*2.10.5. Microtomography of the mandible (Micro-CT)*

(region 3) and inferior borders (region 4) of the jaw bone the MAR (Fig.15A) and BFR (Fig. 15B) were significantly suppressed compared with those in the control group (P < 0.05). Most of the periosteal surfaces in the mandibular regions of the control group showed significant‐ ly higher values recorded for the mineral apposition rate and the bone formation rate when compared to the DM group. These results agree with previous studies that recorded dimin‐ ished lamellar bone formation in DM rats' femur and may suggest an association between the DM condition and the decreased number and function of osteoblasts [16, 19]. The alveolar crest region was the only region that did not show a significant difference in the mineral apposition rate and the bone formation rate parameters among the two groups; this may be attributed to the unique nature of this region exhibiting a highly intensive bone remodeling process especially during the teeth eruption that decreases toward the base of the socket [33], however further studies are needed to elaborate the detailed pattern of bone growth at the

**Figure 15.** A) The changes in mineral apposition rate (MAR) of the mandible between the control group and the DM group. Alveolar crest (region 1, upper 1/2 of the tooth root, near the tooth crown). Alveolar bone (region 2, lower 1/2 of the tooth root, near the root apex). Buccal surface of the jaw bone (region 3). Inferior border of the jaw bone (re‐ gion 4). The data are expressed as means ± SD. N = 5 for each group. \*Significantly different from controls, with (p<0.05). (B) The changes in the bone formation rate (BFR/BS) of the mandible between the control group and the DM group. Alveolar crest (region 1, upper 1/2 of the tooth root, near the tooth crown). Alveolar bone (region 2, lower 1/2 of the tooth root, near the root apex). Buccal surface of the jaw bone (region 3). Inferior border of the jaw bone (region 4). The data are expressed as means ± S.D. N = 5 for each group. \*Significantly different from controls, with (p<0.05).

alveolar crest region.

416 Type 1 Diabetes

Micro-computed tomography (micro-CT) has rapidly become a standard technique for the visualization and quantification of the 3D structure of trabecular bone. Bone architecture and mineralization are generally considered to be important components of bone quality, and determine bone strength in conjunction with bone mineral density.

#### *2.10.6. Protocol adopted to examine the mandible using Micro-CT*

In our study all specimens were imaged by micro-CT (inspeXio SMX-90CT; Shimadzu Sci‐ ence East Corporation, Tokyo, Japan)


**•** Specimens are then dehydrated in an ascending ethanol series and embedded in paraffin

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**•** Serial horizontal sections (5 μm thick parallel to the occlusal plane) are prepared using a

Histological sections are incubated for 30–60 min at 37ºC in a mixture of 0.8% naphthol AS-BI phosphate (Sigma, St Louis, MO, USA), 0.7% fast red violet salt (Sigma, St Louis, MO, USA) and 50mM sodium tartate diluted in 0.2M sodium acetate buffer (pH 5.4). Sections were examined under a light microscope. For the histomorphometric assessment of resorp‐ tion, the number of tartrate-resistant acid phosphatase-positive multinucleated cells (osteo‐ clasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar were counted in each 540 μm x 120 μm area in five consecutive sections, at the middle third of the root selected at least 25μm apart from each specimen (*n* = 5) of each

Bone-resorption activity was assessed by counting the number of tartrate-resistant acid phosphatase-positive multinucleate cells (osteoclasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar (Fig. 18A-D). Statistical analysis demonstrated a significantly higher number of osteoclast cells in the control group when compared with the DM group (*P <* 0*.*05) (Fig. 18 E). Results revealed that the number of os‐ teoclasts was significantly lower in the DM rats than in the controls, in line with previous

microtome (Leica RM 2155,Nussloch,Germany)(Fig.17)

**Figure 17.** Experimental procedure for histological section preparation

(Fig. 17).

*2.10.9. TRAP staining*

group [39, 40].

*2.10.10. Histological analysis*

**Figure 16.** The left mandible was imaged by micro-CT

#### *2.10.7. Microtomography of the DM mandible*

The quantification of micro-CT trabecular bone changes (mean±SD) is shown for the DM and the control groups in (Table 4). All trabecular parameters in both alveolar bone and buc‐ cal surface of jaw bone showed significant changes. Compared with the control group, bone volume fraction (BV/TV) was significantly decreased only in the alveolar bone; however, trabecular thickness (Tb.Th) and trabecular numbers (Tb.N) were significantly decreased both in alveolar and buccal surface of jaw bone, in the DM group. Correspondingly, signifi‐ cantly higher trabecular separation (Tb.Sp) and trabecular space (Tb.S) were revealed both in alveolar and buccal surface of jaw bone for the DM group when compared with that of the control group. Also, the bone surface / bone volume (BS/BV) was significantly increased only in alveolar bone (*P*< 0. 05). These findings indicate deterioration of the bone quality in the DM group. These results agree with other research work suggesting that the glycaemic levels play an important role in modulating the trabecular architecture especially in mandib‐ ular bone [15].

The DM condition resulted in alteration of the trabecular distance and thickness as com‐ pared to the control group indicating profound impact on the histological integrity of the bone. The reduction in trabecular bone volume accompanied by the expansion of the bone marrow space is in agreement with another investigation [37]. In this context, these results may describe a state of osteopenia in experimental diabetic rats, which might be the result of an imbalance between bone formation and resorption

#### *2.10.8. Procedure for preparing mandibles for histological analysis*

**•** Mandibles for all groups were decalcified in 10% EDTA solution pH 7.4 for 5 weeks at 4ºC [38].


**Figure 17.** Experimental procedure for histological section preparation

#### *2.10.9. TRAP staining*

**Figure 16.** The left mandible was imaged by micro-CT

*2.10.7. Microtomography of the DM mandible*

an imbalance between bone formation and resorption

*2.10.8. Procedure for preparing mandibles for histological analysis*

ular bone [15].

418 Type 1 Diabetes

4ºC [38].

The quantification of micro-CT trabecular bone changes (mean±SD) is shown for the DM and the control groups in (Table 4). All trabecular parameters in both alveolar bone and buc‐ cal surface of jaw bone showed significant changes. Compared with the control group, bone volume fraction (BV/TV) was significantly decreased only in the alveolar bone; however, trabecular thickness (Tb.Th) and trabecular numbers (Tb.N) were significantly decreased both in alveolar and buccal surface of jaw bone, in the DM group. Correspondingly, signifi‐ cantly higher trabecular separation (Tb.Sp) and trabecular space (Tb.S) were revealed both in alveolar and buccal surface of jaw bone for the DM group when compared with that of the control group. Also, the bone surface / bone volume (BS/BV) was significantly increased only in alveolar bone (*P*< 0. 05). These findings indicate deterioration of the bone quality in the DM group. These results agree with other research work suggesting that the glycaemic levels play an important role in modulating the trabecular architecture especially in mandib‐

The DM condition resulted in alteration of the trabecular distance and thickness as com‐ pared to the control group indicating profound impact on the histological integrity of the bone. The reduction in trabecular bone volume accompanied by the expansion of the bone marrow space is in agreement with another investigation [37]. In this context, these results may describe a state of osteopenia in experimental diabetic rats, which might be the result of

**•** Mandibles for all groups were decalcified in 10% EDTA solution pH 7.4 for 5 weeks at

Histological sections are incubated for 30–60 min at 37ºC in a mixture of 0.8% naphthol AS-BI phosphate (Sigma, St Louis, MO, USA), 0.7% fast red violet salt (Sigma, St Louis, MO, USA) and 50mM sodium tartate diluted in 0.2M sodium acetate buffer (pH 5.4). Sections were examined under a light microscope. For the histomorphometric assessment of resorp‐ tion, the number of tartrate-resistant acid phosphatase-positive multinucleated cells (osteo‐ clasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar were counted in each 540 μm x 120 μm area in five consecutive sections, at the middle third of the root selected at least 25μm apart from each specimen (*n* = 5) of each group [39, 40].

#### *2.10.10. Histological analysis*

Bone-resorption activity was assessed by counting the number of tartrate-resistant acid phosphatase-positive multinucleate cells (osteoclasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar (Fig. 18A-D). Statistical analysis demonstrated a significantly higher number of osteoclast cells in the control group when compared with the DM group (*P <* 0*.*05) (Fig. 18 E). Results revealed that the number of os‐ teoclasts was significantly lower in the DM rats than in the controls, in line with previous studies on DM rats' mandible [40] and long bones [41, 42]. These results confirm that the de‐ creased rate of bone turnover may be associated with the DM condition.

The studied STZ-DM rats showed significantly reduced growth in most of the craniofacial skeletal units but no significant differences were observed between controls and DM group as regards the remaining craniofacial skeletal units (Sphenoid bone length, posterior neuro‐ cranium height, anterior corpus length, bigonial width and palatal width). Craniofacial growth as a whole was also significantly lower in DM group compared to controls in all three dimensions. Previous study investigated the DM effect exclusively on the growth of the mandible and suggested that the diabetic condition had a differential effect on the oss‐ eous components and / or its associated non-skeletal tissues. They found that the disharmo‐ nious growth of the mandible was due to DM condition and might not be associated with diabetic condition complications such as renal failure, anemia, body weight change or alter‐ ation in the food intake qualities [2]. Thus we hypothesize that the deficiency in the craniofa‐ cial growth in our experiment might be attributed to the diabetic condition in the DM group as it was reported that specific alterations in bone metabolism are associated with DM. Moreover, several pathogenic possibilities have been proposed, such as insulinopenia, bone microangiopathy, impaired regulation of mineral metabolism, alterations in local factors that regulate bone remodeling, and even an intrinsic disorder associated with T1DM [37, 45]. The aforementioned insulin hormone deficiency that is associated with T1DM cases may have direct effect on bone metabolism. It was mentioned in literature that normal insulin hormone level exerts direct anabolic effects on bone cells [37]. Multiple osteoblast-like cell lines express insulin receptors on the cell surface and have a high capacity for insulin bind‐ ing [46]. Moreover, osteoclasts exhibit reduced bone resorption in response to insulin stimu‐ lation [47]. These findings support the idea that the actions of insulin in bone could be mediated directly via stimulation of osteoblasts in combination with inhibition of osteo‐ clasts, [15, 47] and this mechanism of action may explain the retardation of craniofacial

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Diabetes has a deleterious effect on osseous turnover due to decreased osteoblast and osteo‐ clast activities and numbers and, a lower percentage of osteoid surface and osteocalcin syn‐ thesis, as well as increased time for mineralization of osteoid [37]. It was reported that the influence of diabetes on discrete stages of matrix-induced endochondral bone formation could have profound effects on the biomechanical behavior of bone. Also, chondrogenesis and calcification of bone were reduced by 50% in diabetic animals [48]. This was evident in the current study results that showed a significant decrease in the craniofacial linear meas‐

In addition to this, insulin may exert synergistic effects with other anabolic agents in bone, such as parathyroid hormone (PTH) [15, 47]. An animal model of T1DM has frequently demonstrated alteration in bone turnover, retarded growth, increased concentration of PTH, and reduced concentration of 1,25-dihydroxivitamin D [37, 49]. The effects of PTH on the bones are complex; it stimulates resorption or bone formation depending on the concentra‐ tion used, the duration of the exhibition, and the administration method [37, 48, 49, 50]. Al‐ so, 1,25-dihydroxivitamin D, like PTH, belongs to the most important group of bone regulatory hormones. It regulates osteoclastic differentiation from hematopoietic mononu‐

growth in STZ-DM.

urements of the DM group.

clear cells, and osteoblastic functions and activity [37, 51].

**Figure 18.** Osteoclast counts in a horizontal section of the mandibular second molar region stained with Tartarateresistant acid phosphatase (TRAP). (A) Low magnification photograph of the three roots of the second molar stained with TRAP stain. The black rectangle (540 X 120 μm) indicates the area on the distal surface of the alveolar bone adja‐ cent to the middle third of the mesio-buccal root of the second molar in which the osteoclast cells were counted. Bu, buccal; Li, lingual; Me, mesial; Di, distal. (B) The mesio-buccal root of the control rat (original magnification 100X). (C) The mesio-buccal root of the DM rat (original magnification 100X). (D) A schematic drawing showing the observation area on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar in which the osteoclast cells were counted. (E) Statistical analysis demonstrated a significantly higher number of osteoclast cells in the control group when compared with the DM group (*P <* 0*.*05).

#### **2.11. Suggested mechanisms for the effect of diabetic condition on craniofacial complex**

Growth of the craniofacial complex is controlled by genetic and environmental factors [2, 10]. Regulatory mechanisms responsible for normal morphogenesis of the face and head in‐ volve hormones, nutrients, mechanical forces, and various local growth factors. The poor growth and alterations in bone metabolism have been associated with T1DM in both hu‐ mans and experimental animals [2]. Because human studies can be limited by small sample sizes, cross-sectional designs, uncontrolled variables, and often retrospective nature, animal models have been used to yield more rigorous analyses [10]. In our studies we observed the rat growth from the age of 3 weeks old till 7 weeks old. This time period is corresponding to early growth stage in human according to previous craniofacial growth studies [43, 44]. Consequently, in the current study STZ-DM model was used to investigate the effect of T1DM on craniofacial growth.

The studied STZ-DM rats showed significantly reduced growth in most of the craniofacial skeletal units but no significant differences were observed between controls and DM group as regards the remaining craniofacial skeletal units (Sphenoid bone length, posterior neuro‐ cranium height, anterior corpus length, bigonial width and palatal width). Craniofacial growth as a whole was also significantly lower in DM group compared to controls in all three dimensions. Previous study investigated the DM effect exclusively on the growth of the mandible and suggested that the diabetic condition had a differential effect on the oss‐ eous components and / or its associated non-skeletal tissues. They found that the disharmo‐ nious growth of the mandible was due to DM condition and might not be associated with diabetic condition complications such as renal failure, anemia, body weight change or alter‐ ation in the food intake qualities [2]. Thus we hypothesize that the deficiency in the craniofa‐ cial growth in our experiment might be attributed to the diabetic condition in the DM group as it was reported that specific alterations in bone metabolism are associated with DM. Moreover, several pathogenic possibilities have been proposed, such as insulinopenia, bone microangiopathy, impaired regulation of mineral metabolism, alterations in local factors that regulate bone remodeling, and even an intrinsic disorder associated with T1DM [37, 45]. The aforementioned insulin hormone deficiency that is associated with T1DM cases may have direct effect on bone metabolism. It was mentioned in literature that normal insulin hormone level exerts direct anabolic effects on bone cells [37]. Multiple osteoblast-like cell lines express insulin receptors on the cell surface and have a high capacity for insulin bind‐ ing [46]. Moreover, osteoclasts exhibit reduced bone resorption in response to insulin stimu‐ lation [47]. These findings support the idea that the actions of insulin in bone could be mediated directly via stimulation of osteoblasts in combination with inhibition of osteo‐ clasts, [15, 47] and this mechanism of action may explain the retardation of craniofacial growth in STZ-DM.

studies on DM rats' mandible [40] and long bones [41, 42]. These results confirm that the de‐

**Figure 18.** Osteoclast counts in a horizontal section of the mandibular second molar region stained with Tartarateresistant acid phosphatase (TRAP). (A) Low magnification photograph of the three roots of the second molar stained with TRAP stain. The black rectangle (540 X 120 μm) indicates the area on the distal surface of the alveolar bone adja‐ cent to the middle third of the mesio-buccal root of the second molar in which the osteoclast cells were counted. Bu, buccal; Li, lingual; Me, mesial; Di, distal. (B) The mesio-buccal root of the control rat (original magnification 100X). (C) The mesio-buccal root of the DM rat (original magnification 100X). (D) A schematic drawing showing the observation area on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar in which the osteoclast cells were counted. (E) Statistical analysis demonstrated a significantly higher number of osteoclast cells in

**2.11. Suggested mechanisms for the effect of diabetic condition on craniofacial complex**

Growth of the craniofacial complex is controlled by genetic and environmental factors [2, 10]. Regulatory mechanisms responsible for normal morphogenesis of the face and head in‐ volve hormones, nutrients, mechanical forces, and various local growth factors. The poor growth and alterations in bone metabolism have been associated with T1DM in both hu‐ mans and experimental animals [2]. Because human studies can be limited by small sample sizes, cross-sectional designs, uncontrolled variables, and often retrospective nature, animal models have been used to yield more rigorous analyses [10]. In our studies we observed the rat growth from the age of 3 weeks old till 7 weeks old. This time period is corresponding to early growth stage in human according to previous craniofacial growth studies [43, 44]. Consequently, in the current study STZ-DM model was used to investigate the effect of

the control group when compared with the DM group (*P <* 0*.*05).

T1DM on craniofacial growth.

creased rate of bone turnover may be associated with the DM condition.

420 Type 1 Diabetes

Diabetes has a deleterious effect on osseous turnover due to decreased osteoblast and osteo‐ clast activities and numbers and, a lower percentage of osteoid surface and osteocalcin syn‐ thesis, as well as increased time for mineralization of osteoid [37]. It was reported that the influence of diabetes on discrete stages of matrix-induced endochondral bone formation could have profound effects on the biomechanical behavior of bone. Also, chondrogenesis and calcification of bone were reduced by 50% in diabetic animals [48]. This was evident in the current study results that showed a significant decrease in the craniofacial linear meas‐ urements of the DM group.

In addition to this, insulin may exert synergistic effects with other anabolic agents in bone, such as parathyroid hormone (PTH) [15, 47]. An animal model of T1DM has frequently demonstrated alteration in bone turnover, retarded growth, increased concentration of PTH, and reduced concentration of 1,25-dihydroxivitamin D [37, 49]. The effects of PTH on the bones are complex; it stimulates resorption or bone formation depending on the concentra‐ tion used, the duration of the exhibition, and the administration method [37, 48, 49, 50]. Al‐ so, 1,25-dihydroxivitamin D, like PTH, belongs to the most important group of bone regulatory hormones. It regulates osteoclastic differentiation from hematopoietic mononu‐ clear cells, and osteoblastic functions and activity [37, 51].

Moreover, Insulin may indirectly regulate the enhancement of growth hormone (GH) serum concentration by direct regulation of the hepatic growth hormone receptor, this results in abnormalities in the insulin growth factor-1 (IGF-1) in T1DM [52] which consequently may have lead to the retarded growth in uncontrolled DM in the current study.

**3. Effect of Type 1 Diabetes on teeth susceptibility to caries**

the forming front reaches the cervix of the crown [3].

activity of external cariogenic factors [3].

total thickness of enamel or dentin.

Tooth growth begins before birth and continues throughout adolescence. The dental develop‐ ment is a continuous process of tooth initiation, matrix secretion, crown mineralization, den‐ tal eruption, and root completion. Primate dental development begins before birth with initiation of the deciduous dentition, followed by initiation of the permanent dentition. During the process of dental eruption the tooth must move past the bone margin (alveolar eruption) and the gum (gingival eruption) in order to emerge into the oral cavity and eventually into functional occlusion. Dentine is formed when odontoblasts secrete a collagenous matrix predentine which rapidly undergoes mineralization to form primary dentine. Dentine formation begins at the dentine horn underlying the future cusp tip and progresses inward through secretion and downward through extension until it reaches the apex of the root. Enamel is formed when ameloblasts secrete enamel matrix proteins that mineralize into long, thin bundles of hydrox‐ yapatite crystallites known as enamel prisms. As the secretory cells progress outward to‐ ward the future tooth surface, additional adjacent cells are activated through extension until

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The resistance of the dental tissues to caries is determined by the quantitative content of the inorganic and organic matrices. Numerous reports have showed that teeth with a correct macro- and microstructure and a proper degree of mineralization are more resistant to the

Several biological complications in patients suffering from type 1 diabetes mellitus have been widely investigated over the previous years, however, the scientific data available on the possible effects of T1DM on teeth are scant. These data suggest that T1DM condition may be associated with a change in the chemical composition of the teeth or alteration in the

Various clinical studies on children reported high caries prevalence in diabetic children when compared with healthy controls [3]. This high caries prevalence may be associated with factors affecting the tooth structure itself or due to some changes in the oral environ‐

Proper mineralization of teeth during its development is the key factor for the proper resist‐ ance of teeth to cariogenic challenge and thus any metabolic disorder affecting the teeth mineralization during its development may render these teeth more prone to be involved by caries. Rat experimental model provides an excellent model for the study of the various met‐ abolic disorders on the mineralization of teeth hard tissues due the fact that the rats incisors are continuously growing and erupting during its life and thus it is possible to study vari‐ ous effects of metabolic disorders which can be induced artificially during the growth peri‐ od of these rats [53]. The results obtained from the aforementioned experimental rat model can be compared to the results obtained from control rats living under identical situations

ment causing the increase of the susceptibility of the teeth to dental caries.

**3.2. Effects of Type 1 Diabetes on tooth mineral composition**

**3.1. Tooth formation**

In the present study most of the periosteal surfaces in the mandibular regions of the control group showed significantly higher values recorded for the mineral apposition rate and the bone formation rate when compared to the DM group. These results agree with previous studies that recorded diminished lamellar bone formation in DM rats' femur and may sug‐ gest an association between the DM condition and the decreased number and function of osteoblasts [16, 19]. The alveolar crest region was the only region that did not show a signifi‐ cant difference in the mineral apposition rate and the bone formation rate parameters among the two groups; this may be attributed to the unique nature of this region exhibiting a highly intensive bone remodeling process especially during the teeth eruption that de‐ creases toward the base of the socket [33].

A significant decrease of bone volume fraction, trabecular thickness, and trabecular numbeis confirmed by Micro-CT analysis in DM rats, there was Also, it showed a significant increase of the trabecular separation and the trabecular space in the DM group when compared with the control group. This finding indicates deterioration of the bone quality in the DM group. These results agree with other research work suggesting that the glycaemic levels play an important role in modulating the trabecular architecture especially in mandibular bone [15]. In this context, these results may describe a state of osteopenia in experimental diabetic rats, which might be the result of an imbalance between bone formation and resorption.

A histometric evaluation of bone resorption was performed by counting the number of os‐ teoclast cells on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar. These evaluations revealed that the number of osteoclasts was significant‐ ly lower in the DM rats than in the controls, in line with previous studies on DM rats' man‐ dible [40] and long bones [41, 42]. These studies confirm that the decreased rate of bone turnover may be associated with the DM condition.

This deteriorating effect on mandibular bone structure and dynamic bone formation might be be attributed to several pathogenic possibilities, such as insulinopenia, bone microangi‐ opathy, impaired regulation of mineral metabolism, alterations in local factors that regu‐ late bone remodeling, and even an intrinsic disorder associated with DM [12, 37]. However, the detrimental effects observed may not be associated with the significant loss of rats` weights observed in the diabetic group starting from day 14 because previous research [2, 12, 15, 42] showed that the mandibular growth was not affected in normal rats supplied with restricted diet and having same pattern of weight loss resembling weight loss pat‐ tern observed in DM rats.
