon Eclipse 80i light photomicroscope (Nikon Corporation, Tokyo, Japan). For new bone and new vessel formations, morphologic parameters were scored semiquantitatively between 0-3. If no change was observed, it was graded as none (score = 0); in case of changes, intensities were graded as mild (+, score = 1), moderate (++, score = 2), and strong (+++, score = 3). In this context, for osteoblast density, 6 high-powered (×200) areas were randomly selected in each section. Digitized images were obtained from these selected areas using a digital video analysis system. Osteoblast density was evaluated in each area. An average of 6 areas was estimated for each sample. The density of osteoblasts was evaluated as follows: no osteoblast cells: 0, <25% osteoblast cells: 1, 25%-50% osteoblast cells: 2, and >50% osteoblast cells: 3.
Each parameter was assessed by the same histologist, who was unaware of the group assignment of the tissue samples, with samples randomly selected for evaluation (blind evaluation).
Statistical analysis
Data were evaluated in the statistical package program SPSS Statistics for Windows (version 25.0; IBM Corp, Armonk, NY). Descriptive statistics, such as mean, standard deviation, minimum, median, and maximum, were used to define independent variables. The conformity of the data to a normal distribution was assessed using the Shapiro-Wilk test, Q-Q plots, histograms, and boxplots. The homogeneity of variances was evaluated using the Levene test. Comparisons between groups were made using one-way analysis of variance for normally distributed variables, and the Kruskal-Wallis test was used for non-normally distributed variables. The Tukey honest significant difference test was applied as a post-hoc test for normally distributed data, and the Bonferroni-correction Mann-Whitney U test was used for non-normally distributed data. Parameters of new bone formation and new vessel formation were represented as scores showing densities, and group scores were compared with the Fisher exact test. P values of 0.05 were considered statistically significant.
Results
The average blood glucose levels for the experimental groups in our study are as follows: control: 90.1 ± 5.7, DM: 497.2 ± 59.6, expansion: 80.0 ± 9.1, DM + expansion: 231.3 ± 145.2, and DM + expansion + insulin: 175.25 ± 124.7 (mg/dL) ( Fig 3 ).
Blood glucose averages between groups (milligrams per deciliter).
Diastema occurred between the incisors in all expansion groups ( Fig 4 ). Statistical analysis of BV, BV/TV, BS/BV, BMD, Tb.Th, and Tb.Sp results ( Fig 5 ; Table ) showed significant differences between the groups ( P < 0.001). No significant difference was observed between the groups in terms of TV and Tb.N.
Sutural separation of the rat maxilla.
The micro-CT image of the maxillae of the groups (a: control, b: DM, c: expansion, d: DM + expansion, and e: DM + expansion + insulin). The midline diastema and separation of the premaxillary suture seen in the experimental groups indicate that the expansion was successful.
Table
Statistical comparison of BMD, BV, BV/TV, BS/BV, TV, Tb.Th, Tb.Sp, and Tb.N values of the groups
| BMD (g/cm 3) | BV (mm 3) | BV/TV (%) | BS/BV (1/mm) | TV (mm 3) | Tb.Th (mm) | Tb.Sp (mm) | Tb.N (1/mm) | |
|---|---|---|---|---|---|---|---|---|
| Control (1) | 0.311 ± 0.211 | 0.626 ± 0.162 | 39.735 ± 11.164 | 21.921 ± 4.016 | 1.597 ± 0.291 | 0.185 ± 0.04 | 0.25 ± 0.033 | 2.124 ± 0.317 |
| DM control (2) | 0.302 ± 0.164 | 0.884 ± 0.527 | 46.447 ± 17.981 | 18.188 ± 4.96 | 1.868 ± 0.425 | 0.214 ± 0.049 | 0.289 ± 0.115 | 2.129 ± 0.637 |
| Expansion control (3) | 0.400 ± 0.306 | 0.771 ± 0.132 | 48.501 ± 9.201 | 18.855 ± 3.736 | 1.596 ± 0.110 | 0.216 ± 0.039 | 0.242 ± 0.046 | 2.255 ± 0.232 |
| DM + expansion (4) | 1.048 ± 0.231 | 1.271 ± 0.164 | 79.781 ± 10.225 | 9.703 ± 2.374 | 1.594 ± 0.064 | 0.397 ± 0.09 | 0.148 ± 0.033 | 2.081 ± 0.398 |
| DM + expansion + insulin (5) | 1.139 ± 0.338 | 1.363 ± 0.093 | 84.941 ± 8.054 | 8.557 ± 0.72 | 1.614 ± 0.142 | 0.405 ± 0.029 | 0.118 ± 0.015 | 2.103 ± 0.219 |
| P value | 0.001 | 0.001 | 0.001 | 0.001 | 0.184 | 0.001 | 0.001 | 0.880 |
| P value | 1-4, 1-5, 2-4, 2-5, 3-4, and 3-5 | 1-4, 1-5, 3-4, and 3-5 | 1-4, 1-5, 2-4, 2-5, 3-4, and 3-5 | 1-4, 1-5, 2-4, 2-5, 3-4, and 3-5 | 1-4, 1-5, 2-4, 2-5, 3-4, and 3-5 | 1-4, 1-5, 2-5, 3-4, and 3-5 |
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