Type of the Paper (Article Journal of Baghdad College of Dentistry, Vol. 35, No. 2 (2023), ISSN (P): 1817-1869, ISSN (E): 2311-5270 54 Research Article Effects of vitamin D deficiency on bone and root re- sorption post-orthodontic retention in rats Asmaa Murshed Khamees 1*, Dheaa H. Al Groosh 2, Natheer H Al-Rawi 3 1. Ph.D. student, Department of Orthodontic, College of Dentistry, University of Baghdad, Iraq. 2. Professor, Department of Orthodontics, College of Dentistry, University of Baghdad, Iraq. 3. Professor, Department of Oral and Craniofacial Health Sciences, University of Sharjah, UAE. * Correspondence email: d.al-groosh@codental.uobaghdad.edu.iq Abstract: Background: Orthodontic therapy often causes external root resorption. Serum vitamin D (VD) level is important for tooth mineralization and bone remodeling. This study aimed to test the impact of vitamin D (VD) supplements on bone and root remodeling in a vitamin D (VD) deficient rat model following orthodontic retention. Methods and Material: 30 male Wistar rats were divided into three groups: a control group of 10 rats and two exper- imental groups of 10 rats, each with vitamin D deficiency (VDD) induced by a VD-free diet for 21 days. And a third group with VD supplement. All groups received active orthodontic treatment using a modified orthodontic appliance that applied 50 gm of force for 14 days to move the maxillary right first molar mesially, followed by seven days of retention and relapse. The VDD group received no intervention, while the VDS group received 40,000 IU/kg of sys- temic VD3 by intramuscular injection on the first and fifteenth day of orthodontic treatment. Histomorphometric analysis was performed to assess bone and cementum resorption and deposition. Results: The VDD group exhibited a significant increase in bone and root resorp- tion and decreased bone deposition and cementum deposition ratio. In addition, bone depo- sition and the ratio of cementum deposition were substantially greater in the VDS group com- pared to the control group. Conclusion: VDD may increase bone and root cementum resorp- tion and decrease deposition after orthodontic retention, which may play a significant role in relapse after retention. Before beginning orthodontic treatment, routine VD screening may be beneficial. Keywords: Vitamin D, orthodontic, bone, cementum. Introduction Root resorption is a process of root tissue removal that may occur in different areas alongside the root, that is laterally and/or around the apex, and it may be found with severe tissue loss (1). External root resorption is common during orthodontic treatment; however, it has no significant clinical consequences (2, 3) nor other health-related issues such as root fracture, periodontal illness, or early tooth loss (4). Root resorption could be either a physiological or pathological, and the latter may be referred to as orthodontic treatment (5). Orthodontic force enhances an inflammatory process that activates osteoclast cells, which cause the removal of bone and root tissues. This process is followed by a reparative process (1). In addition, root resorption could be associated with other anomalies such as hormonal deficiency and alveolar bone density (6, 7). Vitamin D deficiency (VDD), a common hormonal deficiency problem, (8) increases serum parathyroid hormone, which results in progressive bone loss and mineralisation defects. Epidemiologic studies showed that VDD was associated with low bone mineral density and fracture incidence (9, 10). Vitamin D (VD) serum level is an essential parameter in root and bone remodeling (11). A correlation was observed Received date: 25-12-2022 Accepted date: 02-02-2023 Published date: 15-06-2023 Copyright: © 2023 by the au- thors. The article is published under the terms and condi- tions of the Creative Com- mons Attribution (CC BY) li- cense (https://creativecomons.org/li- censes/by/4.0/). https://doi.org/10.26477 /jbcd.v35i2.3403 mailto:d.al-groosh@codental.uobaghdad.edu.iq https://orcid.org/0000-0001-7120-8720 https://orcid.org/0000-0003-0052-7286 https://orcid.org/0000-0002-7483-6594 https://creativecomons.org/licenses/by/4.0/ https://creativecomons.org/licenses/by/4.0/ https://doi.org/10.26477/jbcd.v35i2.3403 https://doi.org/10.26477/jbcd.v35i2.3403 J. Bagh. Coll. Dent. Vol. 35, No. 2. 2023 Khamees and Al Groosh 55 between root resorption and VD receptor gene polymorphism (12). However, the relationship of VDD and root resorption was inconclusive, probably because of published data with short-term follow-up (13). In the resorption lacunae, fibroblast-like cells from the periodontal ligament invading these lacunae repair the resorbed cementum (14). The first histologic evidence of repair is the presence of a thin, uncalcified cementoid (14). These cells secrete noncollagenous matrix proteins, particularly bone sialoprotein and os- teopontin, filling the cementoblasts to secrete collagen and form a thin cementoid repair matrix (14, 15). Sub- sequently, hydroxyapatite crystals grow between collagen fibrils for mineralisation (16). The reparative ce- mentum is a cellular intrinsic fibre type (14). In contrast, the recently deposited cementum appears as layers (thin layer of acellular cementum) in rats, which covered some of the resorption lacunae (17). This study aimed to assess the amount of bone and root remodeling after orthodontic retention in a VDD- enhanced rat model and to evaluate the effect of VD supplements on VD-deficient rats. Materials and Methods 1. Animal preparation The study was approved by the scientific research and ethics committee at the Department of Ortho- dontics, College of Dentistry/University of Baghdad (Issue no: 177, date 16/1/2020). All experimental pro- cedures were performed in Iraqi Center of Cancer and Medical Genetics Research. Male Wistar rats (n = 30) aged 8–10 weeks and weighing 220–300 g were used in this study. Firstly, the rats were acclimatised for one week before VD induction and subjected to 12/12 h of dark/light cycles at 21 °C ± 2 °C with a relative humidity of 50% ± 10%. Rats were fed a laboratory standard diet and given water ad libitum (18). All the rats followed the same principle. They were weighed. The tail of the rats was coloured (every one special colour). Blood serum was collected by aspiration using a 5 mL sterile syringe (disposable syringe with a needle, 1 mL luer slip, JiangSu JiChun Medical devices, China) after anesthetising with chloroform (Alpha Chemika, India). Serum VD was investigated and assessed using the VD antibody protein ELISA kit following the manufacturer’s instructions (MyBioSource, USA). Afterward, the rats were divided into two groups. The control group without intervention, and the exper- iment group in which the rats were fed a VD-free diet (Casein-free VD, Bio-Serv, USA) for 21 days (19, 20). After 21 days, serum VD was re-investigated, and rats suffering from VDD were divided into three groups: 1. The control group. 2. The VDD group continues deficiency during the treatment period. 3. The VDD group received vitamin D supplementation (VDS) by intramuscular injection on days 1 and 15. However, these doses elevated the VD serum level the same as the control group during orthodontic treatment. 2. Orthodontic treatment stages The rats were anesthetised after being weighed using intramuscular injection of ketamine (87 mg/kg; ket- amine 10%, Alfasan, Woerden, Holland) and xylazine muscle relaxant (10 mg/kg; XYL-M2 injectable so- lution 25 mL, VMD, Arendonk, Belgium) at a 2:1 ratio(21), that is in thigh muscle based on the animal hous- ing guidelines in CCMGR. This process was applied in all orthodontic stages. Interproximal spaces were measured between the distal surface of the first molar and the mesial surface of the second molar by using an interproximal vernier (feeler gauge, ROSHTOO80, Japan), and this process was repeated two times by one operator (22). An orthodontic ligature wire (0.010″, Truforce stainless steel, Ortho Technology, USA) was inserted inter- dentally between the 1st and 2nd right maxillary molars (23) (unilateral orthodontic appliances were bonded, J. Bagh. Coll. Dent. Vol. 35, No. 2. 2023 Khamees and Al Groosh 56 and the left molar serves as the control side) (24). Then, the first maxillary molar was ligated, and a hook, nickel–titanium closed-coil spring was formed (Dentaurum, Rematitan® LITE Tension Spring, Dentau- rum). This hook was inserted, and the other end was attached to the hook formed on maxillary central incisors using a ligature wire (0.012 Kobayashi, Klardent, Sweden) after being etched with acid etch gel (37% Microdont, Brazil). The force delivered with 50 ± 5 gm was adjusted using a digital hand-held force gauge (Sr-1 kg Gray Digital Hanging Scale, American Weigh Scales, GA, USA) to move the 1st molar me- sially. Bonding adhesive was applied (3M ESPE, Adper, single bond 2, USA), and composite filling mate- rials (3M ESPE, Filtek, Z350 XT, Universal Restoration, USA) were adapted and cured according to the manufacturer’s instructions (Fig. 1A) (21). Vitamin D3 (cholecalciferol 300000 I.U.\1 mL, Pisa, Italy) of 40,000 IU/kg was administrated slowly in left thigh muscle on day one and day 15 for the VDD group (25). On day 15, the orthodontic spring was re- moved; spaces were measured, and a retainer was inserted for seven days. Afterward, relapse was allowed for another seven days (Fig. 1 B) (26). Then, an overdose of anaesthesia was given, and the final space was measured. Figure 1: A; Orthodontic appliance bonding, B; Relapse 3. Histological preparation All rats were sacrificed under an overdose of anaesthesia, and the maxilla was placed in 10% natural buff- ered formalin solution (formaldehyde, Scharlab S.L., Spain) for 24 h. The sample was rinsed for 21 days in 10% EDTA solution (pH = 7; BDH Chemicals Ltd. Poole, England), and the solution was continuously changed (21, 27). After decalcification, half of the maxilla (28) underwent dehydration (29) and then embedded in paraffin wax (Leica Biosystems, Richmond, USA) to obtain longitudinal sections of teeth with the adja- cent tissue. Two sections of 5 µm thickness, including the mesial root of the maxillary first molar, were stained using haematoxylin and eosin stain (H&E, Leica Biosystems, Richmond, USA). The pressure and tension sides were photographed under a light microscope (OPTIKA, Microscopes, Italy) with 4×,10× and 40× objective lenses (28) equipped with a photomicroscope (Olympus, Tokyo, Japan). For inter-examiner calibration, the section was randomly selected two times and examined by the same histopathologist under a blind test (30). The bone area was sectioned to rectangles alongside the mesial root of the maxillary right molar, and bone resorption and formation in pressure and tension sides were calculated using Image J processing program (Image J.exe, LOCI, University of Wisconsin) that was developed at the National Institutes of Health (31, 32). The resorbed and newly deposited radicular cementum was detected and measured under a light micro- scope similarly as bone. The resorption area of cementum appears as lacunae, and the severity of root resorption was evaluated on the basis of the semiquantitative scale, in which the number, depth and area of each resorption lacuna were calculated (Fig. 2 A) (17). J. Bagh. Coll. Dent. Vol. 35, No. 2. 2023 Khamees and Al Groosh 57 The measurement of the level of lacuna repair in cementum was dependent on the grading system, and the thickness of the repair cementum was measured in micrometre using a line as the perpendicular dis- tance from the deepest area in the lacuna until the outer surface of the deposited layer. The image above analysis was used for this purpose (Fig. 2 B) (33). Figure 2: A, Resorbed cementum (RC) represented in marked rectangular, white arrows show the thick- ness of resorption. B, Recent deposit cementum (RC) represented in marked rectangular, white arrow shows the thickness of deposit cementum. Statistical analysis The collected data were statistically evaluated by using statistical package for social sciences (IBM® SPSS® version 25). The Shapiro–Wilk test was used to assess the normality of data. ANOVA and Games– Howell tests were used to compare the bone and cementum (resorption and deposition). Results In the VDD group, data revealed a significant decrease in bone formation and an increase in bone resorption. However, bone resorption and formation were comparable between the VDS group and the control group (Table 1). Table (1): Comparison between the bone resorption and deposition among the control, VDD and VDS groups using ANOVA and Games-Howell tests. Side Group Descriptive statistics Comparison No Mean (µm2) SD (µm2) F-test p-value Groups p-value Bone Resorption Control 10 173.5 7.84 112.2 0.000[S]* Control VDD 0.000 [S]* VDD 10 261 24.24 VDS 0.091[NS] VDS 10 164 10.75 VDD VDS 0.000 [S]* Bone deposition Control 10 199.5 6.85 171.4 0.000[S]* Control VDD 0.000 [S]* VDD 10 145.5 6.85 VDS 0.773[NS] VDS 10 202 9.19 VDD VDS 0.000 [S]* SD: standard deviation; NS: Non-Significant; S: Significant at P<0.05. A B J. Bagh. Coll. Dent. Vol. 35, No. 2. 2023 Khamees and Al Groosh 58 The pressure side data revealed that the alveolar bone surface was irregular, with incomplete mineralisa- tion and few resorption cavities (Figs. 3A and B). Both the control and VDS groups experienced the same outcome. In the VDD group, however, the resorption cavity expanded (Fig. 3 C). The tension side of the mesial root in control and VDS groups exhibited a tension periodontal ligament with a distinct reversal line between the basal and new bone (Figs. 4 D, E). In contrast, the tension side of the mesial root in the VDD group exhibited narrow new bone apposition that was separated from the basal bone by a reversal line (Fig. 4 F). Figure 3: Pressure side area of the mesial root of the upper maxillary first molar. PDL: periodontal ligament, yellow arrow: resorption cavities, (A): Control; (B): VDD; (C): VDS group (H &E; 10x). Figure 4: Tension side area of the mesial root of the upper maxillary first molar. Tension periodon- tal ligament(TPDL); new bone (NB); reversal line (arrows); basal bone (BB). Control (D); VDD (E), VDS (F) groups. (H &E; 10x). Similar to bone reaction, root resorption and cementum deposition were significantly increased in the VDD group compared to the control and VDS groups. Table 2 shows no statistically significant difference between the control and VDS groups. Consequently, the ratio of cementum deposition to cementum re- sorption was decreased in the VDD group primarily (Table 3). Table 2: Comparison between cementum resorption and deposition among the control, VDD and VDS groups using ANOVA and Games-Howell tests. Side Group Descriptive statistics Comparison No Mean(µm2) SD(µm2) F-test p-value Groups p-value Cementum Resorption Control 10 15 5.27 204.7 0.000[S]* Control VDD 0.000 [S]* VDD 10 63 9.49 VDS 0.052 [NS] VDS 10 10.1 2.73 VDD VDS 0.000 [S]* Cementum deposition Control 10 6.1 1.10 26.01 0.000[S]* Control VDD 0.001 [S]* VDD 10 20 8.16 VDS 0.765 [NS] VDS 10 6.6 1.96 VDD VDS 0.001 [S]* SD: standard deviation; NS: Non-Significant; S, Significant at p< 0.05. A B C E D F A B C E D F J. Bagh. Coll. Dent. Vol. 35, No. 2. 2023 Khamees and Al Groosh 59 Table 3: Percentage of cementum deposition to cementum resorption with the same group Groups Sample No Mean/ CR Mean/CD CD/CR % Control 10 15 6.1 46 % VDD 10 63 20 31.6% VDS 10 10.1 6.6 68.4% CR: Cementum resorption; CD: Cementum deposition Discussion Vitamin D deficiency is considered a common health-related problem that is faced by people of all ages worldwide (34, 35). It may be associated with low bone mineral density and bone activity (9). In ortho- dontics, retention is an important phase of the treatment (36). However, the prevalence of relapse was re- ported, and only 30%–50% of treated patients maintained the initial acquired satisfactory alignment after 10 years. In addition, only 10% of treated patients maintained a satisfactory alignment after 20 years (37). Although the mechanism of orthodontic relapse remains unknown (38), bone turnover may influence the relapse (39). Previous research has used the local administration of different medications or biologicals to decrease the relapse following active orthodontic treatment (21, 40–42). However, Arqub et al. (2021) reported that local administration may be associated with a short half-life of the agents and may require multiple administration. Therefore, this study adopted the systemic administration of VD (43). The data revealed a decrease in bone resorption and an increase in bone deposition (bone synthesis) in the control and VDS groups. The metabolites of 1α,25-hydroxylated stimulate the expression of osteoblast signature genes, including receptor activators of nuclear factor kappa-B ligand (RANKL) and vitamin D receptors (VDR) (44). The activation of VDR increases the expression level of receptor activator of nuclear factor kappa (RANK) on the osteoclast-progenitor cells surface, which stimulates osteoclastogenesis and bone resorption by binding RANK to RANKL. The expression of VDR on the osteoblast cell represents an activation pathway of bone resorption (45). In addition, VD plays a role in skeleton mineralisation, which has an anabolic activity ingredient that plays an important role in bone synthesis stimulation (46). This finding is consistent with the result of Kale et al. (2004), who found that the local administration of 1,25- dihydroxy cholecalciferol increased and facilitated tooth movement by regulating bone deposition and resorption (40). Moreover, Kawakami and Takano-Yamamoto (2004) reported that the stability of teeth po- sition was improved after orthodontic movement and local administration of VD (47). Furthermore, many authors suggested that VD improves bone health and mineral metabolism (48, 49, 50). The data displayed a significant decrease in root resorption with VDS and an increase in the VDD group. During orthodontic treatment, the generation of proinflammatory mediators under orthodontic forces, such as prostaglandins and leukotrienes, causes tooth movement (51) and cementum loss, which may lead to root resorption (52). This finding is consistent with the results of Seifi et al. (2013), who found that VDD during orthodontic treatment may play a key role in the pathophysiological process of root resorption (53). In addition, Booij Vrieling et al. (2010) reported that active VD signals induce pathophysiological root resorption in cats (54). However, the abovementioned results were contrary to that of Tehranchi et al. (2017), who demonstrated no significant correlation between the VD serum level and external apical root resorption development J. Bagh. Coll. Dent. Vol. 35, No. 2. 2023 Khamees and Al Groosh 60 (55). Moreover, this result was inconsistent with that of Al-Attar and Abid (2022), who demonstrated a non- significant correlation between root resorption and VD serum level after the first three months of ortho- dontic treatment (13). A different method of root resorption assessment could be used, as the latter used a periapical radiograph with a digital sensor as opposed to the gold standard for histological investigation in the current study. Considering that root resorption is common, resorption lacunae were fully or par- tially repaired with new cementum (14). This acellular tissue in rats contrasts with the cellular intrinsic fibre cementum reported in humans (16, 17). The results show a significant increase in cementum deposition in VDD in comparison with other groups. In contrast, the ratio of cementum deposition to resorption cavities decreases in VDD in comparison with other groups. This result is consistent with the process of metabolic activity of VD by the indirect role of VD in the stimulation of osteoclastogenesis through regulating some secondary messengers for expression and interactions amongst inflammatory cells, cytokines, enzymes and clast cells (56), which is consistent with the suggestion that VD deficiency and dietary VD redundancy have an indirect role in the pathophysiology of the regeneration of common permanent tooth in cats (54). Conclusion After orthodontic retention, vitamin D deficiency may be elevated in bone and cementum resorption and decreased in bone deposition. Nonetheless, correction of VDD in rats is associated with bone and cementum remodeling, which may significantly reduce relapse after retention. 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(Crossref) تقويم األسنان في الفئران تثبيت العظام والجذور بعد تأكل نقص فيتامين )د( على ر العنوان: تاثي 2, ضياء حسين الكروش 1الباحثون: اسماء مرشد خميس :الخالصة تاكل الجذرالخارجي أثناء عالج تقويم األسنان ، فقد اقترح أن مستوى فيتامين )د( في المصل ضروري المتصاص وبناء الخلفية: ليس من غير المألوف ويم االسنان في األنسجة الصلبة مثل العظام واألسنان، وقد هدفت هذه الدراسة إلى تقييم نسبة تكوين العظام والجذور فيما بعد فترة التثبيت من عالج تق العظام والجذور. ان المصابة بنقص فيتامين )د( وتقييم تأثير مكمالت فيتامين )د( على إعادة تكويننموذج الفئر فئران ومجموعتين تجريبية )كل 10إلى ثالث مجموعات. مجموعة التحكم Wasterالمواد وطرق العمل: تم تقسيم الجرذان البالغة ثالثون ذكر نوع يوًما. خضعت جميع المجموعات 21فئران( المصابة بنقص فيتامين )د( الناجم عن اطعام الفئران غذاء مخصص خالي من فيتامين )د( لمدة 10واحدة 14جم من القوة لتحريك األضراس األولى اليمنى للفك العلوي األيمن لمدة 50ه لتطبيق لعالج لتقويم األسنان باستخدام جهاز تقويم أسنان معدل تم تركيب وحدة دولية / كجم 40000أيام. مجموعة نقص فيتامين )د( دون تداخل ، بينما تلقت المجموعة المكملة بفيتامين )د( 7أيام ، واالنتكاس 7يوًما ، والتثبيت لي في اليوم األول والخامس عشر من فترة العالج التقويمي. تم إجراء فحص االنسجة لتقييم تاكل ووبناء العظام من فيتامين )د( عن طريق الحقن العض والمالط. كانت نسبة تكوين النتائج: كان هناك زيادة معنوية في تاكل العظام والجذور وانخفاض بناء العظام ونسبة بناء المالط في مجموعة نقص فيتامين )د(. بينما ظام والمالط أعلى بكثير في المجموعة المكملة بفيتامين )د( وكذلك في مجموعة التحكم. الع ا مهًما في : قد يؤدي نقص فيتامين )د( إلى ارتفاع تاكل العظم والجذر، وكذلك تقليل بناء العظم والجذربعد فترة تثبيت التقويم التي قد تلعب دورً االستنتاج ت. قد يكون الفحص الروتيني لفيتامين )د( مفيدًا قبل البدء في عالج تقويم األسنان.زيادة االنتكاس بعد فترة التثبي https://doi.org/10.1111/j.1600-0722.2009.00707.x https://doi.org/10.1016/s0003-9969(03)00201-2