The Histopathological analysis of Proton Pump Inhibitor & H2 receptor antagonist on bone cells in orthodontically induced tooth movement: A Randomised Control animal study

The Histopathological analysis of Proton Pump Inhibitor & H2 receptor antagonist on bone cells in orthodontically induced tooth movement: A Randomised Control animal study | 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 Histopathological analysis of Proton Pump Inhibitor & H2 receptor antagonist on bone cells in orthodontically induced tooth movement: A Randomised Control animal study Manami Das, ashish agrawal, Bipin Rai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7186441/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 Objectives: The primary objective was to assess the effects of ranitidine and esomeprazole on OTM in an animal model via: a) Histopathology: H&E staining for osteoclasts and SATB2 immunostaining for osteoblasts. The secondary objective evaluated its impact on bone remodeling, correlating its effect with serum biochemistry: calcium, magnesium, phosphorus, and creatinine. Materials and Methods : Sixty-nine male Rattus norvegicus (200–250 g, 10 weeks old) were randomly assigned to three groups: control, ranitidine (Group 2), and esomeprazole (Group 3). A 50 g NiTi closed-coil spring was placed between the left central incisor and first molar. Groups 2 and 3 received (30 mg/kg/day) of the respective drug via oral gavage; controls received saline. Blood was collected on days 1 and 42 for serum analysis. On day 42, tooth movement was recorded, the animals were euthanized, and histological analysis was performed to evaluate osteoclasts and osteoblasts. Paired t-tests and one-way ANOVA were used for statistical analysis (P ≤ 0.05). Results : Esomeprazole (Group 3) produced the highest osteoclast count (84 ± 7.1) and greatest tooth movement (0.585 ± 0.021 mm). Control had the highest number of osteoblasts (SATB2), while Groups 2 and 3 showed inhibition. Serum phosphorus significantly decreased in Groups 2 (3.642 ± 1.449) and 3 (5.556 ± 1.595 mg/dL); calcium decreased in Group 3 (9.368 ± 0.328 mg/dL). Conclusion : Esomeprazole (PPI) induced a decrease in serum Ca levels, the highest osteoclastic activity, and OTM, followed by ranitidine (H₂RA), with the control group showing the least. Both PPI and H 2 RA inhibit or reduce osteoblastic activity. Serum biochemistry Esomeprazole Ranitidine Osteoclast osteoblast Orthodontic tooth movement Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Orthodontic tooth movement (OTM) arises from a coordinated cascade of mechanical, cellular, and molecular events. When orthodontic force is applied, mechanotransduction within the periodontal ligament triggers an inflammatory response, releasing mediators such as prostaglandins, cytokines, and growth factors that regulate bone remodeling—resorption on the pressure side and deposition on the tension side, thereby facilitating tooth displacement [ 23 ]. Pharmacologically, bone-resorptive inhibitors—like NSAIDs, bisphosphonates, calcium supplements, and statins—dampen osteoclast activity and slow OTM [ 37 ]. In contrast, agents such as vitamin D, prostaglandin analogs, parathyroid hormone, corticosteroids, and relaxin can enhance osteoclastogenesis or alter extracellular matrix turnover, thus accelerating tooth movement [ 33 ]. Isabelle Graf et al. [ 10 ] investigated orthodontics related to social media by analyzing posts made by patients or peers and found that more positive posts were on Instagram. With increased access to social media, more adult patients are becoming aware of orthodontic treatment options and seeking adjunctive or comprehensive treatment according to their needs [ 8 ]. Christensen et al [ 7 ] documented an increase in the number of adults seeking orthodontic treatment over the years. Adult orthodontics is witnessing increasing demand due to heightened awareness of dental aesthetics and improved oral health consciousness among older individuals [ 31 ]. Adults now actively seek orthodontic treatment not only for aesthetic reasons but also for correcting functional issues such as malocclusion and bite discrepancies [ 12 ]. The growing acceptance and visibility of fixed orthodontic appliances, including tooth-colored brackets and clear aligners, have made treatment more appealing to adult patients [ 17 ]. Additionally, many adults are on long-term medications, such as proton pump inhibitors (PPIs) and H₂ receptor antagonists used for gastric conditions, who may experience altered bone metabolism, potentially affecting the rate and quality of orthodontic tooth movement [ 6 , 3 ]. Understanding the influence of such medications is critical in planning and delivering effective orthodontic care for adult patients. Given the widespread use of over-the-counter medications, accurately documenting patients’ current drug intake (including non-prescription agents) is crucial. This enables orthodontists to anticipate changes in treatment duration, adjust the applied force, and collaborate effectively with other healthcare providers [ 34 ]. Proton pump inhibitors (PPIs) are among the 43 drugs listed in India's National List of Essential Medicines (2011) and are commonly prescribed. Rotman et al. reported PPI use prevalence of 4% in the US (2002) to 9.2% in 2009, with 62.9% of users lacking documented gastrointestinal indications [ 32 ]. Similarly, Jakupi et al. observed a marked increase in PPI usage in Kosovo from 2011 to 2013 [ 15 ]. Animal studies support these dynamics. For example, rats treated with high-dose pantoprazole (200 mg/kg for 6 weeks) showed significantly increased OTM and decreased bone mineral density, indicative of induced osteoporosis [ 36 ]. The cumulative effects of pharmacological agents on tooth movement could be inhibitory, additive, or synergistic [ 2 ]. The doses and protocols vary, making it clinically unreliable. There is limited knowledge about the use of PPIs and H2R antagonists in both growing and non-growing individuals, as well as their effects on orthodontic treatment. Rat models (adult) are frequently used in various research studies, as a specific mathematical formula exists for converting dosages from humans to rats, which enhances the quantitative and qualitative reliability of the study. Therefore, this study is designed to evaluate the effects of commonly used pharmacological agents (such as PPIs and H2R antagonists) on tissue remodelling (histopathological evaluation), the rate of tooth movement in rats, and their correlation with serum biochemical analysis. AIMS & OBJECTIVES The primary objective was to assess the effects of ranitidine and esomeprazole on bone remodelling during OTM in an animal model (Rattus norvegicus Albinus), histologically using H&E staining for osteoclasts and SATB2 immunohistochemical staining for osteoblasts. The secondary objective was to correlate its effect with serum biochemistry (calcium, magnesium, phosphorus, and creatinine) and the rate of tooth movement. MATERIALS & METHOD A Randomized Controlled Trial was approved by the Ethics committee (No. Dean/2021/EC/2721) and was carried out in the Unit of Orthodontics, Faculty of Dental Sciences, for a period of 1 year from 2021 to 2022. All procedures performed in the present research were as per the Animal Research Reporting of in Vivo Experiments (ARRIVE) guidelines [ 18 ] & the National Research Council's Guide for the Care and Use of Laboratory Animals [ 27 ]. A sample size calculation was performed using G*Power 3.1.9.7 targeting a one-way ANOVA with three groups, an alpha of 0.05, and a power of 80%. Assuming a medium-to-large effect size (f = 0.4) based on expected differences in tooth movement and osteoclastic activity, the estimated total sample size was 66 rats (22 per group). A pilot study was conducted on 15 rats before the main experiment to assess feasibility and standardize experimental protocols. All procedures were conducted in accordance with the predefined research design. The results of the pilot study were not included in the main study. However, the experiment was found to be feasible. Thereafter, 69 healthy male rats (Rattus norvegicus Albinus), each weighing 200–250 g and 10 weeks old, were randomly selected from the Animal House of the Institute for the experimental setup. Animals used in previous studies or showing signs of oedema, allergy, alopecia, or cachexia were excluded. All animals were housed individually in plastic cages under a 12:12-hour light–dark cycle, with controlled temperature (23 ± 2°C) and humidity (55 ± 10%). Simple random allocation of the sample was performed manually using Microsoft Excel by generating random numbers with the {=RAND()} function, followed by sorting and sequential group assignment (Control Group I, Experimental Groups II and III, n = 23). Group II received Ranitidine (30 mg/kg), and Group III received Esomeprazole (30 mg/kg) via oral gavage once daily for six weeks, ensuring no spillage during administration. To reduce the risk of NiTi spring displacement during mastication, they were fed a soft diet ad libitum (finely ground standard pellets). The control group (Group 1) received an equivalent volume of saline solution once daily for six weeks, administered at the same time each day as the experimental groups. The dosages of Ranitidine and Esomeprazole were calculated based on a previous study by Nasser S. Alshahrani et al. [ 28 ] in rats to reflect the therapeutic dose administered to humans. RAT MODEL PREPARATION The animals were anaesthetized with intraperitoneal administration of Xylazine Hydrochloride (0.2 mg/kg) and Ketamine Hydrochloride (1 ml/kg) in a ratio of 1:3. Then, a NiTi closed-coil spring (force 50 g) was stretched between the left maxillary first molar and the incisors and ligated with ligature wire (Figure No. 1 a & b). A constant force of 50 mg (Figure No. 1c) was maintained for 6 weeks. Subcutaneous injections of Carprofen (5 mg/kg) were given for analgesia on the day of coil placement and daily for 3 days [ 20 ]. Body weights were checked before and after the experimental period and recorded. Blood samples were collected from the retro-orbital sinus on day 1 and after six weeks (Figure No. 1 d) for biochemical analysis of calcium, magnesium, phosphorus, and creatinine. The samples were coded and labelled for blinding. Topical anaesthesia (Tetracaine Hydrochloride (1%)) was applied, and 1.4 to 1.8 ml of blood was collected (using a sterile haematocrit capillary tube) from each rat, according to the guidelines of the National Institutes of Health (NIH), Institutional Animal Care and Use Committee (IACUC) [ 40 ]. Serum was separated by centrifugation (at 1500 x g) and stored at -70°C until required for bone metabolic marker assays. Total serum calcium, inorganic phosphorus, magnesium, and creatinine levels were measured by a certified laboratory with Architect plus ci4100 (Abbott) using commercial tests (Calcium Architect/Aeroset REF 3L79-21 and 3L79-31 304328/R1, Abbott; Phosphorus Architect REF 7D71 305532/R02, Abbott; Magnesium Architect REF 3P68 305531/R02, Abbott; and Creatinine (enzymatic) REF 8L24-31 and 8L24-41 G10667/R08 B8L240 for use with Architect, Abbot, respectively), performed according to their manufacturers’ instructions. To ensure double blinding, samples were recoded and relabeled, and group allocation was concealed from the individual conducting the histological analysis. The desired tooth movement was measured between the distal surface of the 1st molar and the mesial surface of the 2nd molar using Vernier Callipers, and data were recorded for the control and experimental groups. HISTOLOGIC ANALYSIS To assess histologic changes after 6 weeks, all rats were euthanized with an injection of chemical anesthetics (Pentobarbital − 100 mg/kg, intraperitoneal) [ 20 ], and the left hemimaxillae of all animals were surgically removed after sacrifice. The tissues were then placed in 10% formalin for 10 days. Decalcification was performed using 5% formic acid for at least 7 days, and the samples were embedded in paraffin blocks. STATISTICAL ANALYSIS The data obtained was analysed using SPSS 26 software for statistical analysis to test the significance of the study. Paired T test & one-way ANOVA (analysis of variance) were used to compare data between pre- & post-orthodontic force application. Results are presented as mean ± standard deviation (SD). For all tests, differences with values of P ≤ 0.05 were considered significant, and P ≤ 0.001 was considered highly significant. RESULTS In this study, 69 rats were randomly assigned to three groups: a control group (Group 1) and two experimental groups (Groups 2 and 3). There was no loss of sample during the entire study duration. Administration of PPI and H2R antagonists began on the day orthodontic force (50 mg force applied by a NiTi coil spring) was applied. The drugs were delivered via oral gavage, which did not cause any toxicity such as edema, allergy, alopecia, cachexia, etc., throughout the study. The mean weights were 208.3 g for Group 1, 205.05 g for Group 2, and 210.15 g for Group 3. Additionally, there was no weight gain or loss before or after the study (p ≤ 0.05). Serum Biochemical analysis (Table No. 1&2) The blood from the Control group (Group 1- Orthodontic force and normal saline) was analyzed for serum content. The study found a significant decrease (p ≤ 0.05) in serum magnesium levels (Pre = 2.50 ± .439(SD)mg/dl & Post = 2.15 ± .287(SD)mg/dl) only. No statistical correlation was observed for calcium, phosphorus, and creatinine in this group. However, in Group 2 (Ranitidine with orthodontic force), the decrease in serum magnesium levels (Pre = 2.29 ± .286(SD)mg/dl, Post = 1.96 ± .26978(SD)mg/dl) was statistically significant (p ≤ 0.05), and the decrease in phosphorus levels (Pre = 5.35 ± 1.623(SD)mg/dl, Post = 3.64 ± 1.44930(SD)mg/dl) was highly significant (P ≤ 0.001). Similarly, in Group 3 (Esomeprazole with orthodontic force), there was a statistically significant (P ≤ 0.05) decrease in serum calcium (Pre = 10.02 ± .697(SD)mg/dl, Post = 9.37 ± .32832(SD)mg/dl) and phosphorus levels (Pre = 6.30 ± 1.53189(SD)mg/dl, Post = 5.56 ± 1.59467(SD)mg/dl). Table No. 1 - Comparison of rat serum biochemistry between Group 1,2 & 3(Paired T test) GROUPS BLOOD PARAMETERS ON DAY 1 ON DAY 42 Paired T test (P value) MEAN ± SD MEAN ± SD GROUP 1 (Control) n = 23 Calcium 9.69 ± .286 9.66 ± .328 .853 Magnesium 2.50 ± .439 2.15 ± .287 .044* Phosphorus 5.41 ± 1.440 6.02 ± 1.440 .348 Creatinine 0.42 ± 0.014 0.42 ± 0.017 .226 GROUP 2 (Ranitidine) n = 23 Calcium 9.84 ± .257 9.9360 ± .45164 .329 Magnesium 2.29 ± .286 1.9640 ± .26978 .019* Phosphorus 5.35 ± 1.623 3.6420 ± 1.44930 .001** Creatinine 0.38 ± .078 0.3940 ± .0889 .061 GROUP 3 (Esomeprazole) n = 23 Calcium 10.02 ± .697 9.3680 ± .32832 .005* Magnesium 2.43 ± .323 2.3320 ± .31982 .396 Phosphorus 6.2980 ± 1.5318 5.5560 ± 1.59467 .002* Creatinine 0.608 ± 0.1084 0.601 ± 0.108 .066 Paired t test, Statistically significant = P ≤ 0.05*, Statistically highly significant = P ≤ 0.001** Table No. 2 - Comparison of rat serum calcium, magnesium, phosphorus& creatinine between control & experimental groups (One way ANOVA) Dependent Variable Groups Mean ± SD Significance Tukey’s post hoc analysis Calcium 1 9.6620 ± .32812 .008* 2 > 1 > 3 2 9.9360 ± .45164 3 9.3680 ± .32832 Magnesium 1 2.1510 ± .28726 .031* 3 > 1 > 2 2 1.9640 ± .26978 3 2.3320 ± .31982 Phosphorus 1 6.0160 ± 1.44042 .003* 1 > 3 > 2 2 3.6420 ± 1.44930 3 5.5560 ± 1.5947 Creatinine 1 .4150 ± .04301 .000** 3 > 1 > 2 2 .3800 ± .07803 3 .6080 ± .10840 Statistically significant = P ≤ 0.05*, Highly significant = P ≤ 0.001** Histopathological analysis The highest number of osteoclasts (multinucleated cells forming a resorption lacuna around themselves on the surface of the alveolar bone) was observed in Group 3 (84 ± 7.1 (SD)), which were scattered throughout the mesial and apical surfaces of the first molar roots (Figure No. 2 a & b). The lowest number of osteoclasts was counted in Group 1 (25.4 ± 4.5 (SD)). Tukey’s post hoc analysis suggests that there is a statistically significant (P ≤ 0.001) increase in the number of osteoclasts in Group 3, followed by Group 2, which is higher than Group 1. IHC (SATB2) (Figure No. 3) revealed a predominant presence of osteoblasts and their precursors in Group 1 (Control), which was higher than in the experimental groups. Immunohistochemistry with SATB2 showed osteoblasts scattered across the slide in decreasing order from Group 1 (Control) to the experimental groups (Groups 2 and 3) qualitatively. This observation confirms the inhibitory effect of esomeprazole and ranitidine on osteoblasts, although the mechanism underlying this effect remains unknown. Measurement of Orthodontic tooth movement (OTM) A significant change in OTM was observed in Group 3 (0.5850 ± 0.021 mm) (P ≤ .001), followed by Group 2 (0.3600 ± 0.039 mm), which was greater than Group 1 (0.2880 ± 0.03 mm), confirming the effect of drugs. Based on Table No. 3, it was inferred that the maximum amount of tooth movement occurred under long-term administration of Esomeprazole (Group 3). Table No. 3: Relationship between osteoclast & OTM in control & experimental groups Dependent Variable Group No. of Cells (Mean ± SD) Significance Tukey’s post hoc analysis Osteoclast 1 25.4 ± 4.5 P ≤ .001** 3 > 2 > 1 2 67.2 ± 6.03 3 84 ± 7.1 Dependent Variable Group Distance in mm (Mean ± SD) Significance Tukey’s post hoc analysis OTM 1 0.2880 ± 0.03 P ≤ .001** 3 > 2 > 1 2 0.3600 ± .039 3 0.5850 ± 0.021 One way ANOVA, statistically highly significant = P ≤ 0.001** DISCUSSION There has been a substantial increase in the global use of PPIs and H2R antagonists [ 13 , 9 ] because they more effectively inhibit acid secretion, particularly in adult patients. Both medication types have been shown to have osteoporotic effects when used over extended periods [ 30 ]. Therefore, it was crucial to assess how these drugs impact bone metabolism during orthodontic tooth movement, especially in adults seeking aesthetic correction. Based on physiological factors such as minerals and hormones, the WHO [ 39 ] stated that a 10% increase in peak bone mass could delay the onset of osteoporosis by 13 years. In this animal study using Rattus norvegicus albinus (rats), we compared the effects of PPI and H2R antagonists with a control group to investigate their biochemical roles, indicated by changes in serum calcium, magnesium, phosphorus, and creatinine, and their influence on bone cells during orthodontic tooth movement. Rats were selected as the study model because their entire lifespan correlates with that of humans [ 35 ]. Specifically, one human year roughly equals two rat weeks (13.8 rat days). The study lasted 6 weeks, equivalent to approximately three rat years, aligning with the American Board of Orthodontics (ABO) standards, which average orthodontic treatment times at 24.6 months [ 1 ], or approximately 2.5 to 3 years. Sixty-nine rats (weighing 200–250 g and 10 weeks old) were divided into three groups (Groups 1, 2, and 3). Group 2 received Ranitidine (30 mg/kg), Group 3 received Esomeprazole (30 mg/kg), while the control group (Group 1) was administered an equivalent volume of normal saline via oral gavage. The biological function of bone is to undergo continuous remodeling to maintain a balance between extracellular and intracellular mineral content in the body [ 29 ]. In this study, we observed a significant decrease in serum calcium levels after administering Esomeprazole (30mg/kg) for 6 weeks. This finding aligns with a study by Agnieszka Matuszewska et al. [ 24 ], who reported a decrease in total serum calcium, osteocalcin, and bone mineral density after 12 weeks of PPI administration. According to Fan Li et al. [ 22 ], rats treated with subcutaneous injections of parathyroid hormone (PTH) (4 mg/100 g of body weight for 12 days) showed accelerated orthodontic tooth movement by increasing alveolar bone turnover. Both studies indicate that serum calcium, PPI, and PTH influence tooth movement. PTH secretion is stimulated when serum calcium levels decrease, helping maintain calcium ion homeostasis by increasing osteoclastic activity. This supports our findings, which histologically (via H&E and immunohistochemistry) confirm the role of PPI (Esomeprazole (30 mg/kg)- Group 3) in increasing osteoclastic activity (84 ± 7.1(SD)), decreasing serum calcium levels (Pre = 10.02 ± .697(SD)mg/dl and Post = 9.368 ± .32832(SD)mg/dl), and enhancing orthodontic tooth movement (0.5850 ± 0.021 (SD) mm, P ≤ .001). However, in our study, serum calcium levels did not differ significantly in Ranitidine-treated rats (Group 2), indicating a reduced biological effect compared to Group 3 on calcium metabolism (9.84 ± 0.257 (SD) mg/dL) and orthodontic tooth movement (0.3600 ± 0.039 mm). Magnesium indirectly affects calcium hemostasis by altering PTH secretion. Low magnesium levels stimulate PTH secretion, but very low serum concentrations cause an incomprehensible block [ 38 ]. This block results in hypocalcemia. According to an in vitro study by Camire et al. [ 5 ], magnesium absorption decreases as gastric pH rises from 5 to 7 in subjects treated with omeprazole. This contrasts with our study, where we found no significant change in magnesium levels in the PPI-treated group (P = 0.396), indicating a limited influence of esomeprazole on magnesium metabolism (2.332 ± 3.198 mg/dL). Rats treated with ranitidine showed a significant (P ≤ 0.05) decrease in magnesium levels (2.29 ± 0.286 SD mg/dL). However, this decrease remains within the normal range (1.8-3.038 mg/dl) [ 26 ], supporting the finding of increased osteoclast activity in Group 3 (Esomeprazole 30 mg/kg) compared to Group 2 (Ranitidine 30 mg/kg). The level of phosphorus is significantly (P ≤ 0.05) decreased in Group 2 (Ranitidine- 30mg/kg) & Group 3 (Esomeprazole-30mg/kg) which is in accordance with Agnieszka Matuszewska et al [ 24 ] where rats treated with Ranitidine (10 mg/kg, intraperitoneal) show significant difference in serum concentration of phosphorus. However, the study is in discord with Giorgio G et al. [ 11 ] & Lacrosniere CS et al. [ 21 ], who found that PPI administered for 10 days did not influence phosphorus absorption. This may be due to the study's short duration. We were not able to relate the effect of phosphorus & calcium in ranitidine (Group 2) & esomeprazole (Group 3) treated rats based on bone cell and OTM, as the levels of phosphorus decrease significantly (P ≤ 0.05) in both the experimental groups (Group 2- 3.6420 ± 1.44930 (SD)mg/dl and Group 3- 5.5560 ± 1.59467 (SD)mg/dl) which was similar to decrease in calcium levels (Group 2- 9.9360 ± .45164 (SD)mg/dl and Group 3- 9.3680 ± .32832 (SD)mg/dl). This decrease in levels of both phosphorus can be due to the unknown side effects of esomeprazole and ranitidine. Serum creatine levels were analyzed in our study; however, the levels and changes found between the three groups were not significant, and we were unable to relate their effect on bone metabolism & OTM. Histopathology is regarded as the gold standard [ 4 ] for measuring changes at the cellular level. Haematoxylin & Eosin (first slide) was used to identify bone cells. The number of osteoclasts was counted from H&E-stained slides along the mesial root surface and was highest in the Esomeprazole group (84 ± 7.1 (SD)), followed by the Ranitidine group (67.2 ± 6.03 (SD)), and the control group (25.4 ± 4.5 (SD)). Notably, the increase in osteoclasts in Esomeprazole-treated rats was statistically highly significant (P ≤ 0.001). Since PPIs inhibit gastric H+ -K + ATPase, they also inhibit the vacuolar type of H+-K + ATPase present in osteoclasts [ 25 ]. This causes abnormal osteoclastic bone resorption and, as shown in our study, enhances tooth movement. Multinucleated osteoclasts were visible in H & E stain, but it was difficult to identify osteoblasts. Therefore, sections from each group were analyzed using Immunohistochemistry (IHC). The specific osteoblast marker SATB2 was used to examine the distribution pattern of osteoblasts across all three groups. Qualitatively, osteoblasts appeared scattered throughout the slide, decreasing from Group 1 (Control) to the experimental groups. This may suggest that Ranitidine and Esomeprazole exert an inhibitory effect on osteoblastic activity through unknown mechanisms. Quantitative analysis of osteoblasts was not possible, so definitive conclusions cannot be drawn. Yunju Jo et al [ 16 ] stated that the effect of PPIs on bone metabolism is more evident in the elderly than in younger age groups, as its impact on bone metabolism is compensated in younger individuals. The skeletal response to acid suppressants may differ based on endogenous factors, such as histamines and PTH, as well as dietary components (minerals and vitamin B), but the risk is more pronounced in patients with underlying secondary factors, including renal dysfunction and osteoporosis [ 14 ]. Therefore, the harmful effects of PPIs must be carefully noted when taking a medical history, especially for adult orthodontic patients, to inform them. Further studies should be conducted on younger individuals receiving PPI, especially children and teenagers [ 19 ], since the majority of orthodontic treatment seekers fall into this age group. Consequently, some modifications in force application might be necessary during orthodontic procedures. LIMITATIONS We were unable to determine the effect of phosphorus and calcium on bone cells and OTM because phosphorus levels decreased significantly in both experimental groups. This reduction in calcium and phosphorus may be a result of the effects of Esomeprazole and Ranitidine. Serum creatinine levels were analyzed, but the differences and changes among the three groups were not significant, and we could not determine their impact on bone metabolism and OTM. Further studies are needed for a quantitative analysis of osteoblasts in relation to the use of PPIs and H2R antagonists. CONCLUSION The highest osteoclastic activity and rate of tooth movement were observed in Group 3 (Esomeprazole – PPI), followed by Group 2 (Ranitidine – H2R antagonist), with the lowest in the control group. Esomeprazole significantly affected serum calcium and phosphorus levels, bone cell activity, and orthodontic tooth movement. It induced the highest osteoclast count, promoting bone resorption. However, the associated decrease in serum calcium and phosphorus raises concerns about disrupted mineral balance, indicating that Esomeprazole may not be ideal for encouraging tooth movement. Ranitidine also significantly influenced serum magnesium and phosphorus levels. Although magnesium levels remained within the normal range, phosphorus levels decreased enough to pose a risk of metabolic disturbance. Ranitidine caused noticeable, though less substantial, tooth movement compared to Esomeprazole. Immunohistochemical observations showed the highest osteoblastic activity in the control group, indicating a possible suppressive effect of both Esomeprazole and Ranitidine on osteoblasts. However, since no statistical analysis was conducted for these findings, definitive conclusions could not be drawn. Further research is needed to quantitatively assess the impact of these drugs on osteoblast function. Declarations Author Contributions Manami Das made substantial contributions to the conception or design of the work, drafted the work, revised it critically for important intellectual content, and approved the version to be published. Ashish Agrawal supervised the work being done. Bipin Rai helped in blood extraction experiments. All three authors collectively agreed on the manuscript and its final draft. Acknowledgments We gratefully acknowledge the Department of Interdisciplinary, Institute of Sciences at Banaras Hindu University for their invaluable assistance in conducting experiments and investigative procedures. We also extend our sincere thanks to Dr. Subhash Chandra Gupta, Department of Interdisciplinary, Institute of Sciences, BHU, for his dedicated coordination and support throughout this research. Declaration of Conflicting Interests The authors declared no potential conflicts of interest for the research, authorship, and/or publication of this article. Fundin g: No funding was available for this research. References Aljehani D, Baeshen HA. Effectiveness of the American Board of Orthodontics Discrepancy Index in Predicting Treatment Time J Contemp Dent Pract. 2018;19(6):647-650. Anroop B, Jacob S. A simple practice guide for dose conversion between animals and humans. J Basic Clin Pharm. 2016; 7:27-31. Arias LC, Marquez-Orozco MC. 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A proton pump inhibitor’s effect on bone metabolism mediated by osteoclast action in old age: A prospective randomized study. Gut Liver. 2015;9(5):607-614. Keim RG, Gottlieb EL, Nelson AH, Vogels DS. 2008 JCO Orthodontic Practice Study, Part 1: Trends. J Clin Orthod. 2008;42(11):625-640. Kilkenny C, et al. The ARRIVE guidelines: Animal Research: Reporting In Vivo Experiments. NC3Rs. 2021. Kurth B-M, Schaffrath Rosario A, Hölling H, Kamtsiuris P. Use of orthodontic services by children and adolescents in Germany. J Health Monit. 2019;4(2):24–41. Laferriere C, Daniel SJ. Review of intraperitoneal injection of sodium pentobarbital as a method of euthanasia in laboratory rodents. J Am Assoc Lab Anim Sci. 2020;59(3):254-263. Lacrosniere CS, et al. Hypochlorhydria from short-term omeprazole treatment does not inhibit intestinal absorption of calcium, phosphorus, magnesium, or zinc. J Am Coll Nutr. 1995;14(4):364-368. Li F, et al. Effect of parathyroid hormone on experimental tooth movement in rats. Am J Orthod Dentofacial Orthop. 2013; 144:523-532. Li Y, Jacox LA, Shannyn H, Ching-Chang K. Orthodontic tooth movement: The biology and clinical implications. Kaohsiung J Med Sci. 2018;34(4):207-214. Matuszewska A, et al. Effects of long-term administration of pantoprazole on bone mineral density in young male rats. Pharmacol Rep. 2016;68(5):1060-1064. Mizunashi K, Furukawa Y, Katano K, Abe K. Effect of omeprazole on bone resorption in humans. Calcif Tissue Int. 1993;53(1):21-25. Mulyaningsih N, Lakshmidevi AJ, Soejoko DS, Astuti DA. Serum mineral status and long bone morphometry of ovariectomized rats fed a nano-calcium phosphate diet. Pak J Nutr. 2019; 18:1058-1067. National Research Council. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington, DC: National Academies Press; 2011. Nasser S, et al. Ranitidine impairs bone healing and implant osseointegration in rats’ tibiae. J Oral Maxillofac Surg. 2020;78(8):1389-1398. Papageorgiou SN, Hochli D, Eliades T. Outcomes of comprehensive fixed appliance orthodontic treatment: A systematic review. Korean J Orthod. 2017;47(6):401-413. Park JH, et al. Comparing proton pump inhibitors with histamine-2 receptor blockers regarding the risk of osteoporotic fractures. BMC Geriatr. 2020; 20:407. Proffit WR, Fields HW, Larson B, Sarver DM. Contemporary Orthodontics. 6th ed. Elsevier; 2018. Rotman SR, Bishop TF. Proton pump inhibitor uses in the US ambulatory setting, 2002–2009. PLoS One. 2013;8(2): e56060. Samson DS, Chaithra B, Harshitha V, Mithun K, Abhinay S. Drugs in Orthodontics: A review. Indian J Forensic Med Toxicol. 2018;12(3):138-142. Savarino V, Dulbecco P, De Bortoli N, Ottonello A, Savarino E. The Appropriate Use of Proton Pump Inhibitors (PPIs): A Need for Reappraisal. Eur J Intern Med. 2017; 37: 19–24. Sengupta P. A scientific review of age determination for a laboratory rat: How old is it in comparison with human age? Biomed Int. 2012; 2:81-89. Shirazi M, et al. Pantoprazole, a proton pump inhibitor, increases orthodontic tooth movement in rats. Iran J Basic Med Sci. 2014;17:448-453. Singh A, Gill G, Kaur H, Amhmed M, Jakhu H. Role of osteopontin in bone remodeling and orthodontic tooth movement: a review. Prog Orthod. 2018; 19:18. Vetter T, Lohse MJ. Magnesium and the parathyroid. Curr Opin Nephrol Hypertens. 2002;11(4):403-410. World Health Organization. Assessment of Fracture Risk and Its Application to Screening for ostmenopausal Osteoporosis. WHO Tech Rep Ser. 1994; 843:1-129. Guidelines for Blood Collection in Mice and Rats. Office of Animal Care and Use (OACU). https://oacu.oir.nih.gov/training-resources Additional Declarations No competing interests reported. Supplementary Files ARRIVEGUIDLINES.pdf GRAPHICALABSTRACTPPI.png 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7186441","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":493066539,"identity":"7c197e1c-965b-4fbc-9391-3545f4cc8c71","order_by":0,"name":"Manami Das","email":"","orcid":"","institution":"Banaras Hindu University","correspondingAuthor":false,"prefix":"","firstName":"Manami","middleName":"","lastName":"Das","suffix":""},{"id":493066540,"identity":"654a06f0-7cbb-4680-a785-9df5bf369cd9","order_by":1,"name":"ashish agrawal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYFACHoYDjA3MDAzsPWwQAWaitfCcIUELA1iLRA4bcc7i7z978ODPHdZy/DPfHnvMw2Anz8DOewCvFokbeQmHec+kG0vczks35mFINmxg5kvAb80NHoPDjG2HEzdI55hJ8zAwJzAw8xjg1SF//ozBwZ8gLZJnQFrqCWsxOJBjcIAXpEWCB6TlMGEthjdyDA7ztgH9ciYvTXKOwXHDNkJa5M6fMf74sw0YYu1nj0m8qaiW5+c/g18LujsZGIiMnVEwCkbBKBgF+AAACOw9CY8+U6QAAAAASUVORK5CYII=","orcid":"","institution":"Banaras Hindu University","correspondingAuthor":true,"prefix":"","firstName":"ashish","middleName":"","lastName":"agrawal","suffix":""},{"id":493066541,"identity":"db698d9f-3373-4eaa-ba1d-a98f81a8c630","order_by":2,"name":"Bipin Rai","email":"","orcid":"","institution":"Banaras Hindu University","correspondingAuthor":false,"prefix":"","firstName":"Bipin","middleName":"","lastName":"Rai","suffix":""}],"badges":[],"createdAt":"2025-07-22 11:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7186441/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7186441/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88037040,"identity":"77a74989-4917-450f-b555-0f817ee0a99c","added_by":"auto","created_at":"2025-07-31 16:25:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":389006,"visible":true,"origin":"","legend":"\u003cp\u003e(a) NiTi Closed-coil Spring engaged between the maxillary first molar and the incisors ligated with ligature wire \u0026amp; stabilised with composite (b)Graphical representation of rat model (c) Measurement of 50 mg force with the help of Dontrix gauge. (d) Collection of blood from the retro-orbital sinus for serum biochemical analysis of calcium, magnesium, phosphorus \u0026amp; creatinine.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7186441/v1/a2ad3772a644cae6b84368c4.png"},{"id":88037039,"identity":"19d1e8c1-c139-401d-84a1-18c7f5edad91","added_by":"auto","created_at":"2025-07-31 16:25:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":279114,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Longitudinal section of rat molars (Under H \u0026amp; E stain, Magnification 40 X) (b) H \u0026amp; E stained sections of Group 1, Group 2 \u0026amp; Group 3 showing osteoclasts in the mesial surface of bone (Black arrow = Osteoclast). 1- bone, 2- PDL 3-Tooth (Magnification 400 X).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7186441/v1/cb5898c8c312535835025552.png"},{"id":88037780,"identity":"6723afa2-6a87-4d88-a50e-83254f134163","added_by":"auto","created_at":"2025-07-31 16:33:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":184190,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis by SATB2 (nuclei of osteoblast-stained brown in color) showing the highest number of osteoblasts in Group 1(control) as compared to Group 2(Ranitidine) \u0026amp; Group 3 (Esomeprazole).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7186441/v1/a72e9b511cea3eb68813b8e1.png"},{"id":88037043,"identity":"c33da726-ef24-4b4a-96a2-a8c1865b1a86","added_by":"auto","created_at":"2025-07-31 16:25:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130950,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plot showing relationship between number of osteoclasts \u0026amp; tooth movement (a) Control Group 1 – Normal Saline was administered equal to that of experimental groups (b) Experimental Group 2 - 30mg/kg body weight of Ranitidine was administered (c) Experimental Group 3 - 30mg/kg body weight of Esomeprazole was administered\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7186441/v1/739d43493f5f14732d47c5c9.png"},{"id":90135191,"identity":"16e4d18f-ca89-41d3-af3b-99f47f502b27","added_by":"auto","created_at":"2025-08-29 01:16:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1723512,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7186441/v1/93e6b8df-9230-4d20-9e13-c36364b26646.pdf"},{"id":88039011,"identity":"a042dde5-8095-4d0e-98fe-2016b2dac7eb","added_by":"auto","created_at":"2025-07-31 16:41:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":89417,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVEGUIDLINES.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7186441/v1/d4217736705bfcf4045d76af.pdf"},{"id":88037036,"identity":"f24d9b64-f087-4739-8438-5659a009fd14","added_by":"auto","created_at":"2025-07-31 16:25:10","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":840129,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACTPPI.png","url":"https://assets-eu.researchsquare.com/files/rs-7186441/v1/2dd1a5d1e8c5be47e929687a.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Histopathological analysis of Proton Pump Inhibitor \u0026amp; H2 receptor antagonist on bone cells in orthodontically induced tooth movement: A Randomised Control animal study\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOrthodontic tooth movement (OTM) arises from a coordinated cascade of mechanical, cellular, and molecular events. When orthodontic force is applied, mechanotransduction within the periodontal ligament triggers an inflammatory response, releasing mediators such as prostaglandins, cytokines, and growth factors that regulate bone remodeling—resorption on the pressure side and deposition on the tension side, thereby facilitating tooth displacement [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Pharmacologically, bone-resorptive inhibitors—like NSAIDs, bisphosphonates, calcium supplements, and statins—dampen osteoclast activity and slow OTM [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In contrast, agents such as vitamin D, prostaglandin analogs, parathyroid hormone, corticosteroids, and relaxin can enhance osteoclastogenesis or alter extracellular matrix turnover, thus accelerating tooth movement [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIsabelle Graf et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] investigated orthodontics related to social media by analyzing posts made by patients or peers and found that more positive posts were on Instagram. With increased access to social media, more adult patients are becoming aware of orthodontic treatment options and seeking adjunctive or comprehensive treatment according to their needs [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Christensen et al [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] documented an increase in the number of adults seeking orthodontic treatment over the years. Adult orthodontics is witnessing increasing demand due to heightened awareness of dental aesthetics and improved oral health consciousness among older individuals [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Adults now actively seek orthodontic treatment not only for aesthetic reasons but also for correcting functional issues such as malocclusion and bite discrepancies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The growing acceptance and visibility of fixed orthodontic appliances, including tooth-colored brackets and clear aligners, have made treatment more appealing to adult patients [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, many adults are on long-term medications, such as proton pump inhibitors (PPIs) and H₂ receptor antagonists used for gastric conditions, who may experience altered bone metabolism, potentially affecting the rate and quality of orthodontic tooth movement [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Understanding the influence of such medications is critical in planning and delivering effective orthodontic care for adult patients.\u003c/p\u003e\u003cp\u003eGiven the widespread use of over-the-counter medications, accurately documenting patients’ current drug intake (including non-prescription agents) is crucial. This enables orthodontists to anticipate changes in treatment duration, adjust the applied force, and collaborate effectively with other healthcare providers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Proton pump inhibitors (PPIs) are among the 43 drugs listed in India's National List of Essential Medicines (2011) and are commonly prescribed. Rotman et al. reported PPI use prevalence of 4% in the US (2002) to 9.2% in 2009, with 62.9% of users lacking documented gastrointestinal indications [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, Jakupi et al. observed a marked increase in PPI usage in Kosovo from 2011 to 2013 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Animal studies support these dynamics. For example, rats treated with high-dose pantoprazole (200 mg/kg for 6 weeks) showed significantly increased OTM and decreased bone mineral density, indicative of induced osteoporosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe cumulative effects of pharmacological agents on tooth movement could be inhibitory, additive, or synergistic [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The doses and protocols vary, making it clinically unreliable. There is limited knowledge about the use of PPIs and H2R antagonists in both growing and non-growing individuals, as well as their effects on orthodontic treatment. Rat models (adult) are frequently used in various research studies, as a specific mathematical formula exists for converting dosages from humans to rats, which enhances the quantitative and qualitative reliability of the study. Therefore, this study is designed to evaluate the effects of commonly used pharmacological agents (such as PPIs and H2R antagonists) on tissue remodelling (histopathological evaluation), the rate of tooth movement in rats, and their correlation with serum biochemical analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAIMS \u0026amp; OBJECTIVES\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe primary objective was to assess the effects of ranitidine and esomeprazole on bone remodelling during OTM in an animal model (Rattus norvegicus Albinus), histologically using H\u0026amp;E staining for osteoclasts and SATB2 immunohistochemical staining for osteoblasts. The secondary objective was to correlate its effect with serum biochemistry (calcium, magnesium, phosphorus, and creatinine) and the rate of tooth movement.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"MATERIALS \u0026 METHOD","content":"\u003cp\u003eA Randomized Controlled Trial was approved by the Ethics committee (No. Dean/2021/EC/2721) and was carried out in the Unit of Orthodontics, Faculty of Dental Sciences, for a period of 1 year from 2021 to 2022.\u003c/p\u003e\n\u003cp\u003eAll procedures performed in the present research were as per the Animal Research Reporting of in Vivo Experiments (ARRIVE) guidelines [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e] \u0026amp; the National Research Council\u0026apos;s Guide for the Care and Use of Laboratory Animals [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. A sample size calculation was performed using G*Power 3.1.9.7 targeting a one-way ANOVA with three groups, an alpha of 0.05, and a power of 80%. Assuming a medium-to-large effect size (f\u0026thinsp;=\u0026thinsp;0.4) based on expected differences in tooth movement and osteoclastic activity, the estimated total sample size was 66 rats (22 per group). A pilot study was conducted on 15 rats before the main experiment to assess feasibility and standardize experimental protocols. All procedures were conducted in accordance with the predefined research design. The results of the pilot study were not included in the main study. However, the experiment was found to be feasible.\u003c/p\u003e\n\u003cp\u003eThereafter, 69 healthy male rats (Rattus norvegicus Albinus), each weighing 200\u0026ndash;250 g and 10 weeks old, were randomly selected from the Animal House of the Institute for the experimental setup. Animals used in previous studies or showing signs of oedema, allergy, alopecia, or cachexia were excluded. All animals were housed individually in plastic cages under a 12:12-hour light\u0026ndash;dark cycle, with controlled temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (55\u0026thinsp;\u0026plusmn;\u0026thinsp;10%). Simple random allocation of the sample was performed manually using Microsoft Excel by generating random numbers with the {=RAND()} function, followed by sorting and sequential group assignment (Control Group I, Experimental Groups II and III, n\u0026thinsp;=\u0026thinsp;23). Group II received Ranitidine (30 mg/kg), and Group III received Esomeprazole (30 mg/kg) via oral gavage once daily for six weeks, ensuring no spillage during administration. To reduce the risk of NiTi spring displacement during mastication, they were fed a soft diet ad libitum (finely ground standard pellets). The control group (Group 1) received an equivalent volume of saline solution once daily for six weeks, administered at the same time each day as the experimental groups. The dosages of Ranitidine and Esomeprazole were calculated based on a previous study by Nasser S. Alshahrani et al. [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e] in rats to reflect the therapeutic dose administered to humans.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRAT MODEL PREPARATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animals were anaesthetized with intraperitoneal administration of Xylazine Hydrochloride (0.2 mg/kg) and Ketamine Hydrochloride (1 ml/kg) in a ratio of 1:3. Then, a NiTi closed-coil spring (force 50 g) was stretched between the left maxillary first molar and the incisors and ligated with ligature wire (Figure No. 1 a \u0026amp; b). A constant force of 50 mg (Figure No. 1c) was maintained for 6 weeks. Subcutaneous injections of Carprofen (5 mg/kg) were given for analgesia on the day of coil placement and daily for 3 days [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eBody weights were checked before and after the experimental period and recorded. Blood samples were collected from the retro-orbital sinus on day 1 and after six weeks (Figure No. 1 d) for biochemical analysis of calcium, magnesium, phosphorus, and creatinine. The samples were coded and labelled for blinding. Topical anaesthesia (Tetracaine Hydrochloride (1%)) was applied, and 1.4 to 1.8 ml of blood was collected (using a sterile haematocrit capillary tube) from each rat, according to the guidelines of the National Institutes of Health (NIH), Institutional Animal Care and Use Committee (IACUC) [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. Serum was separated by centrifugation (at 1500 x g) and stored at -70\u0026deg;C until required for bone metabolic marker assays. Total serum calcium, inorganic phosphorus, magnesium, and creatinine levels were measured by a certified laboratory with Architect plus ci4100 (Abbott) using commercial tests (Calcium Architect/Aeroset REF 3L79-21 and 3L79-31 304328/R1, Abbott; Phosphorus Architect REF 7D71 305532/R02, Abbott; Magnesium Architect REF 3P68 305531/R02, Abbott; and Creatinine (enzymatic) REF 8L24-31 and 8L24-41 G10667/R08 B8L240 for use with Architect, Abbot, respectively), performed according to their manufacturers\u0026rsquo; instructions. To ensure double blinding, samples were recoded and relabeled, and group allocation was concealed from the individual conducting the histological analysis. The desired tooth movement was measured between the distal surface of the 1st molar and the mesial surface of the 2nd molar using Vernier Callipers, and data were recorded for the control and experimental groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHISTOLOGIC ANALYSIS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess histologic changes after 6 weeks, all rats were euthanized with an injection of chemical anesthetics (Pentobarbital \u0026minus;\u0026thinsp;100 mg/kg, intraperitoneal) [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], and the left hemimaxillae of all animals were surgically removed after sacrifice. The tissues were then placed in 10% formalin for 10 days. Decalcification was performed using 5% formic acid for at least 7 days, and the samples were embedded in paraffin blocks.\u003c/p\u003e\n\u003ch2\u003eSTATISTICAL ANALYSIS\u003c/h2\u003e\n\u003cp\u003eThe data obtained was analysed using SPSS 26 software for statistical analysis to test the significance of the study. Paired T test \u0026amp; one-way ANOVA (analysis of variance) were used to compare data between pre- \u0026amp; post-orthodontic force application. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). For all tests, differences with values of P\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered significant, and P\u0026thinsp;\u0026le;\u0026thinsp;0.001 was considered highly significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eIn this study, 69 rats were randomly assigned to three groups: a control group (Group 1) and two experimental groups (Groups 2 and 3). There was no loss of sample during the entire study duration. Administration of PPI and H2R antagonists began on the day orthodontic force (50 mg force applied by a NiTi coil spring) was applied. The drugs were delivered via oral gavage, which did not cause any toxicity such as edema, allergy, alopecia, cachexia, etc., throughout the study. The mean weights were 208.3 g for Group 1, 205.05 g for Group 2, and 210.15 g for Group 3. Additionally, there was no weight gain or loss before or after the study (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum Biochemical analysis\u003c/strong\u003e (Table No. 1\u0026amp;2)\u003c/p\u003e\n\u003cp\u003eThe blood from the Control group (Group 1- Orthodontic force and normal saline) was analyzed for serum content. The study found a significant decrease (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) in serum magnesium levels (Pre\u0026thinsp;=\u0026thinsp;2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;.439(SD)mg/dl \u0026amp; Post\u0026thinsp;=\u0026thinsp;2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;.287(SD)mg/dl) only. No statistical correlation was observed for calcium, phosphorus, and creatinine in this group. However, in Group 2 (Ranitidine with orthodontic force), the decrease in serum magnesium levels (Pre\u0026thinsp;=\u0026thinsp;2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;.286(SD)mg/dl, Post\u0026thinsp;=\u0026thinsp;1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;.26978(SD)mg/dl) was statistically significant (p\u0026thinsp;\u0026le;\u0026thinsp;0.05), and the decrease in phosphorus levels (Pre\u0026thinsp;=\u0026thinsp;5.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.623(SD)mg/dl, Post\u0026thinsp;=\u0026thinsp;3.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44930(SD)mg/dl) was highly significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.001). Similarly, in Group 3 (Esomeprazole with orthodontic force), there was a statistically significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) decrease in serum calcium (Pre\u0026thinsp;=\u0026thinsp;10.02\u0026thinsp;\u0026plusmn;\u0026thinsp;.697(SD)mg/dl, Post\u0026thinsp;=\u0026thinsp;9.37\u0026thinsp;\u0026plusmn;\u0026thinsp;.32832(SD)mg/dl) and phosphorus levels (Pre\u0026thinsp;=\u0026thinsp;6.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53189(SD)mg/dl, Post\u0026thinsp;=\u0026thinsp;5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59467(SD)mg/dl).\u003c/p\u003e\n\u003cp\u003eTable No. 1 - Comparison of rat serum biochemistry between Group 1,2 \u0026amp; 3(Paired T test)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGROUPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eBLOOD PARAMETERS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eON DAY 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eON DAY 42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePaired T test\u003c/p\u003e\n \u003cp\u003e(P value)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMEAN\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMEAN\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eGROUP 1\u003c/p\u003e\n \u003cp\u003e(Control)\u003c/p\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCalcium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.69\u0026thinsp;\u0026plusmn;\u0026thinsp;.286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.66\u0026thinsp;\u0026plusmn;\u0026thinsp;.328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.853\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMagnesium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;.439\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;.287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e.044*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhosphorus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.348\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCreatinine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.226\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eGROUP 2\u003c/p\u003e\n \u003cp\u003e(Ranitidine)\u003c/p\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCalcium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.84\u0026thinsp;\u0026plusmn;\u0026thinsp;.257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.9360\u0026thinsp;\u0026plusmn;\u0026thinsp;.45164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.329\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMagnesium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;.286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.9640\u0026thinsp;\u0026plusmn;\u0026thinsp;.26978\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e.019*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhosphorus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.6420\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44930\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e.001**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCreatinine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3940\u0026thinsp;\u0026plusmn;\u0026thinsp;.0889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.061\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eGROUP 3\u003c/p\u003e\n \u003cp\u003e(Esomeprazole)\u003c/p\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCalcium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.02\u0026thinsp;\u0026plusmn;\u0026thinsp;.697\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.3680\u0026thinsp;\u0026plusmn;\u0026thinsp;.32832\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e.005*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMagnesium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;.323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.3320\u0026thinsp;\u0026plusmn;\u0026thinsp;.31982\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.396\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhosphorus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.2980\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.5560\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e.002*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCreatinine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.608\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.601\u0026thinsp;\u0026plusmn;\u0026thinsp;0.108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.066\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003ePaired t test, Statistically significant\u0026thinsp;=\u0026thinsp;P \u0026le;\u0026thinsp;0.05*, Statistically highly significant\u0026thinsp;=\u0026thinsp;P\u0026thinsp;\u0026le;\u0026thinsp;0.001**\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTable No. 2 - Comparison of rat serum calcium, magnesium, phosphorus\u0026amp; creatinine between control \u0026amp; experimental groups (One way ANOVA)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabb\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDependent Variable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSignificance\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s post hoc analysis\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eCalcium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.6620\u0026thinsp;\u0026plusmn;\u0026thinsp;.32812\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e.008*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e2\u0026thinsp;\u0026gt;\u0026thinsp;1\u0026thinsp;\u0026gt;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.9360\u0026thinsp;\u0026plusmn;\u0026thinsp;.45164\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.3680\u0026thinsp;\u0026plusmn;\u0026thinsp;.32832\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eMagnesium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1510\u0026thinsp;\u0026plusmn;\u0026thinsp;.28726\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e.031*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e3\u0026thinsp;\u0026gt;\u0026thinsp;1\u0026thinsp;\u0026gt;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.9640\u0026thinsp;\u0026plusmn;\u0026thinsp;.26978\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.3320\u0026thinsp;\u0026plusmn;\u0026thinsp;.31982\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003ePhosphorus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.0160\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44042\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e.003*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e1\u0026thinsp;\u0026gt;\u0026thinsp;3\u0026thinsp;\u0026gt;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.6420\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44930\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.5560\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5947\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eCreatinine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e.4150\u0026thinsp;\u0026plusmn;\u0026thinsp;.04301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e.000**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e3\u0026thinsp;\u0026gt;\u0026thinsp;1\u0026thinsp;\u0026gt;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e.3800\u0026thinsp;\u0026plusmn;\u0026thinsp;.07803\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e.6080\u0026thinsp;\u0026plusmn;\u0026thinsp;.10840\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eStatistically significant\u0026thinsp;=\u0026thinsp;P \u0026le;\u0026thinsp;0.05*, Highly significant\u0026thinsp;=\u0026thinsp;P \u0026le;\u0026thinsp;0.001**\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe highest number of osteoclasts (multinucleated cells forming a resorption lacuna around themselves on the surface of the alveolar bone) was observed in Group 3 (84\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 (SD)), which were scattered throughout the mesial and apical surfaces of the first molar roots (Figure No. 2 a \u0026amp; b). The lowest number of osteoclasts was counted in Group 1 (25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 (SD)). Tukey\u0026rsquo;s post hoc analysis suggests that there is a statistically significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.001) increase in the number of osteoclasts in Group 3, followed by Group 2, which is higher than Group 1. IHC (SATB2) (Figure No. 3) revealed a predominant presence of osteoblasts and their precursors in Group 1 (Control), which was higher than in the experimental groups. Immunohistochemistry with SATB2 showed osteoblasts scattered across the slide in decreasing order from Group 1 (Control) to the experimental groups (Groups 2 and 3) qualitatively. This observation confirms the inhibitory effect of esomeprazole and ranitidine on osteoblasts, although the mechanism underlying this effect remains unknown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Orthodontic tooth movement (OTM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA significant change in OTM was observed in Group 3 (0.5850\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021 mm) (P\u0026thinsp;\u0026le;\u0026thinsp;.001), followed by Group 2 (0.3600\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039 mm), which was greater than Group 1 (0.2880\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mm), confirming the effect of drugs. Based on Table No. 3, it was inferred that the maximum amount of tooth movement occurred under long-term administration of Esomeprazole (Group 3).\u003c/p\u003e\n\u003cp\u003eTable No. 3: Relationship between osteoclast \u0026amp; OTM in control \u0026amp; experimental groups\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabc\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDependent Variable\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo. of Cells\u003c/p\u003e\n \u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSignificance\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s post hoc analysis\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eOsteoclast\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u0026thinsp;\u0026le;\u0026thinsp;.001**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e3\u0026thinsp;\u0026gt;\u0026thinsp;2\u0026thinsp;\u0026gt;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e67.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDependent Variable\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDistance in mm\u003c/p\u003e\n \u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s post hoc analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eOTM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2880\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u0026thinsp;\u0026le;\u0026thinsp;.001**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e3\u0026thinsp;\u0026gt;\u0026thinsp;2\u0026thinsp;\u0026gt;\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3600\u0026thinsp;\u0026plusmn;\u0026thinsp;.039\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5850\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eOne way ANOVA, statistically highly significant\u0026thinsp;=\u0026thinsp;P\u0026thinsp;\u0026le;\u0026thinsp;0.001**\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThere has been a substantial increase in the global use of PPIs and H2R antagonists [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] because they more effectively inhibit acid secretion, particularly in adult patients. Both medication types have been shown to have osteoporotic effects when used over extended periods [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, it was crucial to assess how these drugs impact bone metabolism during orthodontic tooth movement, especially in adults seeking aesthetic correction. Based on physiological factors such as minerals and hormones, the WHO [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] stated that a 10% increase in peak bone mass could delay the onset of osteoporosis by 13 years. In this animal study using Rattus norvegicus albinus (rats), we compared the effects of PPI and H2R antagonists with a control group to investigate their biochemical roles, indicated by changes in serum calcium, magnesium, phosphorus, and creatinine, and their influence on bone cells during orthodontic tooth movement. Rats were selected as the study model because their entire lifespan correlates with that of humans [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Specifically, one human year roughly equals two rat weeks (13.8 rat days). The study lasted 6 weeks, equivalent to approximately three rat years, aligning with the American Board of Orthodontics (ABO) standards, which average orthodontic treatment times at 24.6 months [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], or approximately 2.5 to 3 years. Sixty-nine rats (weighing 200\u0026ndash;250 g and 10 weeks old) were divided into three groups (Groups 1, 2, and 3). Group 2 received Ranitidine (30 mg/kg), Group 3 received Esomeprazole (30 mg/kg), while the control group (Group 1) was administered an equivalent volume of normal saline via oral gavage.\u003c/p\u003e\u003cp\u003eThe biological function of bone is to undergo continuous remodeling to maintain a balance between extracellular and intracellular mineral content in the body [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, we observed a significant decrease in serum calcium levels after administering Esomeprazole (30mg/kg) for 6 weeks. This finding aligns with a study by Agnieszka Matuszewska et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], who reported a decrease in total serum calcium, osteocalcin, and bone mineral density after 12 weeks of PPI administration. According to Fan Li et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], rats treated with subcutaneous injections of parathyroid hormone (PTH) (4 mg/100 g of body weight for 12 days) showed accelerated orthodontic tooth movement by increasing alveolar bone turnover. Both studies indicate that serum calcium, PPI, and PTH influence tooth movement. PTH secretion is stimulated when serum calcium levels decrease, helping maintain calcium ion homeostasis by increasing osteoclastic activity. This supports our findings, which histologically (via H\u0026amp;E and immunohistochemistry) confirm the role of PPI (Esomeprazole (30 mg/kg)- Group 3) in increasing osteoclastic activity (84\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1(SD)), decreasing serum calcium levels (Pre\u0026thinsp;=\u0026thinsp;10.02\u0026thinsp;\u0026plusmn;\u0026thinsp;.697(SD)mg/dl and Post\u0026thinsp;=\u0026thinsp;9.368\u0026thinsp;\u0026plusmn;\u0026thinsp;.32832(SD)mg/dl), and enhancing orthodontic tooth movement (0.5850\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021 (SD) mm, P\u0026thinsp;\u0026le;\u0026thinsp;.001). However, in our study, serum calcium levels did not differ significantly in Ranitidine-treated rats (Group 2), indicating a reduced biological effect compared to Group 3 on calcium metabolism (9.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.257 (SD) mg/dL) and orthodontic tooth movement (0.3600\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039 mm).\u003c/p\u003e\u003cp\u003eMagnesium indirectly affects calcium hemostasis by altering PTH secretion. Low magnesium levels stimulate PTH secretion, but very low serum concentrations cause an incomprehensible block [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This block results in hypocalcemia. According to an in vitro study by Camire et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], magnesium absorption decreases as gastric pH rises from 5 to 7 in subjects treated with omeprazole. This contrasts with our study, where we found no significant change in magnesium levels in the PPI-treated group (P\u0026thinsp;=\u0026thinsp;0.396), indicating a limited influence of esomeprazole on magnesium metabolism (2.332\u0026thinsp;\u0026plusmn;\u0026thinsp;3.198 mg/dL). Rats treated with ranitidine showed a significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) decrease in magnesium levels (2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.286 SD mg/dL). However, this decrease remains within the normal range (1.8-3.038 mg/dl) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], supporting the finding of increased osteoclast activity in Group 3 (Esomeprazole 30 mg/kg) compared to Group 2 (Ranitidine 30 mg/kg).\u003c/p\u003e\u003cp\u003eThe level of phosphorus is significantly (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) decreased in Group 2 (Ranitidine- 30mg/kg) \u0026amp; Group 3 (Esomeprazole-30mg/kg) which is in accordance with Agnieszka Matuszewska et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] where rats treated with Ranitidine (10 mg/kg, intraperitoneal) show significant difference in serum concentration of phosphorus. However, the study is in discord with Giorgio G et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] \u0026amp; Lacrosniere CS et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], who found that PPI administered for 10 days did not influence phosphorus absorption. This may be due to the study's short duration. We were not able to relate the effect of phosphorus \u0026amp; calcium in ranitidine (Group 2) \u0026amp; esomeprazole (Group 3) treated rats based on bone cell and OTM, as the levels of phosphorus decrease significantly (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) in both the experimental groups (Group 2- 3.6420\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44930 (SD)mg/dl and Group 3- 5.5560\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59467 (SD)mg/dl) which was similar to decrease in calcium levels (Group 2- 9.9360\u0026thinsp;\u0026plusmn;\u0026thinsp;.45164 (SD)mg/dl and Group 3- 9.3680\u0026thinsp;\u0026plusmn;\u0026thinsp;.32832 (SD)mg/dl). This decrease in levels of both phosphorus can be due to the unknown side effects of esomeprazole and ranitidine. Serum creatine levels were analyzed in our study; however, the levels and changes found between the three groups were not significant, and we were unable to relate their effect on bone metabolism \u0026amp; OTM.\u003c/p\u003e\u003cp\u003eHistopathology is regarded as the gold standard [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] for measuring changes at the cellular level. Haematoxylin \u0026amp; Eosin (first slide) was used to identify bone cells. The number of osteoclasts was counted from H\u0026amp;E-stained slides along the mesial root surface and was highest in the Esomeprazole group (84\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 (SD)), followed by the Ranitidine group (67.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.03 (SD)), and the control group (25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 (SD)). Notably, the increase in osteoclasts in Esomeprazole-treated rats was statistically highly significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.001). Since PPIs inhibit gastric H+ -K\u0026thinsp;+\u0026thinsp;ATPase, they also inhibit the vacuolar type of H+-K\u0026thinsp;+\u0026thinsp;ATPase present in osteoclasts [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This causes abnormal osteoclastic bone resorption and, as shown in our study, enhances tooth movement. Multinucleated osteoclasts were visible in H \u0026amp; E stain, but it was difficult to identify osteoblasts. Therefore, sections from each group were analyzed using Immunohistochemistry (IHC). The specific osteoblast marker SATB2 was used to examine the distribution pattern of osteoblasts across all three groups. Qualitatively, osteoblasts appeared scattered throughout the slide, decreasing from Group 1 (Control) to the experimental groups. This may suggest that Ranitidine and Esomeprazole exert an inhibitory effect on osteoblastic activity through unknown mechanisms. Quantitative analysis of osteoblasts was not possible, so definitive conclusions cannot be drawn.\u003c/p\u003e\u003cp\u003eYunju Jo et al [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] stated that the effect of PPIs on bone metabolism is more evident in the elderly than in younger age groups, as its impact on bone metabolism is compensated in younger individuals. The skeletal response to acid suppressants may differ based on endogenous factors, such as histamines and PTH, as well as dietary components (minerals and vitamin B), but the risk is more pronounced in patients with underlying secondary factors, including renal dysfunction and osteoporosis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, the harmful effects of PPIs must be carefully noted when taking a medical history, especially for adult orthodontic patients, to inform them. Further studies should be conducted on younger individuals receiving PPI, especially children and teenagers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], since the majority of orthodontic treatment seekers fall into this age group. Consequently, some modifications in force application might be necessary during orthodontic procedures.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eLIMITATIONS\u003c/strong\u003e\u003cp\u003eWe were unable to determine the effect of phosphorus and calcium on bone cells and OTM because phosphorus levels decreased significantly in both experimental groups. This reduction in calcium and phosphorus may be a result of the effects of Esomeprazole and Ranitidine. Serum creatinine levels were analyzed, but the differences and changes among the three groups were not significant, and we could not determine their impact on bone metabolism and OTM. Further studies are needed for a quantitative analysis of osteoblasts in relation to the use of PPIs and H2R antagonists.\u003c/p\u003e\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe highest osteoclastic activity and rate of tooth movement were observed in Group 3 (Esomeprazole \u0026ndash; PPI), followed by Group 2 (Ranitidine \u0026ndash; H2R antagonist), with the lowest in the control group. Esomeprazole significantly affected serum calcium and phosphorus levels, bone cell activity, and orthodontic tooth movement. It induced the highest osteoclast count, promoting bone resorption. However, the associated decrease in serum calcium and phosphorus raises concerns about disrupted mineral balance, indicating that Esomeprazole may not be ideal for encouraging tooth movement.\u003c/p\u003e\u003cp\u003eRanitidine also significantly influenced serum magnesium and phosphorus levels. Although magnesium levels remained within the normal range, phosphorus levels decreased enough to pose a risk of metabolic disturbance. Ranitidine caused noticeable, though less substantial, tooth movement compared to Esomeprazole.\u003c/p\u003e\u003cp\u003eImmunohistochemical observations showed the highest osteoblastic activity in the control group, indicating a possible suppressive effect of both Esomeprazole and Ranitidine on osteoblasts. However, since no statistical analysis was conducted for these findings, definitive conclusions could not be drawn. Further research is needed to quantitatively assess the impact of these drugs on osteoblast function.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eManami Das made substantial contributions to the conception or design of the work, drafted the work, revised it critically for important intellectual content, and approved the version to be published. Ashish Agrawal supervised the work being done. Bipin Rai helped in blood extraction experiments. All three authors collectively agreed on the manuscript and its final draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the Department of Interdisciplinary, Institute of Sciences at Banaras Hindu University for their invaluable assistance in conducting experiments and investigative procedures. We also extend our sincere thanks to Dr. Subhash Chandra Gupta, Department of Interdisciplinary, Institute of Sciences, BHU, for his dedicated coordination and support throughout this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest for the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundin\u003c/strong\u003eg: No funding was available for this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAljehani D, Baeshen HA. Effectiveness of the American Board of Orthodontics Discrepancy Index in Predicting Treatment Time J Contemp Dent Pract. 2018;19(6):647-650.\u003c/li\u003e\n \u003cli\u003eAnroop B, Jacob S. A simple practice guide for dose conversion between animals and humans. 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NC3Rs. 2021.\u003c/li\u003e\n \u003cli\u003eKurth B-M, Schaffrath Rosario A, H\u0026ouml;lling H, Kamtsiuris P. Use of orthodontic services by children and adolescents in Germany. J Health Monit. 2019;4(2):24\u0026ndash;41.\u003c/li\u003e\n \u003cli\u003eLaferriere C, Daniel SJ. Review of intraperitoneal injection of sodium pentobarbital as a method of euthanasia in laboratory rodents. J Am Assoc Lab Anim Sci. 2020;59(3):254-263.\u003c/li\u003e\n \u003cli\u003eLacrosniere CS, et al. Hypochlorhydria from short-term omeprazole treatment does not inhibit intestinal absorption of calcium, phosphorus, magnesium, or zinc. J Am Coll Nutr. 1995;14(4):364-368.\u003c/li\u003e\n \u003cli\u003eLi F, et al. Effect of parathyroid hormone on experimental tooth movement in rats. Am J Orthod Dentofacial Orthop. 2013; 144:523-532.\u003c/li\u003e\n \u003cli\u003eLi Y, Jacox LA, Shannyn H, Ching-Chang K. Orthodontic tooth movement: The biology and clinical implications. Kaohsiung J Med Sci. 2018;34(4):207-214.\u003c/li\u003e\n \u003cli\u003eMatuszewska A, et al. Effects of long-term administration of pantoprazole on bone mineral density in young male rats. Pharmacol Rep. 2016;68(5):1060-1064.\u003c/li\u003e\n \u003cli\u003eMizunashi K, Furukawa Y, Katano K, Abe K. Effect of omeprazole on bone resorption in humans. Calcif Tissue Int. 1993;53(1):21-25.\u003c/li\u003e\n \u003cli\u003eMulyaningsih N, Lakshmidevi AJ, Soejoko DS, Astuti DA. Serum mineral status and long bone morphometry of ovariectomized rats fed a nano-calcium phosphate diet. Pak J Nutr. 2019; 18:1058-1067.\u003c/li\u003e\n \u003cli\u003eNational Research Council. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington, DC: National Academies Press; 2011.\u003c/li\u003e\n \u003cli\u003eNasser S, et al. Ranitidine impairs bone healing and implant osseointegration in rats\u0026rsquo; tibiae. 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The Appropriate Use of Proton Pump Inhibitors (PPIs): A Need for Reappraisal. Eur J Intern Med. 2017; 37: 19\u0026ndash;24.\u003c/li\u003e\n \u003cli\u003eSengupta P. A scientific review of age determination for a laboratory rat: How old is it in comparison with human age? Biomed Int. 2012; 2:81-89.\u003c/li\u003e\n \u003cli\u003eShirazi M, et al. Pantoprazole, a proton pump inhibitor, increases orthodontic tooth movement in rats. Iran J Basic Med Sci. 2014;17:448-453.\u003c/li\u003e\n \u003cli\u003eSingh A, Gill G, Kaur H, Amhmed M, Jakhu H. Role of osteopontin in bone remodeling and orthodontic tooth movement: a review. Prog Orthod. 2018; 19:18.\u003c/li\u003e\n \u003cli\u003eVetter T, Lohse MJ. Magnesium and the parathyroid. Curr Opin Nephrol Hypertens. 2002;11(4):403-410.\u003c/li\u003e\n \u003cli\u003eWorld Health Organization. Assessment of Fracture Risk and Its Application to Screening for ostmenopausal Osteoporosis. WHO Tech Rep Ser. 1994; 843:1-129.\u003c/li\u003e\n \u003cli\u003eGuidelines for Blood Collection in Mice and Rats. Office of Animal Care and Use (OACU). https://oacu.oir.nih.gov/training-resources\u003c/li\u003e\n\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":"Serum biochemistry, Esomeprazole, Ranitidine, Osteoclast, osteoblast, Orthodontic tooth movement","lastPublishedDoi":"10.21203/rs.3.rs-7186441/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7186441/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives:\u003c/strong\u003e The primary objective was to assess the effects of ranitidine and esomeprazole on OTM in an animal model via: a) Histopathology: H\u0026amp;E staining for osteoclasts and SATB2 immunostaining for osteoblasts. The secondary objective evaluated its impact on bone remodeling, correlating its effect with serum biochemistry: calcium, magnesium, phosphorus, and creatinine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u003c/strong\u003e: Sixty-nine male Rattus norvegicus (200–250 g, 10 weeks old) were randomly assigned to three groups: control, ranitidine (Group 2), and esomeprazole (Group 3). A 50 g NiTi closed-coil spring was placed between the left central incisor and first molar. Groups 2 and 3 received (30 mg/kg/day) of the respective drug via oral gavage; controls received saline. Blood was collected on days 1 and 42 for serum analysis. On day 42, tooth movement was recorded, the animals were euthanized, and histological analysis was performed to evaluate osteoclasts and osteoblasts. Paired t-tests and one-way ANOVA were used for statistical analysis (P ≤ 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Esomeprazole (Group 3) produced the highest osteoclast count (84 ± 7.1) and greatest tooth movement (0.585 ± 0.021 mm). Control had the highest number of osteoblasts (SATB2), while Groups 2 and 3 showed inhibition. Serum phosphorus significantly decreased in Groups 2 (3.642 ± 1.449) and 3 (5.556 ± 1.595 mg/dL); calcium decreased in Group 3 (9.368 ± 0.328 mg/dL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Esomeprazole (PPI) induced a decrease in serum Ca levels, the highest osteoclastic activity, and OTM, followed by ranitidine (H₂RA), with the control group showing the least. Both PPI and H\u003csub\u003e2\u003c/sub\u003eRA inhibit or reduce osteoblastic activity.\u003c/p\u003e","manuscriptTitle":"The Histopathological analysis of Proton Pump Inhibitor \u0026amp; H2 receptor antagonist on bone cells in orthodontically induced tooth movement: A Randomised Control animal study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 16:25:05","doi":"10.21203/rs.3.rs-7186441/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":"90bbb473-614e-4ba0-96cb-e7e3a703d6e5","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-29T01:08:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 16:25:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7186441","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7186441","identity":"rs-7186441","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}