Unintentional Amount of Calcium Hydroxide Extrusion and High Alkalinity Diffusion Distance: An In Vitro Study

Unintentional Amount of Calcium Hydroxide Extrusion and High Alkalinity Diffusion Distance: An In Vitro 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 Unintentional Amount of Calcium Hydroxide Extrusion and High Alkalinity Diffusion Distance: An In Vitro Study Panuroot Aguilar, Anna Chairat, Sivawut Aroonjit, Panupat Phumpatrakom, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8611112/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background High alkalinity diffusion and its timeframe have not been thoroughly studied. This study aimed to evaluate the amount of unintentional calcium hydroxide extrusion and to measure the diffusion distance of high alkalinity from different apical foramen sizes. Methods Sixty-three mandibular premolars were divided into 5 groups based on apical foramen sizes of 20, 30, 45, 55, and 70 (n = 12 each) along with three negative controls. For calcium hydroxide extrusion measurement, pipette tips were used to cover the root apex, allowing the extruded calcium hydroxide to be collected in them. Then, calcium hydroxide was delivered into the canal using a polymer tip in the same manner across all groups. The quantity of calcium hydroxide extrusion was determined by comparing the tip weight before and after calcium hydroxide placement. To assess high alkalinity diffusion (pH 10–11), the same teeth were rinsed and reused by embedding them in 2% agar containing universal pH indicators followed by calcium hydroxide delivery. The extent of high alkalinity diffusion was measured by color extension at 0, 2, 6, and 9 hours. Kruskal-Wallis test was employed to compare the weights of calcium hydroxide extruded beyond the apical foramen among different apical foramen sizes, as well as the diffusion distances of calcium hydroxide over time. Pearson correlations were used to define the relationship between the apical foramen size and the amount of extruded calcium hydroxide. Results The extruded calcium hydroxide was positively correlated with the apical foramen sizes. The high alkalinity distances increased over time. Within two hours, all sizes of apical foramina demonstrated high alkalinity diffusion distances over 10 mm. At nine hours, there was no significant difference among the groups. Conclusions The extent of medicament extrusion was influenced by the size of the apical foramen. High alkalinity can develop within a few hours and may extend several millimeters beyond the physical presence of the medicament. Understanding the relationship between apical foramen size and high alkalinity diffusion aids in assessing the potential for tissue irritation or post operative pain and helps guide safer clinical practices when using calcium hydroxide medicaments. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Calcium hydroxide has been widely used in root canal treatment, particularly as an intracanal medicament to enhance antimicrobial efficacy following root canal preparation and irrigation [ 1 ]. During calcium hydroxide placement into the prepared root canal, the medicament may extrude beyond the root canal into the periapical tissues or periapical lesions unintentionally. Many previous ex vivo or in vitro studies have demonstrated factors influencing the extrusion of calcium hydroxide and the resulting effects on simulated periapical tissues following the extrusion beyond the root apex [ 2 – 6 ]. All forms of calcium hydroxide can extrude beyond the root apex and induce pH changes in simulated periapical areas, with the size of the apical foramen having the most significant impact on pH alterations [ 3 ]. Additionally, calcium ions have also been found to extend beyond the root canal [ 2 , 3 ]. Therefore, alterations in pH within the periapical tissues surrounding the root apex can be anticipated in clinical settings. The amount of extruded calcium hydroxide and the pH of its diffusion may play an important role in post-operative pain. Furthermore, the excessive extrusion of calcium hydroxide during endodontic treatment can lead to periapical tissue damage, often presenting severe pain and, in some cases, accompanying paresthesia, as documented in case reports [ 7 , 8 ]. However, calcium hydroxide extrusion beyond the apex is expected minimally in standard root canal treatment regarding the studies mentioned above. Limited research has examined how these pH changes impact surrounding tissues during endodontic treatment and their influence on treatment outcomes. While previous studies focused primarily on the overall increasing pH of calcium hydroxide diffusion [ 2 – 6 ] the distance of increasing high pH diffusion and the timeframe within which this occurs have yet to be studied. The distance over which elevated pH extends beyond the apex provides insight into which structures or cells may be affected by this high pH and for how long, particularly in the context of periapical lesions. It could also clarify whether the size of the apical foramen influences these effects [ 3 ] and may have clinical implications regarding potential complications during root canal treatment, such as postoperative pain or damage to vital structures including nerves, blood vessels, and bone. Thus, this study aimed to investigate the amount of calcium hydroxide extruded beyond the apical foramen, as well as the patterns of alkaline pH diffusion over time following the placement of calcium hydroxide in root canals, utilizing a semi-solid gel model with varying apical foramen sizes. The null hypotheses of this study were: (i) there are no significant differences in the amount of calcium hydroxide extrusion among different apical foramen sizes, and (ii) there are no significant differences in alkaline pH diffusion among different apical foramen sizes. Methods The study was adhered to the Declaration of Helsinki and approved by the Human Research Ethics Committee of Thammasat University (Science), Thailand (Project no. 069/2565). Sample size calculation Based on the results obtained from our pilot study, an effect size of 0.895 was added to a statistical power of 0.95 and a probability level (α) of 0.05 inputs into F test family for one-way analysis of variance (G*power 3.1 for Macintosh). To detect differences among these groups, the estimated minimum sample size for each group was determined to be five. However, to increase the power of the study, we utilized twelve samples for each experimental group. Sample collection A sound, single-rooted mandibular premolar extracted for orthodontic reasons was selected as the sample. After extraction, the teeth were stored in 0.1% thymol solution. The apical foramen of each tooth was examined to assess deviation from the root apex, which did not exceed 0.5 mm when observed under 10x magnification using a dental operating microscope (Carl Zeiss Meditec A, Jena, Germany). Periapical radiographs were taken to confirm that each selected tooth had a single root canal. A total of 63 mandibular premolars were included. Tooth Preparation Access cavities were prepared for all specimens, and canal patency was confirmed using a size 15 K-file (Kerr Dental™, Uxbridge, UK). The coronal portion of each tooth was removed using a 22-mm diamond disc (Edenta™, Hauptstrasse, Switzerland) to obtain a standardized root length of 13 mm. Canal preparation was performed using a series of rotary instruments. The working length was established at 12 mm. A master apical file of size 40 with a 0.04 taper (Mtwo™, Munich, Germany) was used, with irrigation performed using 2.5% sodium hypochlorite and 17% EDTA solutions. The prepared specimens were randomly allocated into five experimental groups (n = 12 per group), with an additional three teeth serving as the negative control. The apical foramina of each group were prepared with K-files of sizes 20, 30, 45, 55, and 70 under a dental operating microscope. The negative control group underwent only root canal preparation, and the external root surfaces were then sealed with nail polish (Fig. 1 I). All prepared teeth were dried with paper points . Calcium Hydroxide Extrusion Measurement A 10-µL pipette tip was weighed using an analytical balance (Denver Instrument TP-214, Goettingen, Germany) to obtain a baseline weight (Fig. 1II, a). Each prepared tooth was filled with calcium hydroxide paste (Ultracal XS, South Jordan, UT, USA). The prepared tooth was mounted with a pipette tip (Fig. 1II, b). The calcium hydroxide paste was introduced into the root canal using a polymer tip positioned 2 mm short of the working length, slowly injected, and gradually withdrawn until it was 1 mm from the canal orifice The access cavity was sealed with a 1-mm-thick layer of temporary filling material (Cavit™, 3M, Bracknell, UK) shaped to match the access outline and gently compacted with a plastic instrument to avoid excessive pressure. Extrusion of calcium hydroxide beyond the apical foramen was then observed (Fig. 1II, c). Any extruded calcium hydroxide adhering to the root apex was carefully swabbed from the inner side of the pipette tip under a stereomicroscope (Fig. 1II, d). The pipette tip with extruded calcium hydroxide was reweighed (Fig. 1II, e). Periapical Tissue Simulation Agar Periapical tissue was simulated using a 2% plain agar solution prepared by dissolving 4 g of agar powder (Himedia, Maharashtra, India) in 200 mL of boiling distilled water with continuous stirring until fully dissolved. The solution was heated at 95°C for 20 minutes to improve long-term stability and enhance cross-linking of the agar. The clear and uniform agar solution was then transferred to a 70°C water bath. The pH of the agar solution was adjusted to 7 using 1 M NaOH. Thirteen mL of universal indicator (Loba Chemic, Mumbai, India) was added to the agar solution to make 6% indicator in agar solution and the agar exhibited a green color, then poured into transparent plastic cuvettes and allowed to cool at room temperature and stored in 37°C with 100% humidity before using. Alkaline pH Diffusion Distance Measurement The same teeth used for extrusion measurement were rinsed with distilled water through a syringe needle until the outflow was clear and then reused for diffusion measurement. The teeth were labeled according to their apical foramen sizes and stored in distilled water until the experimental begin. A plastic cap was drilled at the center to fit each tooth, and the tooth–cap assembly was positioned in a tube containing agar (Fig. 1III, a-c). A transparent ruler (Grande grid GCT-30, Kyoei Plastic Co., Ltd, Osaka, Japan) was positioned in close contact with the cuvette containing the agar to provide a reference scale. The root length of each specimen was determined prior to the placement of calcium hydroxide. After embedding the tooth in plain agar mixed with a universal indicator, a 1-mm calibration length was established (Fig. 2 I, a). Once calibration was confirmed, the root length was measured by drawing the straight line from the margin of the plastic cap to the root apex (Fig. 2 I, b), and recalculated the root length with the 1-mm reference obtained from the same image. Calcium hydroxide was then placed into the root canal in the same manner as the extrusion test (Fig. 2 II, a, b). Calcium hydroxide paste was extruded, and the measurement of high alkalinity distance diffusion was initiated immediately, designated as 0 hr (Fig. 2 II, c). Photographs were then taken at 2, 6, and 9 hours under identical environmental lighting conditions, with a background illuminated by white light The purple coloration, representing high alkalinity (pH 10–11), was identified according to the pH color scale provided on the bottle of the universal indicator solution (Fig. 2 III). At each time point when a photograph was taken. The 1-mm calibration was reperformed (Fig. 2IV, a). Then, a straight line was drawn from the margin of the plastic cap to the farthest clearly distinguishable boundary of the purple area (Fig. 2IV, b), The diffusion distance of high alkalinity at each time point was calculated by subtracting the initial root length. During the diffusion experiment, the specimens were kept at 37°C under 100% humidity. All images were measured by using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Intra-observer reliability Alkaline pH diffusion distance measurements were performed by a single operator on 30 randomly selected samples and repeated after a three-week interval. Intra-observer reliability was assessed using the intraclass correlation coefficient (ICC) employing a two-way mixed-effects model with absolute agreement. Statistical Analysis Pearson correlations were used to define the relationship between the apical foramen size and the amount of extruded calcium hydroxide. Because the data were not normally distributed the Kruskal-Wallis test was employed to compare the weights of calcium hydroxide extruded beyond the apical foramen among different apical foramen sizes, as well as the diffusion distances of calcium hydroxide over time of different pH levels. Dunn’s test was performed for post hoc comparison. All statistical analyses were performed using SPSS software (SPSS Statistics 25.0, IBM Corp, Armonk, NY), with a significance level set at p < 0.05. Results The amount of calcium hydroxide extruded beyond the apical foramen showed a significant positive correlation with the size of the apical foramen. Pearson correlation (r) = 0.682 with p < 0.001. Specifically, larger apical foramen sizes, such as 70 and 55, demonstrated a significantly greater extrusion of calcium hydroxide compared to smaller sizes, such as 20 and 30 (Fig. 3 ). No extrusion of calcium hydroxide was detected in the negative control group. The diffusion distances of high alkalinity increased over time. Mean ± SD (mm.) of diffusion distances of high alkalinity was showed in Table 1 . The increasing trend was showed in Fig. 4 . At two hours, the high alkalinity diffused more than 10 mm in all apical foramen sizes with no significant differences among the groups. However, at six hours, the specimens with an apical foramen size of 20 exhibited a significant shorter diffusion distance than those with larger apical foramina. At nine hours, no significant differences in diffusion distance were observed among all apical foramen sizes. Table 1 High alkalinity diffusion distances for each apical foramen sizes at different time points Time points 0 Hr 2 Hr 6 Hr 9 Hr Foramen size Mean ± SD (mm.) Mean ± SD (mm.) Mean ± SD (mm.) Mean ± SD (mm.) 20 0.21 ± 0.22 11.94 ± 1.58 13.32 ± 1.17 * 27.04 ± 2.41 30 0.46 ± 0.25 12.55 ± 1.19 21.60 ± 3.33 26.88 ± 2.29 45 0.37 ± 0.32 12.62 ± 1.06 21.31 ± 1.93 26.35 ± 2.52 55 0.66 ± 0.34 15.27 ± 0.69 24.84 ± 2.32 28.92 ± 2.74 70 0.67 ± 0.39 14.58 ± 0.95 23.62 ± 1.18 27.94 ± 2.04 Asterisk indicate statistical significance at p < 0.05 between the groups in the same column Excellent intra-observer reliability was observed (ICC = 0.993; 95% CI: 0.986–0.997). Discussion This study investigated the effect of apical foramen sizes on the amount of calcium hydroxide extrusion and high alkalinity diffusion in a simulated periapical tissue model. The results demonstrated significant differences among the groups. Therefore, the first null hypotheses were rejected. While high alkalinity diffusion distances at the end of the studies showed no statistical differences thus the second null hypothesis was accepted. The heterogeneous structure of periapical lesions, composed of microbial elements, host cells and extracellular matrixes, poses challenges for simulating such conditions. These components are distributed or aggregated based on their function in walling off infections and preventing the spread of pathogens from the root canal system into the jawbone [ 9 ]. Thus, this study aims to evaluate the diffusion distance of alkalinity over time, rather than focusing on the magnitude of pH changes, to elucidate the behaviour of high alkalinity diffusion in periapical tissues, particularly in cases of apical granuloma or periapical abscess—common pathological presentations resulting from inflammation or necrosis of dental pulp tissue [ 10 , 11 ]. Numerous studies have investigated the alkalinity beyond the apical foramen resulting from the use of calcium hydroxide as an intracanal medicament in root canal treatment [ 2 – 4 , 6 ]. Most of these studies have focused on the pH values and the factors influencing these changes, such as the size of the apical foramen [ 3 ], the type of calcium hydroxide used [ 3 , 4 ], and the duration of calcium hydroxide placement within the canal [ 4 , 6 ], allowing for diffusion beyond the apex over varying time intervals. Moreover, various models have been utilized in these studies, including distilled water [ 2 , 4 , 6 ], agar to simulate periapical tissue [ 3 ], real teeth [ 6 ], or artificial root canals made from pipette tip [ 3 ], glass tube [ 4 ] or metal tube [ 2 ]. Despite the variability in methodologies, the results consistently demonstrate an increase in alkalinity beyond apical foramen [ 2 – 4 , 6 ]. The result from this study also demonstrated the correlation with increasing the size of the apical foramen and the amount of calcium hydroxide diffusing beyond the apex. But after a period of time, the size of the apical foramen had no influence on the diffusion distance of alkalinity but increasing in a time-dependent manner. The study revealed rapid diffusion of high pH values from the root apex. The diffusion distance exceeded 10 mm within 2 hours and extended beyond 20 mm at 9 hours. Considering that periapical lesions typically range in volume from 1 mm³ to over 30 mm³, with an average of approximately 4–5 mm³ [ 12 , 13 ], these findings suggest that alkaline diffusion from calcium hydroxide can potentially reach the entire lesion area within a clinically relevant timeframe. It was observed that apical foramen sizes of 20 or more could result in a high pH environment (pH 10–11) within the lesion within 2 hours. Biologically, such high pH levels are reported the cytotoxicity to cells, including periodontal ligament cells [ 14 ], osteoblasts [ 15 ], bone marrow cells [ 16 ] and other structures within the lesion, such as nerves [ 7 ], inflammatory cells [ 17 ], and various microorganisms especially, in biofilm [ 18 – 20 ]. Clinically, unintentional extrusion of materials can lead to severe complications [ 21 – 23 ], particularly in teeth adjacent to vital structures such as the mental foramen or mandibular canal. Our study demonstrated that high alkalinity can be detected up to 20–30 mm from the apical foramen. The radiograph may reveal the physical extent of calcium hydroxide at the apical limit, but its alkalinity extends beyond this visible boundary. Therefore, the use of calcium hydroxide in teeth near vital structures should be approached with extreme caution. Additionally, hydraulic calcium silicate sealers, which can produce calcium hydroxide through hydration reactions, are also prone to extrusion due to their excellent flowability. Consequently, hydraulic calcium silicate sealers should also be used with caution under similar precautions even though recent study has reported high success rates in periapical healing following unintentional extrusion [ 24 ]. Furthermore, our study demonstrated that the diffusion distance of alkalinity is time-dependent, supporting the notion that the degree of nerve injury increases with longer exposure to the irritant as contact time extends [ 25 ]. This study employed agar as a substitute for human tissue due to its similar density to human soft tissue, as used in various experimental models, including radiation studies [ 26 , 27 ] and calcium hydroxide diffusion [ 3 ]. Agar offers advantages over solutions in pH measurement, as it prevents rapid diffusion to equilibrium, which may underestimate pH values. Previous studies primarily used liquid media to simulate the periapical environment, which did not accurately represent conditions within the periodontal ligament or alveolar bone, particularly in cases of apical periodontitis, periapical granuloma or granulation tissue [ 2 , 4 ]. The simulated environments failed to closely mimic the granulation tissue surrounding the root apex. Using semi-solid gel media could provide results more aligned with clinical conditions [ 3 ]. However, the method of obtaining pH values from such media had limitations, as it involved extracting the gel and measuring the pH, which only reflected specific points in time [ 3 ]. The diffusion of alkaline pH from extruded calcium hydroxide should vary depending on the distance from the root apex over different time intervals. Additionally, the shape of the container allows diffusion to extend primarily in a longitudinal direction rather than dispersing in all directions. Results from this study are consistent with previous finding [ 3 ], demonstrating that the amount of calcium hydroxide diffusing correlates with the size of the apical foramen. However, differences in pH values observed across studies [ 2 – 5 ] may stem from variations in measurement techniques. Since this study relied on colour changes to indicate the boundary of areas of the level of alkalinity which followed the colour scale labelled on the bottle of the universal pH indicator as a reference for pH interpretation. The preliminary observations revealed that once the alkaline diffusion occurred, the clearly distinguishable colours were mainly purple and turquoise. According to the manufacturer’s colour chart, the purple range corresponds to pH 10 and 11. In practice, however, it was not possible to distinguish a definite boundary between these two shades of purple within the agar medium. Therefore, both pH 10 and 11 were collectively defined as representing high alkalinity in this study. The dark blue colour, corresponding to pH 9.5, appeared only as a very narrow zone or was absent in some specimens and usually blended indistinctly with the adjacent purple area. As this could lead to unreliable measurement, pH 9.5 was excluded from diffusion distance analysis. Conversely, although the turquoise region (pH 8.5–9) was clearly visible, it was not included in the diffusion analysis, as this zone appeared only as a narrow band surrounding the purple area and was rapidly replaced by the higher-alkalinity purple colour over time. The high alkalinity diffusion distance was not measured directly from the root apex to the boundary of the purple colour zone, because the extruded calcium hydroxide paste obscured the apex once it flowed beyond the root canal. Therefore, the diffusion had to be calculated by subtracting the root length which measured before calcium hydroxide placement, from the total diffusion distance of the purple colour. The study was limited by its experimental timeframe, which was restricted to 9 hours. This limitation was due to some groups exhibiting a plateau in colour change, likely resulting from the height restriction of the plastic cuvettes (35 mm). Teeth with larger apical foramina experienced this limitation more prominently, leading to the decision to focus on a 9-hour observation period. Apical foramina of size 15 or smaller were not included. A pilot study demonstrated that both the high alkalinity diffusion distance and the extrusion experiment produced inconsistent results at size 15 (data not shown). This limitation suggests that apical foramina of size 15 and smaller may not allow predictable hydroxyl ion diffusion or detectable extrusion using the current model, therefore were excluded to ensure methodological reliability. The use of different apical foramen sizes in this study may be applicable to teeth with originally large apical foramina, such as immature teeth, as well as cases with iatrogenic apical enlargement resulting from mechanical instrumentation. As this study was conducted under in vitro conditions, certain limitations remain. Granulation tissue, characterized by high vascularity [ 28 , 29 ], plays a critical role in nutrient supply and tissue repair. The blood flow in granulation tissue is approximately 2–10 times greater than in normal tissues in skin wound [ 20 , 30 , 31 ], which might reduce the duration of high pH levels within periapical lesions. Moreover, the inherent pH of periapical tissues, which ranges from 6 to 7.3 [ 32 , 33 ], might further buffer the high alkalinity. In addition, certain proteins and ions dispersed within the extracellular matrix might also have exhibited these effects also. Consequently, while high alkalinity from calcium hydroxide may diffuse extensively within the lesion and exert cytotoxic effects, this effect is likely transient, allowing adjacent cells to proliferate or migrate to repair or clear necrotic debris like pulpal wound healing [ 34 ]. The delivery of calcium hydroxide in this study was performed twice independently by a single operator to maintain consistency in the technique. However, human error is unavoidable among clinicians, and this may have contributed to variations in the amount of calcium hydroxide extrusion. Periapical lesions are generally expected to be devoid of pathogens due to the accumulation of immune cells. However, apical foramina or accessory canals may harbour microbial biofilms. Pathogens such as Actinomyces , Fusobacterium , and Prevotella [ 35 ] have been found in periapical tissues, particularly in cases of periapical abscesses or sinus tracts [ 36 ]. Although high alkalinity (pH 10–11) can eradicate these pathogens, the transient nature of such high pH levels due to the vascularity of granulation tissue may explain the limited efficacy of calcium hydroxide in some cases. Furthermore, bacteria organized in biofilm structures exhibit resistance to high alkalinity [ 37 ]. Previous study [ 38 ] have shown that the overall healing pattern in lesions overfilled with calcium hydroxide was not significantly different from those in which overfilling did not occur. These outcomes may result from ensuring complete delivery of calcium hydroxide to the full working length to maximize disinfection, rather than keeping the material short of the apex solely out of concern for extrusion. However, direct physical contact between calcium hydroxide and vital structures should be avoided. Thus, interpretation of the results should be applied with caution. In conclusion, the amount of calcium hydroxide extrusion from the root canal is influenced by the size of the apical foramen. Apical foramina of size 20 or larger, either naturally occurring or created by iatrogenic apical perforation, demonstrated an increased tendency for calcium hydroxide extrusion. The high alkalinity environment around the apical foramen was then expected within few hours and extended beyond its physical location. Even slight to moderate extrusion may cause irritation or damage to the periapical tissues and surrounding vital structures; therefore, the use of calcium hydroxide should be approached with caution in cases with large apical foramina. However, the high alkalinity would be transient due to high vascularity and acidity of granulation tissue. It is need for further studies to elucidate the exact alkalinity of periapical lesion in vivo after placing calcium hydroxide into the root canal and also investigate the cellular responses to understanding the healing process under alkaline condition. Declarations Ethics approval and consent to participate Informed consent was deemed unnecessary by the Human Research Ethics Committee of Thammasat University (Science), Thailand (Project no. 069/2565). Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare have no conflicts of interest relevant to this article. Funding This study was supported by Faculty of Dentistry, Thammasat University Fund. Clinical trial number Not applicable Authors' contributions P.A.: Project Administration, Conceptualization, Methodology, Investigation, Data Curation, Formal Analysis, Funding Acquisition, Visualization, Resources, Writing-Original Draft Preparation. 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Endod Dent Traumatol. 1995;11(1):15–9. Brändle N, Zehnder M, Weiger R, Waltimo T. Impact of growth conditions on susceptibility of five microbial species to alkaline stress. J Endod. 2008;34(5):579–82. Caliskan MK, Sen BH. Endodontic treatment of teeth with apical periodontitis using calcium hydroxide: a long-term study. Endod Dent Traumatol. 1996;12(5):215–21. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 13 Feb, 2026 Reviews received at journal 12 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers agreed at journal 01 Feb, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers invited by journal 29 Jan, 2026 Editor assigned by journal 29 Jan, 2026 Editor invited by journal 20 Jan, 2026 Submission checks completed at journal 20 Jan, 2026 First submitted to journal 20 Jan, 2026 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8611112","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583691235,"identity":"3754b1fb-b48e-4574-b7cd-3deebb5e4933","order_by":0,"name":"Panuroot Aguilar","email":"","orcid":"","institution":"Thammasat University","correspondingAuthor":false,"prefix":"","firstName":"Panuroot","middleName":"","lastName":"Aguilar","suffix":""},{"id":583691236,"identity":"a1366eb5-44c4-4737-8461-7eb031a9ee8b","order_by":1,"name":"Anna Chairat","email":"","orcid":"","institution":"Thammasat University","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Chairat","suffix":""},{"id":583691237,"identity":"619076e8-b171-4397-9f78-413d88b1b2bf","order_by":2,"name":"Sivawut Aroonjit","email":"","orcid":"","institution":"Thammasat University","correspondingAuthor":false,"prefix":"","firstName":"Sivawut","middleName":"","lastName":"Aroonjit","suffix":""},{"id":583691238,"identity":"0063d346-883b-4037-b7ee-c2f80b24c184","order_by":3,"name":"Panupat Phumpatrakom","email":"","orcid":"","institution":"Thammasat University","correspondingAuthor":false,"prefix":"","firstName":"Panupat","middleName":"","lastName":"Phumpatrakom","suffix":""},{"id":583691239,"identity":"2e45ed56-2879-47b9-9185-68506a42f40c","order_by":4,"name":"Arnonchai Junsuntonpass","email":"","orcid":"","institution":"Thammasat University","correspondingAuthor":false,"prefix":"","firstName":"Arnonchai","middleName":"","lastName":"Junsuntonpass","suffix":""},{"id":583691240,"identity":"76ca2c40-1ac7-4a40-a222-83de3bcdf3a5","order_by":5,"name":"Sirawut Hiran-Us","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBAC+WYow4C9gY2BoQDCYcanxeAwhGIw4DnABqJBgLEZrxYYZSCRQKwWdt6Hn3nb/jCYSz5+9pjHwI6Bv/0A++MCvH5hN5bmbTNgsJydZm7MY5DMIHEmgbF5Bj5rDrMxSOcCtRjczmGT5jEAevwG0GE8+LUw/wZruXkGpKWeQZ4ILWwQW27wgLQcBjIIaDEAarH+cw7oizNpZpJzDI7zGJ5JbJyNT4t8/zHmmzPK5OQMjh9+JvGmolpO7vjhA5/xOgwKeJAYjA1EaBgFo2AUjIJRgA8AANrYPkFVJPMZAAAAAElFTkSuQmCC","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":true,"prefix":"","firstName":"Sirawut","middleName":"","lastName":"Hiran-Us","suffix":""}],"badges":[],"createdAt":"2026-01-15 13:53:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8611112/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8611112/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101753844,"identity":"d9e747ae-da21-4bce-851a-26ce7bffe24a","added_by":"auto","created_at":"2026-02-03 10:41:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":421170,"visible":true,"origin":"","legend":"\u003cp\u003eTooth preparation, group allocation and experimental procedures.\u003c/p\u003e\n\u003cp\u003eI) Teeth preparation for each group of various sizes of apical foramen (root length (RL), working length (WL). II) The weight measurement of calcium hydroxide extrusion: a) The weight of pipette tip was tared to zero prior measuring the extruded calcium hydroxide paste, b) mounting a tooth with 10-µL pipette tip, c) calcium hydroxide placement and covered with Cavit\u003csup\u003eTM\u003c/sup\u003e, calcium hydroxide paste was extruded into pipette (in the red circle), d) the extruded calcium hydroxide adhering to the root apex, e) pipette tip with extruded calcium hydroxide paste was then weighed again. III) Alkalinity diffusion distance through various sizes of apical foramen: a-b) mounting of the tooth with the perforated plastic cap, c) mounting the tooth into the plastic cuvette containing agar mixed with a universal indicator, d) examples of high-alkalinity diffusion distances at 0, 2, 6, and 9 hours.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8611112/v1/d523dd21fd2eb6cf516ce0db.png"},{"id":101645414,"identity":"80fbe5c4-8f90-451a-84d2-05e69d04c38d","added_by":"auto","created_at":"2026-02-02 08:31:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":695624,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of high alkalinity diffusion distance.\u003c/p\u003e\n\u003cp\u003eI) Measurement of root length: a) establishing calibration of a 1-mm reference length using a ruler by drawing a vertical straight line (yellow line in black circle) between two consecutive 1-mm scale marks on the adjacent ruler, starting from the midpoint of the upper black scale line to the midpoint of the next lower line and then, b) used the 1-mm from calibration to measurement of root length in agar from lower border of plastic cap to the root tip. II) Colour changing in agar after placing calcium hydroxide paste: a-b) calcium hydroxide paste placement, c) example of colour changing after calcium hydroxide extrusion at 0 hr. III) Colour scale of the pH indicator ranging from pH 8.5 to 11 as specified by the manufacturer, with the purple range (pH 10–11) defined as high alkalinity. IV) Measurement the high alkalinity diffusion distance: a) calibration of a 1-mm reference length with the same previous method then, b) used the 1-mm from calibration to measurement of high alkalinity by drawing vertical line from lower border of plastic cap to the farthest boundary at which the purple colour. The high alkalinity diffusion distance was obtained by subtracting the root length from the measured length of the purple diffusion. In this sample, the root length was 6 mm and the purple diffusion length was 18.17 mm; therefore, the high alkalinity diffusion distance was 12.17 mm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8611112/v1/bf9aca1e91d7ba0dde3ece13.png"},{"id":101645415,"identity":"18554337-03fd-4cc5-865f-61621c1edf60","added_by":"auto","created_at":"2026-02-02 08:31:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51676,"visible":true,"origin":"","legend":"\u003cp\u003eThe weight of calcium hydroxide extrusion (mg) of each group from different apical foramen sizes (Different letters indicate significant differences at p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8611112/v1/d92fa0f39bee0450c316b0ab.png"},{"id":101645412,"identity":"b7c8392e-d4c9-4cb9-b71e-f59a71b6c9bf","added_by":"auto","created_at":"2026-02-02 08:31:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109099,"visible":true,"origin":"","legend":"\u003cp\u003eHigh alkalinity diffusion distances of each apical foramen sizes at different time points (Asterisk indicate statistical significance at p \u0026lt; 0.05 between the groups).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8611112/v1/6bdfb2c23dd48e4f39514aaa.png"},{"id":101755779,"identity":"a0cbfef4-f2c9-41c0-94b7-c8b03c420da0","added_by":"auto","created_at":"2026-02-03 10:54:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2039424,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8611112/v1/3ba174a0-9b0d-4806-989b-28c8d0f7f28c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unintentional Amount of Calcium Hydroxide Extrusion and High Alkalinity Diffusion Distance: An In Vitro Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCalcium hydroxide has been widely used in root canal treatment, particularly as an intracanal medicament to enhance antimicrobial efficacy following root canal preparation and irrigation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. During calcium hydroxide placement into the prepared root canal, the medicament may extrude beyond the root canal into the periapical tissues or periapical lesions unintentionally. Many previous \u003cem\u003eex vivo\u003c/em\u003e or \u003cem\u003ein vitro\u003c/em\u003e studies have demonstrated factors influencing the extrusion of calcium hydroxide and the resulting effects on simulated periapical tissues following the extrusion beyond the root apex [\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. All forms of calcium hydroxide can extrude beyond the root apex and induce pH changes in simulated periapical areas, with the size of the apical foramen having the most significant impact on pH alterations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, calcium ions have also been found to extend beyond the root canal [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, alterations in pH within the periapical tissues surrounding the root apex can be anticipated in clinical settings.\u003c/p\u003e \u003cp\u003eThe amount of extruded calcium hydroxide and the pH of its diffusion may play an important role in post-operative pain. Furthermore, the excessive extrusion of calcium hydroxide during endodontic treatment can lead to periapical tissue damage, often presenting severe pain and, in some cases, accompanying paresthesia, as documented in case reports [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, calcium hydroxide extrusion beyond the apex is expected minimally in standard root canal treatment regarding the studies mentioned above. Limited research has examined how these pH changes impact surrounding tissues during endodontic treatment and their influence on treatment outcomes.\u003c/p\u003e \u003cp\u003eWhile previous studies focused primarily on the overall increasing pH of calcium hydroxide diffusion [\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] the distance of increasing high pH diffusion and the timeframe within which this occurs have yet to be studied. The distance over which elevated pH extends beyond the apex provides insight into which structures or cells may be affected by this high pH and for how long, particularly in the context of periapical lesions. It could also clarify whether the size of the apical foramen influences these effects [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and may have clinical implications regarding potential complications during root canal treatment, such as postoperative pain or damage to vital structures including nerves, blood vessels, and bone.\u003c/p\u003e \u003cp\u003eThus, this study aimed to investigate the amount of calcium hydroxide extruded beyond the apical foramen, as well as the patterns of alkaline pH diffusion over time following the placement of calcium hydroxide in root canals, utilizing a semi-solid gel model with varying apical foramen sizes. The null hypotheses of this study were: (i) there are no significant differences in the amount of calcium hydroxide extrusion among different apical foramen sizes, and (ii) there are no significant differences in alkaline pH diffusion among different apical foramen sizes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e The study was adhered to the Declaration of Helsinki and approved by the Human Research Ethics Committee of Thammasat University (Science), Thailand (Project no. 069/2565).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample size calculation\u003c/h2\u003e \u003cp\u003eBased on the results obtained from our pilot study, an effect size of 0.895 was added to a statistical power of 0.95 and a probability level (α) of 0.05 inputs into F test family for one-way analysis of variance (G*power 3.1 for Macintosh). To detect differences among these groups, the estimated minimum sample size for each group was determined to be five. However, to increase the power of the study, we utilized twelve samples for each experimental group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eA sound, single-rooted mandibular premolar extracted for orthodontic reasons was selected as the sample. After extraction, the teeth were stored in 0.1% thymol solution. The apical foramen of each tooth was examined to assess deviation from the root apex, which did not exceed 0.5 mm when observed under 10x magnification using a dental operating microscope (Carl Zeiss Meditec A, Jena, Germany). Periapical radiographs were taken to confirm that each selected tooth had a single root canal. A total of 63 mandibular premolars were included.\u003c/p\u003e\n\u003ch3\u003eTooth Preparation\u003c/h3\u003e\n\u003cp\u003eAccess cavities were prepared for all specimens, and canal patency was confirmed using a size 15 K-file (Kerr Dental\u0026trade;, Uxbridge, UK). The coronal portion of each tooth was removed using a 22-mm diamond disc (Edenta\u0026trade;, Hauptstrasse, Switzerland) to obtain a standardized root length of 13 mm. Canal preparation was performed using a series of rotary instruments. The working length was established at 12 mm. A master apical file of size 40 with a 0.04 taper (Mtwo\u0026trade;, Munich, Germany) was used, with irrigation performed using 2.5% sodium hypochlorite and 17% EDTA solutions. The prepared specimens were randomly allocated into five experimental groups (n\u0026thinsp;=\u0026thinsp;12 per group), with an additional three teeth serving as the negative control. The apical foramina of each group were prepared with K-files of sizes 20, 30, 45, 55, and 70 under a dental operating microscope. The negative control group underwent only root canal preparation, and the external root surfaces were then sealed with nail polish (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). All prepared teeth were dried with paper points .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCalcium Hydroxide Extrusion Measurement\u003c/h3\u003e\n\u003cp\u003eA 10-\u0026micro;L pipette tip was weighed using an analytical balance (Denver Instrument TP-214, Goettingen, Germany) to obtain a baseline weight (Fig.\u0026nbsp;1II, a). Each prepared tooth was filled with calcium hydroxide paste (Ultracal XS, South Jordan, UT, USA). The prepared tooth was mounted with a pipette tip (Fig.\u0026nbsp;1II, b). The calcium hydroxide paste was introduced into the root canal using a polymer tip positioned 2 mm short of the working length, slowly injected, and gradually withdrawn until it was 1 mm from the canal orifice The access cavity was sealed with a 1-mm-thick layer of temporary filling material (Cavit\u0026trade;, 3M, Bracknell, UK) shaped to match the access outline and gently compacted with a plastic instrument to avoid excessive pressure. Extrusion of calcium hydroxide beyond the apical foramen was then observed (Fig.\u0026nbsp;1II, c). Any extruded calcium hydroxide adhering to the root apex was carefully swabbed from the inner side of the pipette tip under a stereomicroscope (Fig.\u0026nbsp;1II, d). The pipette tip with extruded calcium hydroxide was reweighed (Fig.\u0026nbsp;1II, e).\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003ePeriapical Tissue Simulation Agar\u003c/b\u003e\u003c/div\u003e \u003cp\u003ePeriapical tissue was simulated using a 2% plain agar solution prepared by dissolving 4 g of agar powder (Himedia, Maharashtra, India) in 200 mL of boiling distilled water with continuous stirring until fully dissolved. The solution was heated at 95\u0026deg;C for 20 minutes to improve long-term stability and enhance cross-linking of the agar. The clear and uniform agar solution was then transferred to a 70\u0026deg;C water bath. The pH of the agar solution was adjusted to 7 using 1 M NaOH. Thirteen mL of universal indicator (Loba Chemic, Mumbai, India) was added to the agar solution to make 6% indicator in agar solution and the agar exhibited a green color, then poured into transparent plastic cuvettes and allowed to cool at room temperature and stored in 37\u0026deg;C with 100% humidity before using.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAlkaline pH Diffusion Distance Measurement\u003c/h2\u003e \u003cp\u003eThe same teeth used for extrusion measurement were rinsed with distilled water through a syringe needle until the outflow was clear and then reused for diffusion measurement. The teeth were labeled according to their apical foramen sizes and stored in distilled water until the experimental begin. A plastic cap was drilled at the center to fit each tooth, and the tooth\u0026ndash;cap assembly was positioned in a tube containing agar (Fig.\u0026nbsp;1III, a-c). A transparent ruler (Grande grid GCT-30, Kyoei Plastic Co., Ltd, Osaka, Japan) was positioned in close contact with the cuvette containing the agar to provide a reference scale. The root length of each specimen was determined prior to the placement of calcium hydroxide. After embedding the tooth in plain agar mixed with a universal indicator, a 1-mm calibration length was established (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, a). Once calibration was confirmed, the root length was measured by drawing the straight line from the margin of the plastic cap to the root apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, b), and recalculated the root length with the 1-mm reference obtained from the same image.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCalcium hydroxide was then placed into the root canal in the same manner as the extrusion test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e II, a, b). Calcium hydroxide paste was extruded, and the measurement of high alkalinity distance diffusion was initiated immediately, designated as 0 hr (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eII, c). Photographs were then taken at 2, 6, and 9 hours under identical environmental lighting conditions, with a background illuminated by white light The purple coloration, representing high alkalinity (pH 10\u0026ndash;11), was identified according to the pH color scale provided on the bottle of the universal indicator solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eIII).\u003c/p\u003e \u003cp\u003eAt each time point when a photograph was taken. The 1-mm calibration was reperformed (Fig.\u0026nbsp;2IV, a). Then, a straight line was drawn from the margin of the plastic cap to the farthest clearly distinguishable boundary of the purple area (Fig.\u0026nbsp;2IV, b), The diffusion distance of high alkalinity at each time point was calculated by subtracting the initial root length. During the diffusion experiment, the specimens were kept at 37\u0026deg;C under 100% humidity. All images were measured by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIntra-observer reliability\u003c/h3\u003e\n\u003cp\u003eAlkaline pH diffusion distance measurements were performed by a single operator on 30 randomly selected samples and repeated after a three-week interval. Intra-observer reliability was assessed using the intraclass correlation coefficient (ICC) employing a two-way mixed-effects model with absolute agreement.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003ePearson correlations were used to define the relationship between the apical foramen size and the amount of extruded calcium hydroxide. Because the data were not normally distributed the Kruskal-Wallis test was employed to compare the weights of calcium hydroxide extruded beyond the apical foramen among different apical foramen sizes, as well as the diffusion distances of calcium hydroxide over time of different pH levels. Dunn\u0026rsquo;s test was performed for post hoc comparison. All statistical analyses were performed using SPSS software (SPSS Statistics 25.0, IBM Corp, Armonk, NY), with a significance level set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe amount of calcium hydroxide extruded beyond the apical foramen showed a significant positive correlation with the size of the apical foramen. Pearson correlation (r)\u0026thinsp;=\u0026thinsp;0.682 with p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Specifically, larger apical foramen sizes, such as 70 and 55, demonstrated a significantly greater extrusion of calcium hydroxide compared to smaller sizes, such as 20 and 30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No extrusion of calcium hydroxide was detected in the negative control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe diffusion distances of high alkalinity increased over time. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (mm.) of diffusion distances of high alkalinity was showed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The increasing trend was showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. At two hours, the high alkalinity diffused more than 10 mm in all apical foramen sizes with no significant differences among the groups. However, at six hours, the specimens with an apical foramen size of 20 exhibited a significant shorter diffusion distance than those with larger apical foramina. At nine hours, no significant differences in diffusion distance were observed among all apical foramen sizes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHigh alkalinity diffusion distances for each apical foramen sizes at different time points\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eTime points\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0 Hr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 Hr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 Hr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 Hr\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForamen size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (mm.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (mm.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (mm.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (mm.)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27.04\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27.94\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eAsterisk indicate statistical significance at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 between the groups in the same column\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExcellent intra-observer reliability was observed (ICC\u0026thinsp;=\u0026thinsp;0.993; 95% CI: 0.986\u0026ndash;0.997).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study investigated the effect of apical foramen sizes on the amount of calcium hydroxide extrusion and high alkalinity diffusion in a simulated periapical tissue model. The results demonstrated significant differences among the groups. Therefore, the first null hypotheses were rejected. While high alkalinity diffusion distances at the end of the studies showed no statistical differences thus the second null hypothesis was accepted.\u003c/p\u003e \u003cp\u003eThe heterogeneous structure of periapical lesions, composed of microbial elements, host cells and extracellular matrixes, poses challenges for simulating such conditions. These components are distributed or aggregated based on their function in walling off infections and preventing the spread of pathogens from the root canal system into the jawbone [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, this study aims to evaluate the diffusion distance of alkalinity over time, rather than focusing on the magnitude of pH changes, to elucidate the behaviour of high alkalinity diffusion in periapical tissues, particularly in cases of apical granuloma or periapical abscess\u0026mdash;common pathological presentations resulting from inflammation or necrosis of dental pulp tissue [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous studies have investigated the alkalinity beyond the apical foramen resulting from the use of calcium hydroxide as an intracanal medicament in root canal treatment [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Most of these studies have focused on the pH values and the factors influencing these changes, such as the size of the apical foramen [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], the type of calcium hydroxide used [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and the duration of calcium hydroxide placement within the canal [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], allowing for diffusion beyond the apex over varying time intervals. Moreover, various models have been utilized in these studies, including distilled water [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], agar to simulate periapical tissue [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], real teeth [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], or artificial root canals made from pipette tip [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], glass tube [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] or metal tube [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite the variability in methodologies, the results consistently demonstrate an increase in alkalinity beyond apical foramen [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The result from this study also demonstrated the correlation with increasing the size of the apical foramen and the amount of calcium hydroxide diffusing beyond the apex. But after a period of time, the size of the apical foramen had no influence on the diffusion distance of alkalinity but increasing in a time-dependent manner.\u003c/p\u003e \u003cp\u003eThe study revealed rapid diffusion of high pH values from the root apex. The diffusion distance exceeded 10 mm within 2 hours and extended beyond 20 mm at 9 hours. Considering that periapical lesions typically range in volume from 1 mm\u0026sup3; to over 30 mm\u0026sup3;, with an average of approximately 4\u0026ndash;5 mm\u0026sup3; [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], these findings suggest that alkaline diffusion from calcium hydroxide can potentially reach the entire lesion area within a clinically relevant timeframe. It was observed that apical foramen sizes of 20 or more could result in a high pH environment (pH 10\u0026ndash;11) within the lesion within 2 hours. Biologically, such high pH levels are reported the cytotoxicity to cells, including periodontal ligament cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], osteoblasts [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], bone marrow cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and other structures within the lesion, such as nerves [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], inflammatory cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and various microorganisms especially, in biofilm [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Clinically, unintentional extrusion of materials can lead to severe complications [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], particularly in teeth adjacent to vital structures such as the mental foramen or mandibular canal. Our study demonstrated that high alkalinity can be detected up to 20\u0026ndash;30 mm from the apical foramen. The radiograph may reveal the physical extent of calcium hydroxide at the apical limit, but its alkalinity extends beyond this visible boundary. Therefore, the use of calcium hydroxide in teeth near vital structures should be approached with extreme caution. Additionally, hydraulic calcium silicate sealers, which can produce calcium hydroxide through hydration reactions, are also prone to extrusion due to their excellent flowability. Consequently, hydraulic calcium silicate sealers should also be used with caution under similar precautions even though recent study has reported high success rates in periapical healing following unintentional extrusion [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, our study demonstrated that the diffusion distance of alkalinity is time-dependent, supporting the notion that the degree of nerve injury increases with longer exposure to the irritant as contact time extends [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study employed agar as a substitute for human tissue due to its similar density to human soft tissue, as used in various experimental models, including radiation studies [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and calcium hydroxide diffusion [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Agar offers advantages over solutions in pH measurement, as it prevents rapid diffusion to equilibrium, which may underestimate pH values. Previous studies primarily used liquid media to simulate the periapical environment, which did not accurately represent conditions within the periodontal ligament or alveolar bone, particularly in cases of apical periodontitis, periapical granuloma or granulation tissue [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The simulated environments failed to closely mimic the granulation tissue surrounding the root apex. Using semi-solid gel media could provide results more aligned with clinical conditions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the method of obtaining pH values from such media had limitations, as it involved extracting the gel and measuring the pH, which only reflected specific points in time [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The diffusion of alkaline pH from extruded calcium hydroxide should vary depending on the distance from the root apex over different time intervals. Additionally, the shape of the container allows diffusion to extend primarily in a longitudinal direction rather than dispersing in all directions. Results from this study are consistent with previous finding [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], demonstrating that the amount of calcium hydroxide diffusing correlates with the size of the apical foramen. However, differences in pH values observed across studies [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] may stem from variations in measurement techniques.\u003c/p\u003e \u003cp\u003eSince this study relied on colour changes to indicate the boundary of areas of the level of alkalinity which followed the colour scale labelled on the bottle of the universal pH indicator as a reference for pH interpretation. The preliminary observations revealed that once the alkaline diffusion occurred, the clearly distinguishable colours were mainly purple and turquoise. According to the manufacturer\u0026rsquo;s colour chart, the purple range corresponds to pH 10 and 11. In practice, however, it was not possible to distinguish a definite boundary between these two shades of purple within the agar medium. Therefore, both pH 10 and 11 were collectively defined as representing high alkalinity in this study. The dark blue colour, corresponding to pH 9.5, appeared only as a very narrow zone or was absent in some specimens and usually blended indistinctly with the adjacent purple area. As this could lead to unreliable measurement, pH 9.5 was excluded from diffusion distance analysis. Conversely, although the turquoise region (pH 8.5\u0026ndash;9) was clearly visible, it was not included in the diffusion analysis, as this zone appeared only as a narrow band surrounding the purple area and was rapidly replaced by the higher-alkalinity purple colour over time.\u003c/p\u003e \u003cp\u003eThe high alkalinity diffusion distance was not measured directly from the root apex to the boundary of the purple colour zone, because the extruded calcium hydroxide paste obscured the apex once it flowed beyond the root canal. Therefore, the diffusion had to be calculated by subtracting the root length which measured before calcium hydroxide placement, from the total diffusion distance of the purple colour.\u003c/p\u003e \u003cp\u003eThe study was limited by its experimental timeframe, which was restricted to 9 hours. This limitation was due to some groups exhibiting a plateau in colour change, likely resulting from the height restriction of the plastic cuvettes (35 mm). Teeth with larger apical foramina experienced this limitation more prominently, leading to the decision to focus on a 9-hour observation period.\u003c/p\u003e \u003cp\u003eApical foramina of size 15 or smaller were not included. A pilot study demonstrated that both the high alkalinity diffusion distance and the extrusion experiment produced inconsistent results at size 15 (data not shown). This limitation suggests that apical foramina of size 15 and smaller may not allow predictable hydroxyl ion diffusion or detectable extrusion using the current model, therefore were excluded to ensure methodological reliability. The use of different apical foramen sizes in this study may be applicable to teeth with originally large apical foramina, such as immature teeth, as well as cases with iatrogenic apical enlargement resulting from mechanical instrumentation.\u003c/p\u003e \u003cp\u003eAs this study was conducted under in vitro conditions, certain limitations remain. Granulation tissue, characterized by high vascularity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], plays a critical role in nutrient supply and tissue repair. The blood flow in granulation tissue is approximately 2\u0026ndash;10 times greater than in normal tissues in skin wound [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which might reduce the duration of high pH levels within periapical lesions. Moreover, the inherent pH of periapical tissues, which ranges from 6 to 7.3 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], might further buffer the high alkalinity. In addition, certain proteins and ions dispersed within the extracellular matrix might also have exhibited these effects also. Consequently, while high alkalinity from calcium hydroxide may diffuse extensively within the lesion and exert cytotoxic effects, this effect is likely transient, allowing adjacent cells to proliferate or migrate to repair or clear necrotic debris like pulpal wound healing [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The delivery of calcium hydroxide in this study was performed twice independently by a single operator to maintain consistency in the technique. However, human error is unavoidable among clinicians, and this may have contributed to variations in the amount of calcium hydroxide extrusion.\u003c/p\u003e \u003cp\u003ePeriapical lesions are generally expected to be devoid of pathogens due to the accumulation of immune cells. However, apical foramina or accessory canals may harbour microbial biofilms. Pathogens such as \u003cem\u003eActinomyces\u003c/em\u003e, \u003cem\u003eFusobacterium\u003c/em\u003e, and \u003cem\u003ePrevotella\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] have been found in periapical tissues, particularly in cases of periapical abscesses or sinus tracts [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Although high alkalinity (pH 10\u0026ndash;11) can eradicate these pathogens, the transient nature of such high pH levels due to the vascularity of granulation tissue may explain the limited efficacy of calcium hydroxide in some cases. Furthermore, bacteria organized in biofilm structures exhibit resistance to high alkalinity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Previous study [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] have shown that the overall healing pattern in lesions overfilled with calcium hydroxide was not significantly different from those in which overfilling did not occur. These outcomes may result from ensuring complete delivery of calcium hydroxide to the full working length to maximize disinfection, rather than keeping the material short of the apex solely out of concern for extrusion. However, direct physical contact between calcium hydroxide and vital structures should be avoided. Thus, interpretation of the results should be applied with caution.\u003c/p\u003e \u003cp\u003eIn conclusion, the amount of calcium hydroxide extrusion from the root canal is influenced by the size of the apical foramen. Apical foramina of size 20 or larger, either naturally occurring or created by iatrogenic apical perforation, demonstrated an increased tendency for calcium hydroxide extrusion. The high alkalinity environment around the apical foramen was then expected within few hours and extended beyond its physical location. Even slight to moderate extrusion may cause irritation or damage to the periapical tissues and surrounding vital structures; therefore, the use of calcium hydroxide should be approached with caution in cases with large apical foramina. However, the high alkalinity would be transient due to high vascularity and acidity of granulation tissue. It is need for further studies to elucidate the exact alkalinity of periapical lesion \u003cem\u003ein vivo\u003c/em\u003e after placing calcium hydroxide into the root canal and also investigate the cellular responses to understanding the healing process under alkaline condition.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was deemed unnecessary by the Human Research Ethics Committee of Thammasat University (Science), Thailand (Project no. 069/2565).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare have no conflicts of interest relevant to this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Faculty of Dentistry, Thammasat University Fund.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP.A.:\u0026nbsp;\u003c/strong\u003eProject Administration, Conceptualization, Methodology, Investigation, Data Curation, Formal Analysis, Funding Acquisition, Visualization, Resources, Writing-Original Draft Preparation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.C.:\u003c/strong\u003e Conceptualization, Methodology, Investigation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS.A.:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Investigation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP.P.:\u003c/strong\u003e Writing-Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.J.:\u0026nbsp;\u003c/strong\u003eWriting-Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS.H.:\u0026nbsp;\u003c/strong\u003eConceptualization, Data Curation, Formal Analysis, Writing-Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to express their sincere gratitude to Dr. Paopanga Montanapisut and Ms. Harikarn Mungpayabarn for their technical expertise and assistance with the laboratory procedures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60 cases of endodontic therapy. Int Endod J. 1985;18(1):35\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHosoya N, Takahashi G, Arai T, Nakamura J. Calcium concentration and pH of the periapical environment after applying calcium hydroxide into root canals in vitro. J Endod. 2001;27(5):343\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobert GH, Liewehr FR, Buxton TB, McPherson JC. 3rd. Apical diffusion of calcium hydroxide in an in vitro model. J Endod. 2005;31(1):57\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZmener O, Pameijer CH, Banegas G. An in vitro study of the pH of three calcium hydroxide dressing materials. Dent Traumatol. 2007;23(1):21\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBallal NV, Shavi GV, Kumar R, Kundabala M, Bhat KS. In vitro sustained release of calcium ions and pH maintenance from different vehicles containing calcium hydroxide. J Endod. 2010;36(5):862\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNadar A, Muliya VS, Pai S, Pentapati KC. A comparative evaluation of calcium ion release and pH change using calcium hydroxide nanoparticles as intracanal medicament with different vehicles - An in vitro study. J Conserv Dent. 2023;26(1):47\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahravan A, Jalali S, Mozaffari B, Pourdamghan N. Overextension of nonsetting calcium hydroxide in endodontic treatment: literature review and case report. Iran Endod J. 2012;7(2):102\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Bruyne MA, De Moor RJ, Raes FM. Necrosis of the gingiva caused by calcium hydroxide: a case report. Int Endod J. 2000;33(1):67\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStashenko P. Role of immune cytokines in the pathogenesis of periapical lesions. Endod Dent Traumatol. 1990;6(3):89\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamachandran Nair PN, Pajarola G, Schroeder HE. Types and incidence of human periapical lesions obtained with extracted teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996;81(1):93\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicucci D, Mannocci F, Ford TR. A study of periapical lesions correlating the presence of a radiopaque lamina with histological findings. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101(3):389\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanomyong D, Arayasantiparb R, Sirakulwat K, Kasemsuwan J, Chirarom N, Laopan N, Lapthanasupkul P. Association between Clinical/Radiographic Characteristics and Histopathological Diagnoses of Periapical Granuloma and Cyst. Eur J Dent. 2023;17(4):1241\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNatkin E, Oswald RJ, Carnes LI. The relationship of lesion size to diagnosis, incidence, and treatment of periapical cysts and granulomas. Oral Surg Oral Med Oral Pathol. 1984;57(1):82\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SHPK, Yoo HK, Shin SH. Biological effects of pH concentration on cultured human periodontal ligament cell activity in vitro. J Periodontal Implant Sci. 1995;25(3):540\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArnett T. Regulation of bone cell function by acid-base balance. Proc Nutr Soc. 2003;62(2):511\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohn DH, Sarmadi M, Helman JI, Krebsbach PH. Effects of pH on human bone marrow stromal cells in vitro: implications for tissue engineering of bone. J Biomed Mater Res. 2002;60(2):292\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLardner A. The effects of extracellular pH on immune function. J Leukoc Biol. 2001;69(4):522\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvans M, Davies JK, Sundqvist G, Figdor D. Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Int Endod J. 2002;35(3):221\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChavez de Paz LE. Redefining the persistent infection in root canals: possible role of biofilm communities. J Endod. 2007;33(6):652\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDistel JW, Hatton JF, Gillespie MJ. Biofilm formation in medicated root canals. J Endod. 2002;28(10):689\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGluskin AH, Lai G, Peters CI, Peters OA. The double-edged sword of calcium hydroxide in endodontics: Precautions and preventive strategies for extrusion injuries into neurovascular anatomy. J Am Dent Assoc. 2020;151(5):317\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin Y, Roh BD, Kim Y, Kim T, Kim H. Accidental injury of the inferior alveolar nerve due to the extrusion of calcium hydroxide in endodontic treatment: a case report. Restor Dent Endod. 2016;41(1):63\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontenegro Fonseca J, Rangel Palmier N, Amaral-Silva GK, Aristizabal Arboleda LP, Affonso Almeida JF, de Goes MF, Agustin Vargas P, Ajudarte Lopes M, Santos-Silva AR. Massive extrusion of calcium hydroxide paste containing barium sulphate during endodontic treatment. Aust Endod J. 2020;46(2):257\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBamrungwong J, Ratisoontorn C, Hiran-Us S, Sinsareekul C. Outcomes and prognostic factors of endodontically treated teeth with unintentional root canal sealer extrusion: A retrospective cohort study. J Dent. 2025;158:105804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRice DD, Grandhi A, Roque-Torres GD, Oyoyo U, Bakland LK. The Effect of Calcium Hydroxide Pastes on Isolated Vital Nerve Fibers. J Endod. 2024;50(3):355\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJawli A, Aldehani W, Nabi G, Huang Z. Tissue-Mimicking Material Fabrication and Properties for Multiparametric Ultrasound Phantoms: A Systematic Review. Bioeng (Basel) 2024;11(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRabie M, Ahmed M, Mann A, Singhal S. Ultrasound Tissue Mimicking Materials Using 2% Agar based phantom. The International Conference on Electrical Engineering 2014(9):1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerar AM, Bondor CI, Matroş L, C\u0026acirc;mpian RS. Radiological, histological and immunohistochemical evaluation of periapical inflammatory lesions. Rom J Morphol Embryol. 2016;57(2):419\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoi C, Negruțiu ML, Roi A, Riviș M, Luca RE, Raica M, Ceaușu RA, Motofelea AC, Gaje PN. Human Periapical Odontogenic Granulomas: Aspects of Microvessel Density (MVD), Heterogeneity of Blood Vessels and Mast Cells Density (MCD). Biomedicines 2023;11(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRendell MS, Milliken BK, Finnegan MF, Finney DA, Healy JC. The skin blood flow response in wound healing. Microvasc Res. 1997;53(3):222\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRendell MS, Johnson ML, Smith D, Finney D, Capp C, Lammers R, Lancaster S. Skin blood flow response in the rat model of wound healing: expression of vasoactive factors. J Surg Res. 2002;107(1):18\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCormick JE, Weine FS, Maggio JD. Tissue pH of developing periapical lesions in dogs. J Endod. 1983;9(2):47\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNekoofar MH, Namazikhah MS, Sheykhrezae MS, Mohammadi MM, Kazemi A, Aseeley Z, Dummer PM. pH of pus collected from periapical abscesses. Int Endod J. 2009;42(6):534\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchr\u0026ouml;der U. Effects of calcium hydroxide-containing pulp-capping agents on pulp cell migration, proliferation, and differentiation. J Dent Res 1985;64 Spec 541-8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBronzato JD, Bomfim RA, Hayasida GZP, C\u0026uacute;ri M, Estrela C, Paster BJ, Gomes B. Analysis of microorganisms in periapical lesions: A systematic review and meta-analysis. Arch Oral Biol. 2021;124:105055.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiger R, Manncke B, Werner H, L\u0026ouml;st C. Microbial flora of sinus tracts and root canals of non-vital teeth. Endod Dent Traumatol. 1995;11(1):15\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBr\u0026auml;ndle N, Zehnder M, Weiger R, Waltimo T. Impact of growth conditions on susceptibility of five microbial species to alkaline stress. J Endod. 2008;34(5):579\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaliskan MK, Sen BH. Endodontic treatment of teeth with apical periodontitis using calcium hydroxide: a long-term study. Endod Dent Traumatol. 1996;12(5):215\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8611112/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8611112/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHigh alkalinity diffusion and its timeframe have not been thoroughly studied. This study aimed to evaluate the amount of unintentional calcium hydroxide extrusion and to measure the diffusion distance of high alkalinity from different apical foramen sizes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSixty-three mandibular premolars were divided into 5 groups based on apical foramen sizes of 20, 30, 45, 55, and 70 (n\u0026thinsp;=\u0026thinsp;12 each) along with three negative controls. For calcium hydroxide extrusion measurement, pipette tips were used to cover the root apex, allowing the extruded calcium hydroxide to be collected in them. Then, calcium hydroxide was delivered into the canal using a polymer tip in the same manner across all groups. The quantity of calcium hydroxide extrusion was determined by comparing the tip weight before and after calcium hydroxide placement. To assess high alkalinity diffusion (pH 10\u0026ndash;11), the same teeth were rinsed and reused by embedding them in 2% agar containing universal pH indicators followed by calcium hydroxide delivery. The extent of high alkalinity diffusion was measured by color extension at 0, 2, 6, and 9 hours. Kruskal-Wallis test was employed to compare the weights of calcium hydroxide extruded beyond the apical foramen among different apical foramen sizes, as well as the diffusion distances of calcium hydroxide over time. Pearson correlations were used to define the relationship between the apical foramen size and the amount of extruded calcium hydroxide.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe extruded calcium hydroxide was positively correlated with the apical foramen sizes. The high alkalinity distances increased over time. Within two hours, all sizes of apical foramina demonstrated high alkalinity diffusion distances over 10 mm. At nine hours, there was no significant difference among the groups.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe extent of medicament extrusion was influenced by the size of the apical foramen. High alkalinity can develop within a few hours and may extend several millimeters beyond the physical presence of the medicament. Understanding the relationship between apical foramen size and high alkalinity diffusion aids in assessing the potential for tissue irritation or post operative pain and helps guide safer clinical practices when using calcium hydroxide medicaments.\u003c/p\u003e","manuscriptTitle":"Unintentional Amount of Calcium Hydroxide Extrusion and High Alkalinity Diffusion Distance: An In Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 08:31:06","doi":"10.21203/rs.3.rs-8611112/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-13T12:08:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T20:30:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T13:53:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T05:58:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288188612581683805094795110865657549166","date":"2026-02-02T11:06:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1257062065440841611372337435321815033","date":"2026-02-01T05:22:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203566208451147258120641072991226432543","date":"2026-01-30T01:52:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-29T19:26:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-29T14:03:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-20T09:03:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T08:08:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2026-01-20T07:54:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3e9d26e-58d3-4f77-ba05-23fc5efe453e","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T13:25:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 08:31:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8611112","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8611112","identity":"rs-8611112","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}