Specific aspects of the radiographic morphometric and densitometric characteristics of mandible of the rats with experimental diabetes mellitus after tooth extraction

Materials and methods. Studies were conducted on 120 male Wistar rats, 8–10 months old, divided into eight groups of 15 animals each. The experimental diabetes mellitus was modelled in 45 animals by a single administration of streptozotocin interperitoneally (SIGMA Chemical, the USA) at a dose of 50 mg/kg, diluted in 0.5 ml of 0.1 M citrate buffer (pH 4.5) ex tempore, on the 21st day after its induction the level of fasting glucose was determined (by the glucose oxidise method using standard test strips Test Strip II, glucometer Glucocard, Japan), which was 24.7 ± 2.2 mM/l. The extraction of the first low molar of the right mandible was realised using thiopental anaesthesia (40 mg / kg dose) with additional local infiltration anaesthesia with Ubistesine (3M Deutschland GmbH, Germany).On the 1st, 7th, and 14th day, dates that correspond to the experiment after the extraction of the tooth, the rats were decapitated using thiopental anaesthesia (dose 40 mg/kg).The visiography of the mandible was performed using a computerized 3D CBCT tomograph Panoura 18S Panoramic 3D. With the help of digital image analysis the radiographic density of tissues of the alveolar socket of the removed 1st molar, crown of the 1st, 2nd and 3rd molars, their root and dental septum of the alveolar crest were determined.

Diabetes mellitus (DM) is regarded today as a metabolic disease, which is considered to be a modulator of endodontic infections [1], is responsible for altering the immune and inflammatory responses, impedes the healing process, contributes to damage of organs and tissues of the body, including tooth pulp and periapical tissues [2,3]. Moreover, numerous clinical observations have shown that DM can not only affect the pathogenesis of dental nosologies, but the bidirectional mutually confounding connection between apical periodontitis, periostitis, gingivitis and the systemic diabetes effects has been also proven. It has been established that in case of DM patients, on the background of an infectious process in the maxillofacial area were noted to have more evident resorption of the bone and hard tissues of the tooth, sluggish inflammatory processes in the periapical area and periodontal [1][2][3][4][5][6].
A cross-sectional study realized among the patients with diabetes showed that the worst periapical status correlated with a higher level of glycated hemoglobin (HbA1c), and each further examination after endodontic treatment was increasing the level of HbA1c [7]. In addition, the presence of DM in a patient was connected with a decrease of the successful therapy results [8]. There are reports of cases that mark a sudden increase of glucose level in the blood of patients during exacerbation of a combined endodontic-periodontal lesion [17].

The aim
The aim of the work is to study the radiographic characteristics of the dental septum of the alveolar crest of the mandible, crown and root of the low molars of rats with experimental streptozotocin diabetes at different times of the post-extraction period (the 1st, 7th and 14th day).

Materials and methods
The experimental part of the research was carried out in accordance with the national "Common Ethical Principles of Animal Experiments" (Ukraine, 2001), which are coordinated with Council Directive 2010/63EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. The study protocol is agreed with the local ethics committee. Studies were conducted on 120 male Wistar rats, 8-10 months old, divided into eight groups of 15 animals each: -the 1 st group -rats that did not have the extraction of a tooth (Control-0); -the 2 nd group -rats that were removed from the experiment a day after the extraction of the tooth (Control-1); -the 3 rd group -rats that were removed from the experiment 7 days after the extraction of the tooth (Control-7); -the 4 th group -rats that were removed from the experiment 14 days after the extraction of the tooth (Control-14); -the 5 th group -a group of rats with experimental streptozotocin diabetes that did not have the extraction of a tooth (Diabetes-0); -the 6 th group -a group of rats with experimental streptozotocin diabetes that were removed from the experiment a day after the extraction of the tooth (Diabetes-1); -the 7 th group -a group of rats with experimental streptozotocin diabetes that were removed from the experiment 7 days after the extraction of the tooth (Diabetes-7); -the 8 th group -a group of rats with experimental streptozotocin diabetes that were removed from the experiment 14 days after the extraction of the tooth (Diabetes-14).
In groups of rats with experimental diabetes mellitus (EDM), which was modelled by a single administration of streptozotocin interperitoneally (SIGMA Chemical, the USA) at a dose of 50 mg/kg, diluted in 0.5 ml of 0.1 M citrate buffer (pH 4.5) ex tempore, on the 21 st day after its induction was determined the level of fasting glucose (by the glucose oxidise method using standard test strips Test Strip II, glucometer Glucocard, Japan), which was 24.7 ± 2.2 mM/l.
In the morning at 10:00 AM the extraction of the first low molar of the right mandible of the rats was carried out in six groups of 45 control animals and 45 animals with simulated EDM using thiopental anaesthesia (40 mg/kg dose) with additional local infiltration anaesthesia with Ubistesine (3M Deutschland GmbH, Germany).
On the 1 st , 7 th , and 14 th day, dates that correspond to the experiment after the extraction of the tooth, the rats were decapitated using thiopental anaesthesia (dose 40 mg/kg).
Radiographic images of the semi-mandibles of rats were taken after the removing of the animals from the experiment by single-step decapitation under thiopental anaesthesia (40 mg/kg body weight). The jaw bone was immediately separated from animal's head, soft tissues were removed and placed in a cold NaCl solution of 0.9 %. The visiography of the mandible was performed no later than 2-3 hours after the release of the gross specimen. Images of the semi-mandibles were obtained using a computerized 3D CBCT tomograph Panoura 18S Panoramic 3D.
For digital image analysis and determination of the radiographic density of the mandible tissues in the obtained images in an interactive mode the "areas of interest" were distinguished, which corresponds to the alveolar socket of the removed 1 st molar, crown of the 1 st , 2 nd and 3 rd molars, their root and dental septum of the alveolar crest (interalveolar and interradicular septa (IIS)) (Fig. 1).
The prepared images were calculated using software Axio Vision 4.8.2 (Carl Zeiss, Germany) and Excel 7.0.
Radiographic density (RD) was calculated using the formula: where RD is the radiographic density of the mandible tissues; AOI -"area of interest"; BG is the radiographic density of the "background area" of the selected area of the mandible soft tissues without bone formations.
Statistical calculations were performed in Microsoft Excel 2016 spreadsheet (Microsoft Corp., USA). For all the indicators the value of the arithmetic mean of the sample (M), its dispersions, and the mean error (m) were calculated. To identify the significance of differences in the results of research in the experimental and control groups of rats, the Student's coefficient (t) was calculated, and after that the probability of the difference in samples (P) and the confidence interval of the average as per the Student's tables of distribution were determined. The differences of values for which Pst < 0.05 were considered valid [9,10].

Results
Extraction of the 1 st low molar from the rats of the control group on the 1 st day of the post-extraction period led to a significant decrease in RD for more than 2.7 times in the root area of the extracted tooth and 65 % in the IIS area, which is connected not only with the extraction of tooth bone tissue, but also with an active inflammatory process, leading to bone tissue resorption ( Table 1).
On the 7 th day after the extraction, the RD index in the area of the tooth root did not significantly differ from the values of the previous term group, whereas in the IIS area the RD values permitted to suggest a decrease in resorptive processes in the mandible bone tissue and a partial recovery of mineralization processes, that can be confirmed by the increase of RD by 44.1 % comparing to the 1 st day of the post-extraction period ( Table 1).
The 14 th day of observation showed, comparing to the previous period, the 7 th day, an increase in the RD index in the 1 st molar root area by 53.8 % and the absence of significant differences in the IIS area. It should be noted that in this period of complete restoration of bone density to the indicators of the "Control-0" group did not occur, because RD in the area of the root remained less by 1.67 times, and in the area of IIS -by 11 %, which is an important evidence of the ongoing reconstructive processes in the area of the alveolar socket ( Table 1).
It is known that tooth extraction is accompanied not only by local inflammatory processes in the area of the alveolar socket, the associated changes in nearby teeth, which are characterized by the presence of a pain component, the development of periapical periodontitis, the occurrence of pathological mobility, are proved [11].
The analysis of RD indices located near to the extracted tooth of the 2 nd and 3 rd molars of the control group rats showed that its extraction could not but affect the state of the bone tissue of IIS. Already on the 1 st day there was a decrease in radiological density of the IIS of the 2 nd molar by 24.4 %, and on the 3 rd by 16.8 %. Moreover, the RD of the crown of the 2 nd molar showed its decrease by 21.9 %, which is most likely due to bone tissue resorption due to the activity of the inflammatory process, disruption of local trophism, changes in the pH of the oral fluid [12]. It should be noted that the identified process is reversible because already on the 7 th day, a full recovery of the values up to the control indicators was observed, while the RD indicator on the 14 th day of the post-extraction period did not have significant differences in the studied areas ( Table 1).
A comparative analysis of the radiological density of the mandible tissues in the area of the 1 st -3 rd molars of the rats with experimental streptozotocin diabetes of The colour designations of the "areas of interest" for digital analysis of the radiographic density of the parameters under study (the crown of the tooth -blue, the root of the toothyellow, the interalveolar and interradicular septa -orange, the area of alveolar socket -red).

A B
Оригінальні дослідження the group without extraction of the tooth showed that the formed hyperglycaemia leads to a significant decrease in RD in the area of IIS of all three molars: by 27.5 % for the 1 st , 20.6 % for the 2 nd and 21.5 % for the 3 rd . Moreover, the RD index of the 1 st molar root area was also 10.3 % lower than the values of the corresponding control and this allows suggesting their increased resorptive processes in the bone tissue (Tables 1, 2). Extraction of the 1 st molar of the rats with EDM on the 1 st day led to the expected decrease in radiological density in the area of its root, but it increased in the IIS area by 22.0 %. A similar pattern was observed in the IIS area of the 2 nd and 3 rd molars -the RD index was increased by 21.8 % and 18.3 %, respectively, which is a result of the development of the resorptive phase of the inflammatory reaction with tissue infiltration not only in the remote molar area, but also its considerable prevalence on the adjacent teeth ( Table 2).
On the 7 th day of the post-extraction period rats with EDM, in contrast to the indicators of the control rats of the corresponding age, had a decrease in radiographic density in the area of the IIS of the 2 nd and 3 rd molars by 24.5 % and 36.0 %, respectively, with its decrease in the root area of the 2 nd molar by 13.5 % (Tables 1, 2). The 14 th day's observations showed that in this period there were significant structural changes in the studied areas, i.e. the inflammatory process did not end, but on the contrary, there was a decrease in the RD index in the alveolar socket and IIS of the 1 st molar by 28.6 % and 33.0 %, respectively. Obvious resorptive processes were revealed during the study of the radiological density of parts of the 2 nd molar, with a decrease in RD in the crown area by 8.2 %, tooth root by 19.9 %, IIS -by 18.2 %. The resorptive processes also affected the 3 rd molar, since a decrease in the RD of its root by 15.8 % was noted ( Table 2).

Discussion
During the realized study it was found that diabetes mellitus makes a significant contribution not only to the course of the wound process after tooth extraction, but also its role was shown in the pathogenesis of the complications formation due to violation of the resorption-reparative relationship in the area of the alveolar socket and adjacent molars.
It is important to note that the features of the course of the wound process on the background of diabetes mellitus have already been repeatedly discussed. Most researchers identify several key factors that are crucial in the formation of postoperative complications in patients with diabetes. Among them it is necessary to single out the pre-existing ones, which change the direction, duration and staging of the inflammatory reaction, and newly formed during tissue trauma associated with an imbalance of enzyme systems and local immune responses [13].
According to the results of numerous clinical observations and experimental studies, pathological changes  in bone tissue, significant microcirculatory disorders, and neurotrophic disorders were identified among the preexis ting adverse factors affecting the course of the postoperative period of patients with DM. At the same time, it is believed that osteopenia and osteoporosis, as the most frequent pathological changes in bone tissue, have the diffuse character, since both cancellous and tubular bones "suffer" [14]. Conducted histomorphometric studies have shown that in case of diabetes mellitus type 1 there is a reduced bone formation with a decrease in metabolic processes in it. In the experiment on rats, it was found out that with simulated streptozotocin diabetes in bone tissue, there is a decrease in the content of non-mineralized matrix and the number of osteoclasts [15]. In the study realized by us the indisputable confirmation of emerging osteoporosis with bone resorption was the decrease in the radiological density of the tooth root of the 1 st molar, as the most traumatized and loaded rats' tooth, and IIS in the area of the 1 st -3 rd molars.
Another important fact that was stated in the work was the confirmation of the DM effect on the inflammatory process after tooth extraction. It was found out that persistent hyperglycaemia, a key pathogenetic factor of disease complication, significantly changes the nature of the wound process, which according to most researchers, is associated with impaired stages of inflammation phases, their duration and effectiveness. In particular in the works of O. E. Lutsevich et al. it was shown that the wound process on the background of diabetes mellitus is characterized by pronounced macro-and microcirculatory disorders, the presence of microthrombus, the formation of a sludge-phenomenon, dystrophic and necrotic processes, the predominance of the alterative component over the reparative, the inhibition of cell proliferation, the suppression of phagocyte activity of leukocytes, incomplete phagocytosis, decrease in general and local immunological reactivity [16].
Studied in the work features of the dynamics of changes in indicators of radiological bone tissues density of the mandible after tooth extraction on the background of DM in different periods of the post-extraction period were indirect confirmation of the above mentioned. Thus, an increase and not the decrease of the RD index on the 1 st day in the alveolar socket area and the adjacent 2 nd and 3 rd molars may be a consequence of significant infiltration associated with the development of marked secondary alteration. The final stage of the resorptive phase of the local inflammatory reaction also had its own characteristics. Thus, in the control on the 7 th day of the post-extraction period, that correspond to this phase [17], almost complete recovery of RD is observed, whereas on the background of diabetes mellitus, on the contrary, the radiological density of the studied areas decreased, and this process continued on the 14 th day. The established fact indicates the progression of resorptive processes against the background of low proliferative activity. In addition to the identified features of the inflammatory process, the study showed the role of EDM in the formation of long-term complications in the form of increased mobility of adjacent teeth. This was evidenced by the decrease and not the increase of the RD index in the IIS area, which, being part of a tooth support apparatus, normally not only ensures the stability of the tooth position in the bone, their trophic function has also been proven, due to abundant vascularisation and the large number of nerve endings [17].

Conclusions
1. Tooth extraction from rats with normal indices of carbohydrate metabolism is accompanied by dynamic changes in radiological density not only in the extraction area of the 1 st molar (it decreases on the 1 st day in the area of the root by more than 2.7 times and by 65 % in the area of interalveolar and interradicular septa with its restore to the 14 th day). It was established that the pre-existing early resorptive phase of the local inflammatory reaction ends by the 7 th day of the post-extraction period, changing to the reparative by the 14 th day.
2. The development of experimental diabetes mellitus in rats leads to an increase in resorptive processes in the mandible bone tissue, which is manifested by a decrease in RD in the area of the 1 st molar root by 2.27 times along with its increase in the IIS area of all three low molars by 22.0 %, 21.8 % and 18.3 % respectively.
3. Experimental diabetes mellitus contributes to the particular course of the wound process after tooth extraction. At the same time, it is an important pathogenetic link for the formation of complications due to the disturbance of the resorption-reparative relationship in the area of the alveolar socket and the adjacent molars.