The Effect of Vitamin a as a Teratogenic Agent in Condylar Bone Growth

The Effect of Vitamin a as a Teratogenic Agent in Condylar Bone Growth | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Effect of Vitamin a as a Teratogenic Agent in Condylar Bone Growth Shimaa Abdel Shafy, Shadia Hussein Abdel majeed, Mounir Mahmoud El –Adawy, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6487311/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Vitamin A is present in food such as meat, dairy products, and vegetables. A control balanced diet maintains the nutritional needs of vitamin A. Over consumption of products as well as supplementations with vitamin A may increase the risk of hypervitaminosis A. Hypervitaminosis A has been associated with thinning of the long bones and reduction of bone formation. Objectives Investigate the impact of both optimum and excessive vitamin A dosages on the development of the mandibular condyle in Wister albino rats. Materials and Methods Forty –eight healthy offspring’s male Wister albino rats were used in this study. The rats were divided into two groups: Group I (control group) 24 rats were fed a regular diet containing an optimum dose of vitamin A. Group II (study group) 24 rats were fed a regular diet supplemented with an overdose of vitamin A. In both groups, 8 rats were euthanized after 2, 4, and 6 months respectively. Blood samples were collected from all rats following the last vitamin A dosage to measure its concentration in serum. The condyles were dissected removed and processed for histomorphometric and histological examination. Results In contrast to the control group, the study group's condylar zone structure was shown to be disrupted during the histological analysis. Conclusions Hypervitaminosis A could increase condylar bone resorption and diminish endochondral bone growth. Clinical Relevance: Vitamin A supplementation must be under medical control to avoid growth anomalies and subsequent malocclusion. vitamin A teratogenic condylar bone growth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 INTRODUCTION Vitamin A is a member of the fat-soluble vitamins. There are two types of vitamin A (VA) found in food: provitamin A carotenoids and preformed VA (retinol or retinyl esters)[ 1 – 3 ]. Retinol is derived from animal sources such as eggs, liver, milk, and fortified cereals [ 4 ], while Carotenoids originated from plant sources such as carrots, collards, spinach, and squash[ 1 , 2 ]. Vitamin A is required in tiny amounts for various biological processes, such as immunity, growth, differentiation of cells, reproduction, embryological development, and vision [ 3 ]. Lipids aid in the intestinal absorption of vitamin A, which is mostly stored in the liver, kidney, and adipose tissues [ 5 ]. The primary cause of blindness is vitamin A deficiency, which increases the risk of infectious diseases and diarrheal illness. [ 6 ], whereas the prevalence of spontaneous bone fractures, aberrant skeletal growth in children, and increased bone turnover are all associated with hypervitaminosis A[ 7 ]. As vitamin A is quickly absorbed and eliminated, Acute toxicity can result from exposure to high doses for brief periods or chronic toxicity can result from much lower intake for extended periods [ 8 ]. The retinoid form of vitamin A is represented by vitamin A supplements [ 9 ]. It has been discovered that the retinoid type of vitamin A is more likely than the carotenoid type to produce vitamin A toxicity. The retinoid type is absorbed easily at rates of 70–90%, whereas the carotenoids type is absorbed significantly less efficiently at rates of 20–50%. Although the carotenoid form is less harmful than the retinoid type, it has been discovered that too much beta-carotene in the diet can cause carotenodermia [ 7 ]. Carotenodermia is a skin disorder marked by an orange-yellow coloring [ 10 , 11 ]. Increased periosteal bone resorption and decreased bone formation have been associated with the thinning of long bones in rodents with hypervitaminosis A [ 12 ]. Moreover, rats with hypervitaminosis A develop mandibular and scapular perforations [ 13 ]. In humans, Hypervitaminosis A has been linked to bone pain, hypercalcemia, and accelerated bone resorption. Long-term retinoid therapy has also resulted in increasing calcification of ligaments, remodeling anomalies of long bones, and osteoporosis in patients[ 14 ]. The mandibular condyle is a growth site in humans from birth to adulthood[ 15 ]. Regional adaptive growth, movable articulation, and endochondral bone formation are all provided by the mandibular condylar cartilage[ 16 ]. To the best of our knowledge, Vitamin A has been demonstrated in numerous studies to have an impact on bone growth and production, but its impact on the cartilaginous mandibular condyle has not yet been studied. The goal of the current study is to determine how vitamin A dosage, both excessive and optimum, affects the growth of the mandibular condyle in Wister albino rats across age groups, which is connected with the stages of mandibular condyle growth in humans. MATERIALS AND METHODS Material Forty –eight healthy offspring male albinos Wister rats (weighting 5- 7 grams)[17] were used. Based on the estimation of the sample size, the number of these animals was determined. The Medical Research Institute at Alexandria University provided the animals. Three rats per cage, with a light-dark cycle (L:D 12:12, with lights on at 7:00 am), were kept in specially constructed wire mesh bottom cages. Throughout the entire trial time, they had unrestricted access to food and tap water. The experiments complied with the ARRIVE guidelines and were approved by the Alexandria University Faculty of Dentistry's Research Ethics Committee. Power Analysis and the Sample Size Calculator were used to determine the study sample (PASS 2020)[18]. The total required sample size = number per group × number of follow-ups= 15 × 3= 45 rats. The rats included in the study were in a healthy state[19] with a weight range of (5-7grams)[17]. Previously used in experimental studies or have any medical problem or wound were excluded [19]. Study design This study was in vivo experimental controlled animal study. Experimental groups Forty –eight healthy offspring’s rats were randomly divided using computer-generated randomization software into 3 groups, each animal was given a unique identification number , and allocation to groups was performed using random numbers generated in Microsoft Excel : Group I (control group): 24rats were given a regular diet containing an optimum dose of vitamin A (12 IU /g of each pellet)[12, 20]. Group II(study group): 24 rats were given a regular diet supplemented with an overdose of vitamin A (1700 IU /g of each pellet)[12, 20]. In both groups, 8 rats were euthanized after 2, 4, and 6 months from the beginning of the study respectively. Although complete blinding was not feasible throughout all phases of the study, efforts were made to reduce bias wherever possible. Investigators involved in outcome assessment and data analysis were blinded to group allocation. However, due to the nature of the interventions, animal caretakers and researcher were not blinded Methods Administration of vitamin A [20] : Vitamin A was purchased in the form of A-Viton capsule (Kahira Pharmaceutical & Chemical Industries Company. Cairo, Egypt), each capsule consisted of 50000IU of vitamin palmitate, which is equal to 50 mg RE (RE=retinol equivalent, RE=3.333IUof vitamin A). Vitamin A doses were introduced in the diet at a concentration of 12 IU /g in each pellet as an optimum dose of vitamin A for the control group and at a concentration of 1700 IU /g in each pellet as an overdose of vitamin A for experimental group [12, 21]. Retinyl palmitate (A-Viton), a type of vitamin A, was added to the pelleted diet of rats. The content of each capsule was in the form of oil-soluble material. It was dissolved in vegetable oil and then sprayed on a diet on which each gram of pelleted diet contained (12 IU) as an optimum dose of vitamin A for the control group and (1700 IU) as an overdose of vitamin A for the experimental group. Rats started to receive food 3 weeks after birth. Quantitative determination of serum vitamin A concentrations [22, 23] : By the end of the experimental period and before euthanization, Blood was taken from the rats' orbital plexus the day after the last dosage of vitamin A was given to each group. The concentrations of vitamin A in serum were determined for all samples using rat vitamin A enzyme-linked immunosorbent assay kit (ELISA kit). In group I & group II, 8 rats were euthanized after 2, 4, and 6 months respectively. The rats were euthanized by overdose of diethyl ether [24]. Light microscopic study: The mandibular condyles of each rat were fixed in 10% neutral buffered formalin for 24h [25]. Using a microtome, tissue blocks were cut coronally at a thickness of 5 µm [26] Hematoxylin and Eosin (H&E) staining was applied to sections for general examination [25], trichrome stain for demonstration of lamellar and non-lamellar bone[27]and toluidine blue for demonstration of calcified cartilage and chondroitin sulfate [28]. Histomorphometric analysis [29] : Computer-assisted histomorphometry was performed to measure the bone surface area of the condyle, and thickness of chodroblastic and hypertrophic layers among groups. Histomorphometric analysis of tissues is based on quantitative measurements of microscopic organization and structure [30]. Percentage of bone surface area [31, 32] : The total surface area of the condylar bone with the standardized field was measured using the ROI manager and recorded. The bone surface area was then calculated by subtraction of the bone marrow spaces and cartilage area from the total surface area of the condyle. The percentage of bone was then calculated followed by statistical analysis and graphical representation by bar chart. Thickness of chondroblastic and hypertrophic zone [30, 33, 34]: The condylar head was divided into four regions: (1) the anterior, (2) superior, (3) postero-superior, and (4) posterior (Fig. 1). A fifth region was added to make regions equally distributed. Five measurements were taken for chodroblastic and hypertrophic layer in each section (Fig. 1). The average of five measurements, dispersed equally across each region, was obtained. This was followed by statistical analysis and a bar chart for graphical depiction. Statistical analysis: Data obtained from biochemical analysis and histomorphometric analysis was fed to the computer using SPSS software package version 23. The Shapiro-Wilk test, box plots, and descriptive statistics were used to assess the normality of the blood vitamin A concentration, hypertrophic layer thickness, chondroblastic layer thickness, and condyle bone surface area. Variables were not normally distributed except for the bone surface area of the condyle. In addition to mean and standard deviation (SD), the data were primarily displayed using the median, interquartile range (IQR), minimum, and maximum values. Groups were compared regarding concentration of serum vitamin A, hypertrophic layer thickness, and chondroblastic layer thickness using Mann Whitney U test at each time point while intra-group comparisons were done using the Friedman test and followed by pairwise comparisons when results were significant. Differences in bone surface area of the condyle between groups were compared using an independent t-test and repeated measures ANOVA were applied to assess differences across time intervals. P-value of 0.05 was chosen as the significance level. Each test had two tails. RESULTS Serum vitamin A concentration (immuno-chemistry assay) Table 1 and Fig. 2 show a comparison between control and study groups regarding the concentration of serum vitamin A after 2, 4, and 6 months. Comparing the study group to the control group, the study group's mean serum vitamin A concentration was substantially higher (p = 0.021, 0.021, and 0.021respectively).In both the control and study groups; there was no discernible difference between intervals (p = 0.368, 0.105 respectively). Table 1 Comparison between the control and study groups regarding the concentration of serum vitamin A Control Group Study Group Test ( P value) 2 months Mean (SD) 182.25 (28.65) 302.25 (48.40) 2.309 (0.021*) Median (IQR) 194.0 (48.0) 303.50 (91.0) Min - Max 140.0–201.0 242.0–360.0 4 months Mean (SD) 235.50 (4.04) 341.0 (76.95) 2.309 (0.021*) Median (IQR) 236.50 (8.0) 345.0 (143.0) Min - Max 230.0–239.0 243.0–431.0 6 months Mean (SD) 232.50 (57.82) 417.50 (57.87) 2.309 (0.021*) Median (IQR) 228.50 (108.0) 423.0 (107.0) Min - Max 178.0–295.0 345.0–470.0 Test ( P value) 2.00 (0.368) 4.500 (0.105) *Statistically significant difference at p value ≤ 0.05 Histological results After 2 months; in the control group (optimum dose of vitamin A) , the condyle showed normal structure of condylar zones. The fibrocartilage zone was composed of dense collagen fiber with scattered flat cells. The proliferative cells zone was composed of flattened and polygonal cells with large nuclei. The chondroblastic zone showed mature and spherical chondrocytes. The hypertrophic zone showed larger chondrocytes with the disappearance of nuclei. The erosive layer blended into the hypertrophic zone and was composed of numerous chondroclasts resorbed in the calcified matrix. The osteogenic zone showed bone trabeculae surrounding normal bone marrow (Fig. 3 ). After 2 months; in the study group (hypervitaminosis A group) , the condyle revealed thin chondroblastic zones and thick hypertrophic zones with signs of degeneration in comparison to the control group. The erosive zone revealed more chondroclasts than the control group (Fig. 4 a, 4 b). The osteogenic zone revealed thin trabeculae of cancellous bone with irregular outlines lined by numerous osteoclasts in Howship’s lacunae and wide bone marrow (Fig. 4 a, 4 c). After 4 months; in the control group (optimum dose of vitamin A) , the condyle showed the normal structure of cartilaginous zones in addition to thick trabeculae of cancellous bone surrounding cellular bone marrow. The bone trabeculae of the condyle showed fan shaped appearance (Fig. 5 ). After 4 months; in the study group (hypervitaminosis A group) , the condyle revealed wide chondroblastic zones and thin hypertrophic zones with signs of degeneration and complete disappearance of chondrocytes in comparison to the control group (Fig. 6 a, 6 b). The osteogenic zone revealed thin trabeculae of cancellous bone with irregular outlines lined by numerous osteoclasts in Howship’s lacunae and wide bone marrow (Fig. 6 c). After 6 months; in the control group (optimum dose of vitamin A) , the condyle showed mature cartilaginous zones. The osteogenic zone showed mature trabeculae with normal size of bone marrow ( Fig. 7 ). After 6 months; in the study group (hypervitaminosis A group) , the condyle revealed wide chondroblastic zones and thin hypertrophic zones with signs of degeneration in comparison to the control group (Fig. 8 a). The erosive zone revealed more chondroclasts than the control group ( Fig. 8 b ) . The osteogenic zone revealed thinner trabeculae of cancellous bone with irregular outlines ( Fig. 8 c ). In the osteogenic zone, the control group displayed replacement of cancellous bone by compact bone in comparison to the study group while in the study group, there were numerous areas of woven bone adjacent to a thin layer of mature cancellous bone (Fig. 9 ). Concerning the chondroitin sulfate in the intercellular matrix of chondroblastic and hypertrophic cell zones, (Fig. 11 ) shows that the study group's intercellular matrix of cartilage had a lower metachromatic reaction than the control groups (Fig. 10 ). Histomorphometric analysis A) The thickness of the chondroblastic layer: Table 2 and Fig. 12 a show a comparison between the control and study groups regarding the thickness of the chondroblastic layer. In the 2-month group: when compared to the control group, the study group's chondroblastic layer thickness decreased, and this difference was statistically significant. (p = 0.005). In the 4-month group: there was no statistically significant difference between the groups (p = 0.328).In the 6-month group: the study group showed an increase in thickness of the chondroblastic layer and it was statistically significant (p = 0.002). In control groups: there was no significant difference between intervals (p = 0.968).In study groups: there was a significant difference between intervals (p = 0.002). Table 2 Comparison between the control and study groups regarding the thickness of the chondroblastic layer. Control Group Study Group Test (P value) 2 months Mean (SD) 0.021 (0.004) 0.012 (0.004) 2.785 (0.005*) Median (IQR) 0.020 (0.006) 0.013 (0.007) Min - Max 0.014–0.027 0.007–0.020 4 months Mean (SD) 0.028 (0.017) 0.031 (0.011) 0.999 (0.328) Median (IQR) 0.019 (0.031) 0.026 (0.018) Min - Max 0.012–0.053 0.019–0.049 6 months Mean (SD) 0.022 (0.006) 0.046 (0.016) 2.941 (0.002*) Median (IQR) 0.020 (0.008) 0.046 (0.027) Min - Max 0.015 (0.034) 0.023–0.070 Test (P value) 0.065 (0.968) 13.000 (0.002*) Pairwise - P1 = 0.008*, P2 = 0.008*, P3 = 0.363 *Statistically significant difference at p value ≤ 0.05 P1: comparison between 2 months and 4 months, P2: comparison between 2 months and 6 months, P3: comparison between 4 months and 6 months. B) Thickness of hypertrophic zone: Table 3 and Fig. 12 b show a comparison between control and study groups regarding the hypertrophic zone. In the 2-month group: the study group showed an increase in the thickness of the hypertrophic layer in comparison to the control group and it was statistically significant (p = 0.001).In the 4 and 6-month groups: the study group showed a reduction in the thickness of the hypertrophic layer in comparison to the control group and it was statistically significant (p = 0.016 and 0.005 respectively). In control groups: there was a significant difference between intervals (p = 0.002). In study groups: there was a significant difference between intervals (p = 0.001). Table 3 Comparison between the control and study groups regarding the thickness of the hypertrophic layer. Control Group (n = 8) Study Group (n = 8) Test ( P value) 2 months Mean (SD) 0.034 (0.006) 0.059 (0.017) 3.151 (0.001*) Median (IQR) 0.034 (0.009) 0.060 (0.025) Min - Max 0.025–0.043 0.040–0.093 4 months Mean (SD) 0.071 (0.021) 0.044 (0.012) 2.415 (0.016*) Median (IQR) 0.076 (0.037) 0.044 (0.020) Min - Max 0.042–0.103 0.027–0.062 6 months Mean (SD) 0.044 (0.011) 0.027 (0.004) 2.793 (0.005*) Median (IQR) 0.046 (0.021) 0.027 (0.005) Min - Max 0.027–0.055 0.021–0.033 Test (P value) 12.250 (0.002*) 13.613 (0.001*) Pairwise P 1 = 0.001* , P 2 = 0.240, P 3 = 0.240 P 1 = 0.312, P 2 = 0.001* , P 3 = 0.137 *Statistically significant difference at p value ≤ 0.05P1: comparison between 2 months and 4 months, P2: comparison between 2 months and 6 months, P3: comparison between 4 months and 6 months. C) The percentage of bone surface area of the condyle: Table 4 and Fig. 12 c show a comparison between control and study groups regarding the percentage of bone surface area of the condyle. In 2,4and 6-month groups: The percentages of the bone surface area were greater in the control group as compared to the study group and it was statistically significant (p = 0.031, 0.0001, and 0.001respectively).In control groups: there was a significant difference between intervals (p = 0.122). In study groups; there was no significant difference between intervals (p = 0.122). Table 4 Comparison between the control and study groups regarding the percentage of bone surface area of the condyle Control Group Study Group Test ( P value) 2 months Mean (SD) 53.37 (5.48) 42.71 (11.31) 2.399 (0.031*) Median (IQR) 53.48 (5.85) 45.25 (16.40) Min - Max 42.86–61.33 22.67–57.41 4 months Mean (SD) 57.97 (9.61) 41.37 (4.02) 4.509 (< 0.0001*) Median (IQR) 57.18 (15.27) 42.58 (6.74) Min - Max 42.16–71.59 34.21–45.27 6 months Mean (SD) 66.81 (3.22) 49.32 (10.54) 4.489 (0.001*) Median (IQR) 65.96 (4.94) 48.80 (12.75) Min - Max 63.72–73.19 29.23–64.62 Test ( P value) 10.787 (0.001*) 2.459 (0.122) Pairwise P 1 = 0.549, P 2 = 0.0001* , P 3 = 0.129 - *Statistically significant difference at p value ≤ 0.05.P1: comparison between 2 and 4 months, P2: comparison between 2 and 6 months, P3: comparison between 4 months and 6 months. DISCUSSION Vitamin A has been recognized to be essential for normal growth and bone health. However, many undesirable effects were associated with an overdose of vitamin A [ 8 , 35 ]. Rats were euthanized after two, four, and six months. These time intervals were designed in correlation with different age groups of humans. Two months of rats’ age is correlated with 6 years of humans. Four months of rats’ age is correlated with 12 years of humans (mixed dentition stage). Six months of rats’ age is correlated with 18 years of adult humans [ 36 ]. The quantitative determination of serum vitamin A concentrations exhibited a significantly higher level of vitamin A in the study groups compared to rats in the control groups. These findings were in agreement with Lind et al[ 20 ], who recorded elevated levels of serum vitamin A in the study group compared to the control group. In control groups (2 months), the condylar cartilage exhibited normal structure and thickness of the condylar zones. The cells maintained their normal morphology and organization. The osteogenic layer showed well-developed trabeculae of cancellous bone surrounded by the normal size of bone marrow. In the experimental group (2 months), condylar cartilage exhibited a thinner chondroblastic zone and thicker hypertrophic zone as compared with the control group. A histomorphometric study of the thickness of the hypertrophic and chondroblastic layers validated these findings. This could be explained on the basis that, vitamin A induces differentiation and maturation of chondrocytes. Additionally, soeta et al[ 37 ], reported that an overdose of vitamin A first disrupted the development of chondrocytes and increased the hypertrophic chondrocytes. Moreover, Azimpour and Mortazavi et al [ 38 ], reported that hypervitaminosis A inhibited chondrogenesis. The inhibitory effect of over-dose of vitamin A is due to the blockage of the production of glycosaminoglycans by chondrocytes[ 39 ]. Furthermore, the osteogenic layer of the experimental group showed an irregular outline with wide bone marrow. The reduced thickness of bony trabeculae was confirmed by histomorphometric analysis of the total surface area of bone and could be explained that Excessive vitamin A promotes bone resorption and hinders bone formation. These effects are expected to promote bone loss, contributing to the development of osteoporosis and fractures[ 40 , 41 ]. Moreover, some studies reported that retinol can accelerate bone resorption through stimulation of parathyroid hormone secretion (PTH) and disturbance of vitamin D metabolism[ 42 , 43 ]. It had been also reported by Lind et al., that excessive retinol reduces bone diameter through alteration of bone mineral metabolism[ 21 ]. However, other studies reported that there was no association between fracture risk and vitamin A [ 5 , 44 ]. Additionally, it was also reported that retinoic acid (RA) inhibits osteoclastogenesis in pure (pre) osteoclastic cultures[ 45 , 46 ]. This variable effect may be a result of dose-response route and duration of administration or species difference. After 4 months, the control showed a normal structure and thickness of and thickness of condylar zones. The osteogenic layer showed well-formed bone trabeculae with the normal size of bone marrow. The results obtained from the experimental group after 4 months revealed a thicker chondroblastic zone and thinner hypertrophic zone as compared to the control group and were confirmed by histomorphometric analysis of the thickness of chondroblastic and hypertrophic layers. The marked increase in the chondroblastic zone is related to a compensatory mechanism for the premature closure of the hypertrophic zone. These findings are in accordance with Onder et al[ 47 ], who reported a reduction in the height of the hypertrophic zone and an increase in the height of the proliferative zone in rats exposed to a high dose of vitamin A.Thickening of chondroblastic zone and thinning of hypertrophic zone related to hypervitaminosis A may be due to retardation of transition from chondroblastic zone to the hypertrophic zone. This retardation delays endochondral bone formation. Tearing of hypertrophic cells with signs of degeneration were observed. These findings are similar to Soeta et al[ 48 ]., who clarified that hypervitaminosis A induced chondroclast cells to absorb the calcified cartilage matrix and result in the erosions of the hypertrophic zone. As regards to osteogenic zone, thin bone trabeculae with wide bone marrow were observed as a sign of osteoporosis and confirmed by histomorphometric analysis of the total surface area of the bone. Similar findings had been recorded by Lind et al.,[ 21 ] who reported thinning of long bone and an increase of osteoclasts upon the administration of vitamin A in overdose. In humans, it has been reported that excessive vitamin A has been correlated with the acceleration of skeletal bone resorption [ 49 ]. In the control group after 6 months, well-formed bone trabeculae in the osteogenic layer with normal structure and thickness of the other condylar zones were observed with further maturation of the condyle and was confirmed by histomorphometric analysis of the condyles A dramatic reduction in the thickness of the hypertrophic zone and increase of chondroblastic zone with wide bone marrow and high osteoclastic activity were established in the study group at the end of this study. Several studies have reported that an overdose of vitamin A causes bone weakening due to increased periosteal bone resorption and decreased bone formation [ 5 , 50 ]. Persistent numerous islands of woven bone had been detected in study groups as compared to control groups and may be due to that hypervitaminosis A leads to delay of bone maturation. These results follow Lind et al, [ 12 ]who found that vitamin A inhibits osteoblast mineralization by decreasing the production of Runx2 and Osterix. (factors required for osteoblast maturation). As concerning the intercellular matrix of chondroblastic and hypertrophic cell zones, there was a reduction of metachromatic reaction of the intercellular matrix in the study group in comparison to the control group. These findings could be explained that hypervitaminosis A inhibits the formation of cartilage matrix by chondrocytes and these findings agreed with studies that reported that hypervitaminosis A reduces the production of proteoglycans by chondrocytes[ 38 , 51 ]. The decrease may be caused by an impairment of core protein synthesis, a change in glycosaminoglycan chain production, or decreased sulphation. In the present study, the control group showed the normal structure of cartilage and bone, while the study groups showed marked osteoporosis and growth arrest of the mandibular condyle. This effect could be explained by the effect of vitamin A on the actions of vitamin D. Vitamin A may inhibit vitamin D absorption, transport, or conversion to its active form, or it may promote vitamin D metabolic breakdown[ 43 ]. Because the major function of vitamin D is to improve the absorption of calcium in the gut, the optimum dose of vitamin A leads to decreased serum calcium levels [ 43 ]. Low serum calcium levels increase the production of parathyroid hormone (PTH), which helps to restore normal serum calcium levels by releasing calcium from the bone, promoting bone turnover and osteoporosis[ 42 ]. It has been observed that parathyroid hormone (PTH) is a strong inhibitor of chondrocyte maturation and suppresses cell hypertrophy which leads to inhibition of the endochondral bone formation and growth arrest[ 52 ]. CONCLUSION over dose of vitamin A inhibits bone formation and diminishes endochondral bone growth by premature closure of the condylar cartilage which leads to teratogenicity of mandibular condyle so taking vitamin A must be under medical control. Declarations ACKNOWLEDGEMENTS : The authors thank Master Walid, the laboratory technician in the Department of Oral Biology, Faculty of Dentistry, Alexandria University for his efforts in preparing and dyeing the slides. AUTHOR CONTRIBUTION: S.H. planned the experiments. S.A. and M.M. carried out the experiments. S.H. and H.S. analyzed and confirmed all data. S.H. prepared the manuscript. S.H. and H.S. confirmed the manuscript. All authors reviewed the manuscript, and S.H.made a final approval. FUNDING: NO funding to conduct this study. Data availability : The datasets used or analyzed during the current study are available from corresponding authors on reasonable request and her email is [email protected] .com Ethics approval : The study was carried out after receiving approval from the Research of Animal Ethics Committee., Faculty of Dentistry, Alexandria University. (Approval number: 0159 and IORG is 0008839). Aprotocol (including the research question, key design features, and analysis plan) was prepared before the study, and this protocol was registered at Faculty of Dentistry, Alexandria University. Conflict of interest No conflicts of interest. References D’Ambrosio DN, Clugston RD, Blaner WS. Vitamin A metabolism: an update. Nutrients. 2011;3:63–103. http://dx.doi.org/10.3390/nu3010063 . Harrison EH. Mechanisms involved in the intestinal absorption of dietary vitamin A and provitamin A carotenoids. 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Lack of a relation between vitamin and mineral antioxidants and bone mineral density: results from the Women’s Health Initiative–. Am J Clin Nutr. 2005;82:581–8. https://doi.org/10.1093/ajcn.82.3.581 . Conaway HH, Persson E, Halén M, Granholm S, Svensson O, Pettersson U, Lie A, Lerner UH. Retinoids inhibit differentiation of hematopoetic osteoclast progenitors. FASEB J. 2009;23:3526–38. https://doi.org/10.1096/fj.09-132548 . Hu L, Lind T, Sundqvist A, Jacobson A, Melhus H. Retinoic acid increases proliferation of human osteoclast progenitors and inhibits RANKL-stimulated osteoclast differentiation by suppressing RANK. PLoS ONE. 2010;5:e13305. https://doi.org/10.1371/journal.pone.0013305 . Onder S, Bilgili SG, Bulut G, Celik HT, Oguztuzun S, Onder H, Calka O, Karadag AS. Biochemical and histopathologic assessment of effects of acitretin on epiphyseal growth plate in rats. Adv Dermatology Allergology/Postȩpy Dermatologii i Alergologii. 2020;37:346. https://doi.org/10.5114/ada.2020.95983 . SOETA S, KODAKA MORIR, T., NAITO, Y., and, TANIGUCHI K. Histological disorders related to the focal disappearance of the epiphyseal growth plate in rats induced by high dose of vitamin A. J Vet Med Sci. 2000;62:293–9. https://doi.org/10.1292/jvms.62.293 . Kawahara TN, Krueger DC, Engelke JA, Harke JM, Binkley NC. Short-term vitamin A supplementation does not affect bone turnover in men. J Nutr. 2002;132:1169–72. https://doi.org/10.1093/jn/132.6.1169 . Kneissel M, Studer A, Cortesi R, Šuša M. Retinoid-induced bone thinning is caused by subperiosteal osteoclast activity in adult rodents. Bone. 2005;36:202–14. https://doi.org/10.1016/j.bone.2004.11.006 . Kirimoto A, Takagi Y, Ohya K, Shimokawa H. Effects of retinoic acid on the differentiation of chondrogenic progenitor cells, ATDC5. J Med Dent Sci. 2005;52:153–62. http://dx.doi.org/10.1136/ard.2008.095414 . Li X, Schwarz EM, Zuscik MJ, Rosier RN, Ionescu AM, Puzas JE, Drissi H, Sheu T-J, O’Keefe RJ. Retinoic acid stimulates chondrocyte differentiation and enhances bone morphogenetic protein effects through induction of Smad1 and Smad5. Endocrinology 2003; 144 , 2514–2523. https://doi.org/10.1210/en.2002-220969 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6487311","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453959695,"identity":"e0c94664-249e-442e-b0f1-db8803a1444f","order_by":0,"name":"Shimaa Abdel Shafy","email":"data:image/png;base64,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","orcid":"","institution":"Alexandria University","correspondingAuthor":true,"prefix":"","firstName":"Shimaa","middleName":"Abdel","lastName":"Shafy","suffix":""},{"id":453959696,"identity":"d580f0b1-2d37-48ec-989b-6492a000493d","order_by":1,"name":"Shadia Hussein Abdel majeed","email":"","orcid":"","institution":"Alexandria University","correspondingAuthor":false,"prefix":"","firstName":"Shadia","middleName":"Hussein Abdel","lastName":"majeed","suffix":""},{"id":453959700,"identity":"541f5d16-6e33-45ae-8158-37cd9239d157","order_by":2,"name":"Mounir Mahmoud El –Adawy","email":"","orcid":"","institution":"Alexandria University","correspondingAuthor":false,"prefix":"","firstName":"Mounir","middleName":"Mahmoud El","lastName":"–Adawy","suffix":""},{"id":453959701,"identity":"c6b4094c-c37b-4c85-8f40-835b60507268","order_by":3,"name":"Hagar Sherif","email":"","orcid":"","institution":"Alexandria University","correspondingAuthor":false,"prefix":"","firstName":"Hagar","middleName":"","lastName":"Sherif","suffix":""}],"badges":[],"createdAt":"2025-04-20 04:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6487311/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6487311/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82365236,"identity":"f10ce0bc-2d2d-460c-b71d-57c161832593","added_by":"auto","created_at":"2025-05-09 12:39:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":452157,"visible":true,"origin":"","legend":"\u003cp\u003eChondroblastic zone denoted by black lines; hypertrophic layer thickness denoted by yellow lines .H\u0026amp;E, x100.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/cebc230dba40e951c8abf420.png"},{"id":82364286,"identity":"d1669ab7-9003-4ba1-a933-93316cd16076","added_by":"auto","created_at":"2025-05-09 12:31:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103940,"visible":true,"origin":"","legend":"\u003cp\u003eMean concentration of serum vitamin A in the control and study groups.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/c311d4926da5f54a6c232745.png"},{"id":82364299,"identity":"64afd1f4-aae9-45dc-a7bf-1a6c1a75ae96","added_by":"auto","created_at":"2025-05-09 12:31:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":652880,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph (control group after 2 months), (3b, 3c) are inset (1, 2) of (3a) respectively. The condyle showed a normal structure of condylar zones. C: hyaline cartilage BM: bone marrow B: trabecular bone AD: articular disc. F: fibrocartilage zone, P: proliferative zone, C: chondroblastic zone, H: hypertrophic zone. Note the chondroclasts in the erosive zone (arrows in 3b). Bone marrow lined by active osteoblasts (arrows in 3c) (H\u0026amp;E, (3aX100), (3b, 3c) X400).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/9e079b7a0a5df0629a2e39f2.png"},{"id":82364292,"identity":"f69efb41-33ce-4ad4-aa29-e8121ae68c33","added_by":"auto","created_at":"2025-05-09 12:31:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":660702,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph (study group after 2 months), (4a, 4b) are insets (1, 2) of (4a) respectively. The condyle revealed thin chondroblastic zones and thick hypertrophic zones. The osteogenic zone revealed thin trabeculae of cancellous bone: hyaline cartilage BM: bone marrow B: trabecular bone AD: articular disc. F: fibrocartilage zone, P: proliferative zone, C: chondroblastic zone, H: hypertrophic zone. Note the sign of degeneration of chondrocytes (dotted arrows in b) and the erosive zone shows numerous chondroclasts (arrows in 4b). Thin bone trabeculae studded with osteoclasts in Howship’s lacunae (arrows in c). (H\u0026amp;E, (4aX100), (4b, 4c) X400).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/1b1874e86d93e45beb799464.png"},{"id":82365235,"identity":"30720429-a28a-4daa-83af-304e9e02892c","added_by":"auto","created_at":"2025-05-09 12:39:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":572153,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph control group (optimum dose of vitamin A, after 4 months), (5a, 5b) are insets (1, 2) of (5a) respectively. The condyle showed the normal structure of cartilaginous zones in addition to thick trabeculae of cancellous bone C: hyaline cartilage BM: bone marrow B: trabecular bone. F: fibrocartilage zone, P: proliferative zone, C: chondroblastic zone, H: hypertrophic zone. Thin bone trabeculae studded with osteoclasts in Howship’s lacunae (arrows in 5b). Note Bone marrow lined by active osteoblasts (arrows in 5c) (H\u0026amp;E, (5aX100), (5b, 5c) X400).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/2953ad1c0ccc5be93d763d06.png"},{"id":82364291,"identity":"64a57401-cf84-46b0-8b8e-595f048281b0","added_by":"auto","created_at":"2025-05-09 12:31:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":540082,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph study group (overdose of vitamin A, after 4 months), (6a, 6b) are insets (1, 2) of (6a) respectively. The condyle revealed wide chondroblastic zones and thin hypertrophic zones. The osteogenic zone revealed thin trabeculae of cancellous bone. C: hyaline cartilage BM: bone marrow B: trabecular bone. F: fibrocartilage zone, P: proliferative zone, C: chondroblastic zone, H: hypertrophic zone. Note thin bone trabeculae studded with osteoclasts in Howship’s lacunae (arrows in 6c). (H\u0026amp;E, (6aX100), (6b, 6c) X400)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/7026d84476ae6437a3ca4790.png"},{"id":82365763,"identity":"9c738259-2d9f-43d0-83d7-40442c162f59","added_by":"auto","created_at":"2025-05-09 12:47:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":487657,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph (control group (optimum dose of vitamin A, after 6 months), (7a, 7b) are inset (1, 2) of 7a respectively. The condyle showed mature cartilaginous zones. The osteogenic zone showed mature trabeculae. C: hyaline cartilage BM: bone marrow B: trabecular bone AD: articular disc. F: fibrocartilage zone, P: proliferative zone, C: chondroblastic zone, H: hypertrophic zone. (H\u0026amp;E, (7aX100), (7b, 7c) X400)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/482acf78a675aadd4c109dc2.png"},{"id":82365237,"identity":"22e32f96-a845-421a-b363-154ee5228f20","added_by":"auto","created_at":"2025-05-09 12:39:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":561528,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph study group (overdose of vitamin A, after 6 months), (8a, 8b) are insets (1, 2) of (8a) respectively. The condyle revealed wide chondroblastic zones and thin hypertrophic zones. The osteogenic zone revealed thinner trabeculae of cancellous bone. C: hyaline cartilage BM: bone marrow B: trabecular bone. C: chondroblastic zone, H: hypertrophic zone. Note the erosive zone reveals numerous chondroclasts (arrows in 8b). (H\u0026amp;E, (8aX100), (8b, 8c) X400).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/b06c10027da455c82e6b084b.png"},{"id":82365241,"identity":"4dd93674-c8ce-48b1-9cf4-fcddfeec4af9","added_by":"auto","created_at":"2025-05-09 12:39:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":501553,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph, (9a, 9b): control and study group after 2 months respectively. (9c, 9d): control and study group after 4 months respectively. (9f, 9g): control and study group after 6 months respectively. Trichrome stain, x100\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/89814b41e5c57dbd65d2a685.png"},{"id":82366918,"identity":"c20458a9-9bff-4539-9653-47d4e40c15db","added_by":"auto","created_at":"2025-05-09 12:55:04","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":588470,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph, control groups after 2, 4and 6 months 10b is an inset of 10a, 10d is an inset of 10c and 10g is an inset of 10f. Toluidine blue stain (10a, 10c and 10f): x100. (10b.10d and 10g): x400\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/d6723543cfaa03c1ab53d527.png"},{"id":82364302,"identity":"af181baa-d259-4516-9e00-643d1379ecc1","added_by":"auto","created_at":"2025-05-09 12:31:04","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":579760,"visible":true,"origin":"","legend":"\u003cp\u003eLight photomicrograph, study groups after 2, 4and 6 months. .11b is inset of 11a, 11d is inset of 11c and 11g is inset of 11f. Toluidine blue stain (11a, 11c and 11f): x100. (11b.11d and 11g): x400\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/dadca5c976e681e88c938c59.png"},{"id":82364315,"identity":"5068a4e7-18be-4342-9146-55c5a87e134c","added_by":"auto","created_at":"2025-05-09 12:31:04","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":165470,"visible":true,"origin":"","legend":"\u003cp\u003ea): Comparison between the control and study groups of the thickness of the chondroblastic layer.\u003c/p\u003e\n\u003cp\u003eb) Comparison between the control and study groups regarding the thickness of the hypertrophic layer.\u003c/p\u003e\n\u003cp\u003ec) Comparison between the control and study groups regarding the percentage of bone surface area of the condyle.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/79ea0d06be6a49bcadc326d0.png"},{"id":83824691,"identity":"9104a7f0-fc87-4272-b9a0-2991e71fa83b","added_by":"auto","created_at":"2025-06-03 09:47:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7351446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6487311/v1/52214e99-5a64-4a58-b1b8-2ab70baea343.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Effect of Vitamin a as a Teratogenic Agent in Condylar Bone Growth\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eVitamin A is a member of the fat-soluble vitamins. There are two types of vitamin A (VA) found in food: provitamin A carotenoids and preformed VA (retinol or retinyl esters)[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRetinol is derived from animal sources such as eggs, liver, milk, and fortified cereals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], while Carotenoids originated from plant sources such as carrots, collards, spinach, and squash[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVitamin A is required in tiny amounts for various biological processes, such as immunity, growth, differentiation of cells, reproduction, embryological development, and vision [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLipids aid in the intestinal absorption of vitamin A, which is mostly stored in the liver, kidney, and adipose tissues [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe primary cause of blindness is vitamin A deficiency, which increases the risk of infectious diseases and diarrheal illness. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], whereas the prevalence of spontaneous bone fractures, aberrant skeletal growth in children, and increased bone turnover are all associated with hypervitaminosis A[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs vitamin A is quickly absorbed and eliminated, Acute toxicity can result from exposure to high doses for brief periods or chronic toxicity can result from much lower intake for extended periods [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe retinoid form of vitamin A is represented by vitamin A supplements [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It has been discovered that the retinoid type of vitamin A is more likely than the carotenoid type to produce vitamin A toxicity. The retinoid type is absorbed easily at rates of 70\u0026ndash;90%, whereas the carotenoids type is absorbed significantly less efficiently at rates of 20\u0026ndash;50%. Although the carotenoid form is less harmful than the retinoid type, it has been discovered that too much beta-carotene in the diet can cause carotenodermia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Carotenodermia is a skin disorder marked by an orange-yellow coloring [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Increased periosteal bone resorption and decreased bone formation have been associated with the thinning of long bones in rodents with hypervitaminosis A [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, rats with hypervitaminosis A develop mandibular and scapular perforations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn humans, Hypervitaminosis A has been linked to bone pain, hypercalcemia, and accelerated bone resorption. Long-term retinoid therapy has also resulted in increasing calcification of ligaments, remodeling anomalies of long bones, and osteoporosis in patients[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mandibular condyle is a growth site in humans from birth to adulthood[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Regional adaptive growth, movable articulation, and endochondral bone formation are all provided by the mandibular condylar cartilage[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, Vitamin A has been demonstrated in numerous studies to have an impact on bone growth and production, but its impact on the cartilaginous mandibular condyle has not yet been studied.\u003c/p\u003e \u003cp\u003eThe goal of the current study is to determine how vitamin A dosage, both excessive and optimum, affects the growth of the mandibular condyle in Wister albino rats across age groups, which is connected with the stages of mandibular condyle growth in humans.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eMaterial\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty \u0026ndash;eight healthy offspring male albinos Wister rats (weighting 5- 7 grams)[17] were used. Based on the estimation of the sample size, the number of these animals was determined. The Medical Research Institute at Alexandria University provided the animals. Three rats per cage, with a light-dark cycle (L:D 12:12, with lights on at 7:00 am), were kept in specially constructed wire mesh bottom cages. Throughout the entire trial time, they had unrestricted access to food and tap water. The experiments complied with the ARRIVE guidelines and were approved by the Alexandria University Faculty of Dentistry\u0026apos;s Research Ethics Committee.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePower Analysis and the Sample Size Calculator were used to determine the study sample (PASS 2020)[18]. The total required sample size = number per group \u0026times; number of follow-ups= 15 \u0026times; 3= 45 rats. The rats included in the study were in a healthy state[19] with a weight range of (5-7grams)[17]. \u0026nbsp;Previously used in experimental studies or have any medical problem or wound were excluded [19].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was in vivo experimental controlled animal study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental groups\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty \u0026ndash;eight healthy offspring\u0026rsquo;s rats were randomly divided using computer-generated randomization software into 3 groups, each animal was given a unique identification number , and allocation to groups was performed using random numbers generated in Microsoft Excel : \u0026nbsp; Group I (control group): 24rats were given a regular diet containing an optimum dose of vitamin A (12 IU /g of each pellet)[12, 20]. Group II(study group): 24 rats were given a regular diet supplemented with an overdose of vitamin A (1700 IU /g of each pellet)[12, 20]. In both groups, 8 rats were euthanized after 2, 4, and 6 months from the beginning of the study respectively. Although complete blinding was not feasible throughout all phases of the study, efforts were made to reduce bias wherever possible. Investigators involved in outcome assessment and data analysis were blinded to group allocation. However, due to the nature of the interventions, animal caretakers and researcher were not blinded\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdministration of vitamin A\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e[20]\u003c/strong\u003e\u003cstrong\u003e:\u003csup\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVitamin A was purchased in the form of A-Viton capsule (Kahira Pharmaceutical \u0026amp; Chemical Industries Company. Cairo, Egypt), each capsule consisted of 50000IU of vitamin palmitate, which is equal to 50 mg RE (RE=retinol equivalent, RE=3.333IUof vitamin A). \u0026nbsp;Vitamin A doses were introduced in the diet at a concentration of 12 IU /g in each pellet as an optimum dose of vitamin A for the control group and at a concentration of 1700 IU /g in each pellet as an overdose of vitamin A for experimental group [12, 21].\u003c/p\u003e\n\u003cp\u003eRetinyl palmitate (A-Viton), a type of vitamin A, was added to the pelleted diet of rats. The content of each capsule was in the form of oil-soluble material. It was dissolved in vegetable oil and then sprayed on a diet on which each gram of pelleted diet contained (12 IU) as an optimum dose of vitamin A for the control group and (1700 IU) as an overdose of vitamin A for the experimental group. Rats started to receive food 3 weeks after birth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative determination of serum vitamin A concentrations\u003c/strong\u003e\u003cstrong\u003e[22, 23]\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy the end of the experimental period and before euthanization, Blood was taken from the rats\u0026apos; orbital plexus the day after the last dosage of vitamin A was given to each group. The concentrations of vitamin A in serum were determined for all samples using rat vitamin A enzyme-linked immunosorbent assay kit (ELISA kit).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn group I \u0026amp; group II, 8 rats were euthanized after 2, 4, and 6 months respectively. The rats were euthanized by overdose of diethyl ether [24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLight microscopic study:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mandibular condyles of each rat were fixed in 10% neutral buffered formalin for 24h [25]. Using a microtome, tissue blocks were cut coronally at a thickness of 5 \u0026micro;m [26] Hematoxylin and Eosin (H\u0026amp;E) staining was applied to sections for general examination [25], trichrome stain for demonstration of lamellar and non-lamellar bone[27]and toluidine blue for demonstration of calcified cartilage and chondroitin sulfate [28].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistomorphometric analysis\u003c/strong\u003e\u003cstrong\u003e[29]\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eComputer-assisted histomorphometry was performed to measure the bone surface area of the condyle, and thickness of chodroblastic and hypertrophic layers among groups. Histomorphometric analysis of tissues is based on quantitative measurements of microscopic organization and structure [30].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePercentage of bone surface area\u003c/strong\u003e\u003cstrong\u003e[31, 32]\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThe total surface area of the condylar bone with the standardized field was measured using the ROI manager and recorded. The bone surface area was then calculated by subtraction of the bone marrow spaces and cartilage area from the total surface area of the condyle. The percentage of bone was then calculated followed by statistical analysis and graphical representation by bar chart.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThickness of chondroblastic and hypertrophic zone\u003c/strong\u003e [30, 33, 34]:\u003c/p\u003e\n\u003cp\u003eThe condylar head was divided into four regions: (1) the anterior, (2) superior, (3) postero-superior, and (4) posterior (Fig. 1). A fifth region was added to make regions equally distributed. Five measurements were taken for chodroblastic and hypertrophic layer in each section (Fig. 1). The average of five measurements, dispersed equally across each region, was obtained. This was followed by statistical analysis and a bar chart for graphical depiction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData obtained from biochemical analysis and histomorphometric analysis was fed to the computer using SPSS software package version 23. The Shapiro-Wilk test, box plots, and descriptive statistics were used to assess the normality of the blood vitamin A concentration, hypertrophic layer thickness, chondroblastic layer thickness, and condyle bone surface area. Variables were not normally distributed except for the bone surface area of the condyle. In addition to mean and standard deviation (SD), the data were primarily displayed using the median, interquartile range (IQR), minimum, and maximum values. Groups were compared regarding concentration of serum vitamin A, hypertrophic layer thickness, and chondroblastic layer thickness using Mann Whitney U test at each time point while intra-group comparisons were done using the Friedman test and followed by pairwise comparisons when results were significant. Differences in bone surface area of the condyle between groups were compared using an independent t-test and repeated measures ANOVA were applied to assess differences across time intervals. P-value of 0.05 was chosen as the significance level. Each test had two tails.\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSerum vitamin A concentration (immuno-chemistry assay)\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show a comparison between control and study groups regarding the concentration of serum vitamin A after 2, 4, and 6 months. Comparing the study group to the control group, the study group's mean serum vitamin A concentration was substantially higher (p\u0026thinsp;=\u0026thinsp;0.021, 0.021, and 0.021respectively).In both the control and study groups; there was no discernible difference between intervals (p\u0026thinsp;=\u0026thinsp;0.368, 0.105 respectively).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison between the control and study groups regarding the concentration of serum vitamin A\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStudy Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003cp\u003e(\u003cem\u003eP\u003c/em\u003e value)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e182.25 (28.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e302.25 (48.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2.309\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0.021*)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e194.0 (48.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e303.50 (91.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e140.0\u0026ndash;201.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e242.0\u0026ndash;360.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e235.50 (4.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e341.0 (76.95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2.309\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0.021*)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e236.50 (8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e345.0 (143.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e230.0\u0026ndash;239.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e243.0\u0026ndash;431.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMean (SD)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e232.50 (57.82)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e417.50 (57.87)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003e2.309\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0.021*)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e228.50 (108.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e423.0 (107.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e178.0\u0026ndash;295.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e345.0\u0026ndash;470.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003cp\u003e(\u003cem\u003eP\u003c/em\u003e value)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003cp\u003e(0.368)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.500\u003c/p\u003e \u003cp\u003e(0.105)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Statistically significant difference at \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHistological results\u003c/h2\u003e \u003cp\u003e \u003cb\u003eAfter 2 months; in the control group (optimum dose of vitamin A)\u003c/b\u003e, the condyle showed normal structure of condylar zones. The fibrocartilage zone was composed of dense collagen fiber with scattered flat cells. The proliferative cells zone was composed of flattened and polygonal cells with large nuclei. The chondroblastic zone showed mature and spherical chondrocytes. The hypertrophic zone showed larger chondrocytes with the disappearance of nuclei. The erosive layer blended into the hypertrophic zone and was composed of numerous chondroclasts resorbed in the calcified matrix. The osteogenic zone showed bone trabeculae surrounding normal bone marrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAfter 2 months; in the study group (hypervitaminosis A group)\u003c/b\u003e, the condyle revealed thin chondroblastic zones and thick hypertrophic zones with signs of degeneration in comparison to the control group. The erosive zone revealed more chondroclasts than the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The osteogenic zone revealed thin trabeculae of cancellous bone with irregular outlines lined by numerous osteoclasts in Howship\u0026rsquo;s lacunae and wide bone marrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAfter 4 months; in the control group (optimum dose of vitamin A)\u003c/b\u003e, the condyle showed the normal structure of cartilaginous zones in addition to thick trabeculae of cancellous bone surrounding cellular bone marrow. The bone trabeculae of the condyle showed fan shaped appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAfter 4 months; in the study group (hypervitaminosis A group)\u003c/b\u003e, the condyle revealed wide chondroblastic zones and thin hypertrophic zones with signs of degeneration and complete disappearance of chondrocytes in comparison to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The osteogenic zone revealed thin trabeculae of cancellous bone with irregular outlines lined by numerous osteoclasts in Howship\u0026rsquo;s lacunae and wide bone marrow (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e \u003cb\u003eAfter 6 months; in the control group (optimum dose of vitamin A)\u003c/b\u003e, the condyle showed mature cartilaginous zones. The osteogenic zone showed mature trabeculae with normal size of bone marrow \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eAfter 6 months; in the study group (hypervitaminosis A group)\u003c/b\u003e, the condyle revealed wide chondroblastic zones and thin hypertrophic zones with signs of degeneration in comparison to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The erosive zone revealed more chondroclasts than the control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. The osteogenic zone revealed thinner trabeculae of cancellous bone with irregular outlines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the osteogenic zone, the control group displayed replacement of cancellous bone by compact bone in comparison to the study group while in the study group, there were numerous areas of woven bone adjacent to a thin layer of mature cancellous bone (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConcerning the chondroitin sulfate in the intercellular matrix of chondroblastic and hypertrophic cell zones,\u003c/p\u003e \u003cp\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e) shows that the study group's intercellular matrix of cartilage had a lower metachromatic reaction than the control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistomorphometric analysis\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eA) The thickness of the chondroblastic layer:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ea show a comparison between the control and study groups regarding the thickness of the chondroblastic layer. In the 2-month group: when compared to the control group, the study group's chondroblastic layer thickness decreased, and this difference was statistically significant. (p\u0026thinsp;=\u0026thinsp;0.005). In the 4-month group: there was no statistically significant difference between the groups (p\u0026thinsp;=\u0026thinsp;0.328).In the 6-month group: the study group showed an increase in thickness of the chondroblastic layer and it was statistically significant (p\u0026thinsp;=\u0026thinsp;0.002). In control groups: there was no significant difference between intervals (p\u0026thinsp;=\u0026thinsp;0.968).In study groups: there was a significant difference between intervals (p\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison between the control and study groups regarding the thickness of the chondroblastic layer.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStudy Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003cp\u003e(P value)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.021 (0.004)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.012 (0.004)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2.785\u003c/p\u003e \u003cp\u003e(0.005*)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.020 (0.006)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.013 (0.007)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.014\u0026ndash;0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.007\u0026ndash;0.020\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.028 (0.017)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.031 (0.011)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.999\u003c/p\u003e \u003cp\u003e(0.328)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.019 (0.031)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.026 (0.018)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.012\u0026ndash;0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.019\u0026ndash;0.049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.022 (0.006)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.046 (0.016)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2.941\u003c/p\u003e \u003cp\u003e(0.002*)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.020 (0.008)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.046 (0.027)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.015 (0.034)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.023\u0026ndash;0.070\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003cp\u003e(P value)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.065\u003c/p\u003e \u003cp\u003e(0.968)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.000\u003c/p\u003e \u003cp\u003e(0.002*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePairwise\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP1\u0026thinsp;=\u0026thinsp;0.008*, P2\u0026thinsp;=\u0026thinsp;0.008*, P3\u0026thinsp;=\u0026thinsp;0.363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Statistically significant difference at p value\u0026thinsp;\u0026le;\u0026thinsp;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eP1: comparison between 2 months and 4 months, P2: comparison between 2 months and 6 months, P3: comparison between 4 months and 6 months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eB) Thickness of hypertrophic zone:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eb show a comparison between control and study groups regarding the hypertrophic zone. In the 2-month group: the study group showed an increase in the thickness of the hypertrophic layer in comparison to the control group and it was statistically significant (p\u0026thinsp;=\u0026thinsp;0.001).In the 4 and 6-month groups: the study group showed a reduction in the thickness of the hypertrophic layer in comparison to the control group and it was statistically significant (p\u0026thinsp;=\u0026thinsp;0.016 and 0.005 respectively). In control groups: there was a significant difference between intervals (p\u0026thinsp;=\u0026thinsp;0.002). In study groups: there was a significant difference between intervals (p\u0026thinsp;=\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison between the control and study groups regarding the thickness of the hypertrophic layer.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl Group\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStudy Group\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003cp\u003e(\u003cem\u003eP\u003c/em\u003e value)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.034 (0.006)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.059 (0.017)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3.151\u003c/p\u003e \u003cp\u003e(0.001*)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.034 (0.009)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.060 (0.025)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.025\u0026ndash;0.043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.040\u0026ndash;0.093\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.071 (0.021)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.044 (0.012)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2.415\u003c/p\u003e \u003cp\u003e(0.016*)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.076 (0.037)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.044 (0.020)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.042\u0026ndash;0.103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.027\u0026ndash;0.062\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.044 (0.011)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.027 (0.004)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2.793\u003c/p\u003e \u003cp\u003e(0.005*)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.046 (0.021)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.027 (0.005)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.027\u0026ndash;0.055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.021\u0026ndash;0.033\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003cp\u003e(P value)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.250\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0.002*)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.613\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0.001*)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePairwise\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.001*\u003c/b\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.240, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.312, \u003cb\u003eP\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.001*\u003c/b\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Statistically significant difference at \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.05P1: comparison between 2 months and 4 months, P2: comparison between 2 months and 6 months, P3: comparison between 4 months and 6 months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eC) The percentage of bone surface area of the condyle:\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ec show a comparison between control and study groups regarding the percentage of bone surface area of the condyle. In 2,4and 6-month groups: The percentages of the bone surface area were greater in the control group as compared to the study group and it was statistically significant (p\u0026thinsp;=\u0026thinsp;0.031, 0.0001, and 0.001respectively).In control groups: there was a significant difference between intervals (p\u0026thinsp;=\u0026thinsp;0.122). In study groups; there was no significant difference between intervals (p\u0026thinsp;=\u0026thinsp;0.122).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison between the control and study groups regarding the percentage of bone surface area of the condyle\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStudy Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003cp\u003e(\u003cem\u003eP\u003c/em\u003e value)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.37 (5.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.71 (11.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.399\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0.031*)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.48 (5.85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.25 (16.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.86\u0026ndash;61.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.67\u0026ndash;57.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.97 (9.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.37 (4.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.509\u003c/p\u003e \u003cp\u003e\u003cb\u003e(\u0026lt;\u0026thinsp;0.0001*)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.18 (15.27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.58 (6.74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.16\u0026ndash;71.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.21\u0026ndash;45.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.81 (3.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.32 (10.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.489\u003c/p\u003e \u003cp\u003e\u003cb\u003e(0.001*)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.96 (4.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.80 (12.75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMin - Max\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63.72\u0026ndash;73.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.23\u0026ndash;64.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTest\u003c/p\u003e \u003cp\u003e(\u003cem\u003eP\u003c/em\u003e value)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.787\u003c/p\u003e \u003cp\u003e(0.001*)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.459\u003c/p\u003e \u003cp\u003e(0.122)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePairwise\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.549, \u003cb\u003eP\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u0026thinsp;\u003cb\u003e=\u0026thinsp;0.0001*\u003c/b\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Statistically significant difference at \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.05.P1: comparison between 2 and 4 months, P2: comparison between 2 and 6 months, P3: comparison between 4 months and 6 months.\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eVitamin A has been recognized to be essential for normal growth and bone health. However, many undesirable effects were associated with an overdose of vitamin A [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Rats were euthanized after two, four, and six months. These time intervals were designed in correlation with different age groups of humans. Two months of rats\u0026rsquo; age is correlated with 6 years of humans. Four months of rats\u0026rsquo; age is correlated with 12 years of humans (mixed dentition stage). Six months of rats\u0026rsquo; age is correlated with 18 years of adult humans [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe quantitative determination of serum vitamin A concentrations exhibited a significantly higher level of vitamin A in the study groups compared to rats in the control groups. These findings were in agreement with Lind et al[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], who recorded elevated levels of serum vitamin A in the study group compared to the control group.\u003c/p\u003e \u003cp\u003eIn control groups (2 months), the condylar cartilage exhibited normal structure and thickness of the condylar zones. The cells maintained their normal morphology and organization. The osteogenic layer showed well-developed trabeculae of cancellous bone surrounded by the normal size of bone marrow. In the experimental group (2 months), condylar cartilage exhibited a thinner chondroblastic zone and thicker hypertrophic zone as compared with the control group. A histomorphometric study of the thickness of the hypertrophic and chondroblastic layers validated these findings. This could be explained on the basis that, vitamin A induces differentiation and maturation of chondrocytes. Additionally, soeta et al[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], reported that an overdose of vitamin A first disrupted the development of chondrocytes and increased the hypertrophic chondrocytes. Moreover, Azimpour and Mortazavi et al [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], reported that hypervitaminosis A inhibited chondrogenesis. The inhibitory effect of over-dose of vitamin A is due to the blockage of the production of glycosaminoglycans by chondrocytes[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the osteogenic layer of the experimental group showed an irregular outline with wide bone marrow. The reduced thickness of bony trabeculae was confirmed by histomorphometric analysis of the total surface area of bone and could be explained that Excessive vitamin A promotes bone resorption and hinders bone formation. These effects are expected to promote bone loss, contributing to the development of osteoporosis and fractures[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Moreover, some studies reported that retinol can accelerate bone resorption through stimulation of parathyroid hormone secretion (PTH) and disturbance of vitamin D metabolism[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. It had been also reported by Lind et al., that excessive retinol reduces bone diameter through alteration of bone mineral metabolism[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, other studies reported that there was no association between fracture risk and vitamin A [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Additionally, it was also reported that retinoic acid (RA) inhibits osteoclastogenesis in pure (pre) osteoclastic cultures[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This variable effect may be a result of dose-response route and duration of administration or species difference.\u003c/p\u003e \u003cp\u003eAfter 4 months, the control showed a normal structure and thickness of and thickness of condylar zones. The osteogenic layer showed well-formed bone trabeculae with the normal size of bone marrow. The results obtained from the experimental group after 4 months revealed a thicker chondroblastic zone and thinner hypertrophic zone as compared to the control group and were confirmed by histomorphometric analysis of the thickness of chondroblastic and hypertrophic layers. The marked increase in the chondroblastic zone is related to a compensatory mechanism for the premature closure of the hypertrophic zone. These findings are in accordance with Onder et al[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], who reported a reduction in the height of the hypertrophic zone and an increase in the height of the proliferative zone in rats exposed to a high dose of vitamin A.Thickening of chondroblastic zone and thinning of hypertrophic zone related to hypervitaminosis A may be due to retardation of transition from chondroblastic zone to the hypertrophic zone. This retardation delays endochondral bone formation. Tearing of hypertrophic cells with signs of degeneration were observed. These findings are similar to Soeta et al[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]., who clarified that hypervitaminosis A induced chondroclast cells to absorb the calcified cartilage matrix and result in the erosions of the hypertrophic zone.\u003c/p\u003e \u003cp\u003eAs regards to osteogenic zone, thin bone trabeculae with wide bone marrow were observed as a sign of osteoporosis and confirmed by histomorphometric analysis of the total surface area of the bone. Similar findings had been recorded by Lind et al.,[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] who reported thinning of long bone and an increase of osteoclasts upon the administration of vitamin A in overdose. In humans, it has been reported that excessive vitamin A has been correlated with the acceleration of skeletal bone resorption [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the control group after 6 months, well-formed bone trabeculae in the osteogenic layer with normal structure and thickness of the other condylar zones were observed with further maturation of the condyle and was confirmed by histomorphometric analysis of the condyles A dramatic reduction in the thickness of the hypertrophic zone and increase of chondroblastic zone with wide bone marrow and high osteoclastic activity were established in the study group at the end of this study. Several studies have reported that an overdose of vitamin A causes bone weakening due to increased periosteal bone resorption and decreased bone formation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePersistent numerous islands of woven bone had been detected in study groups as compared to control groups and may be due to that hypervitaminosis A leads to delay of bone maturation. These results follow Lind et al, [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]who found that vitamin A inhibits osteoblast mineralization by decreasing the production of Runx2 and Osterix. (factors required for osteoblast maturation).\u003c/p\u003e \u003cp\u003eAs concerning the intercellular matrix of chondroblastic and hypertrophic cell zones, there was a reduction of metachromatic reaction of the intercellular matrix in the study group in comparison to the control group. These findings could be explained that hypervitaminosis A inhibits the formation of cartilage matrix by chondrocytes and these findings agreed with studies that reported that hypervitaminosis A reduces the production of proteoglycans by chondrocytes[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The decrease may be caused by an impairment of core protein synthesis, a change in glycosaminoglycan chain production, or decreased sulphation.\u003c/p\u003e \u003cp\u003eIn the present study, the control group showed the normal structure of cartilage and bone, while the study groups showed marked osteoporosis and growth arrest of the mandibular condyle. This effect could be explained by the effect of vitamin A on the actions of vitamin D. Vitamin A may inhibit vitamin D absorption, transport, or conversion to its active form, or it may promote vitamin D metabolic breakdown[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Because the major function of vitamin D is to improve the absorption of calcium in the gut, the optimum dose of vitamin A leads to decreased serum calcium levels [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Low serum calcium levels increase the production of parathyroid hormone (PTH), which helps to restore normal serum calcium levels by releasing calcium from the bone, promoting bone turnover and osteoporosis[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been observed that parathyroid hormone (PTH) is a strong inhibitor of chondrocyte maturation and suppresses cell hypertrophy which leads to inhibition of the endochondral bone formation and growth arrest[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eover dose of vitamin A inhibits bone formation and diminishes endochondral bone growth by premature closure of the condylar cartilage which leads to teratogenicity of mandibular condyle so taking vitamin A must be under medical control.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe\u0026nbsp;authors thank Master Walid, the laboratory technician in the Department of Oral Biology, Faculty of Dentistry, Alexandria University for his efforts in preparing and dyeing the slides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTION:\u003c/strong\u003eS.H. planned the experiments. S.A. and M.M. carried out the experiments. S.H. and H.S. analyzed and confirmed all data. S.H. prepared the manuscript. S.H. and H.S. confirmed the manuscript. All authors reviewed the manuscript, and S.H.made a final approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING:\u003c/strong\u003e NO funding to conduct this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: The datasets used or analyzed during the current study are available from corresponding authors on reasonable request and her email is [email protected] .com\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e: The study was carried out after receiving approval from the Research of Animal Ethics Committee., Faculty of Dentistry, Alexandria University. (Approval number: 0159 and IORG is 0008839).\u003c/p\u003e\n\u003cp\u003eAprotocol (including the research question, key design features, and analysis plan) was prepared before the study, and this protocol was registered at Faculty of Dentistry, Alexandria University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eD\u0026rsquo;Ambrosio DN, Clugston RD, Blaner WS. Vitamin A metabolism: an update. Nutrients. 2011;3:63\u0026ndash;103. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.3390/nu3010063\u003c/span\u003e\u003cspan address=\"10.3390/nu3010063\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrison EH. 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Endocrinology 2003;\u003cem\u003e144\u003c/em\u003e, 2514\u0026ndash;2523. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/en.2002-220969\u003c/span\u003e\u003cspan address=\"10.1210/en.2002-220969\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"vitamin A, teratogenic, condylar bone, growth","lastPublishedDoi":"10.21203/rs.3.rs-6487311/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6487311/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eVitamin A is present in food such as meat, dairy products, and vegetables. A control balanced diet maintains the nutritional needs of vitamin A. Over consumption of products as well as supplementations with vitamin A may increase the risk of hypervitaminosis A. Hypervitaminosis A has been associated with thinning of the long bones and reduction of bone formation.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eInvestigate the impact of both optimum and excessive vitamin A dosages on the development of the mandibular condyle in Wister albino rats.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eForty \u0026ndash;eight healthy offspring\u0026rsquo;s male Wister albino rats were used in this study. The rats were divided into two groups: Group I (control group) 24 rats were fed a regular diet containing an optimum dose of vitamin A. Group II (study group) 24 rats were fed a regular diet supplemented with an overdose of vitamin A. In both groups, 8 rats were euthanized after 2, 4, and 6 months respectively. Blood samples were collected from all rats following the last vitamin A dosage to measure its concentration in serum. The condyles were dissected removed and processed for histomorphometric and histological examination.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn contrast to the control group, the study group's condylar zone structure was shown to be disrupted during the histological analysis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eHypervitaminosis A could increase condylar bone resorption and diminish endochondral bone growth.\u003c/p\u003e\u003ch2\u003eClinical Relevance:\u003c/h2\u003e \u003cp\u003eVitamin A supplementation must be under medical control to avoid growth anomalies and subsequent malocclusion.\u003c/p\u003e","manuscriptTitle":"The Effect of Vitamin a as a Teratogenic Agent in Condylar Bone Growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 12:30:59","doi":"10.21203/rs.3.rs-6487311/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd2965c9-b51e-4b13-9de9-42907d4beb8f","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T09:39:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-09 12:30:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6487311","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6487311","identity":"rs-6487311","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}