The Effects of Mineral Trioxide Aggregate and Second-Generation Autologous Growth Factor on Pulpotomy

The Effects of Mineral Trioxide Aggregate and Second-Generation Autologous Growth Factor on Pulpotomy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Effects of Mineral Trioxide Aggregate and Second-Generation Autologous Growth Factor on Pulpotomy Ayca Kurt, Ahter Sanal Cıkman, Emre Balaban, Zeynep Gumrukcu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4165836/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Aug, 2024 Read the published version in BMC Oral Health → Version 1 posted 13 You are reading this latest preprint version Abstract The aim of our study is to see the effect of Mineral Trioxide Aggregate (MTA), bioactive endodontic cement, and Concentrated Growth Factor (CGF), second-generation autologous growth factor, on pulpotomy-induced pulp inflammation. The study used the maxillary anterior central teeth of thirty-six young male Sprague Dawley rats. In total, 72 maxillary central teeth were included. We randomly assigned 48 teeth to two groups (12 rats/group; 24 teeth/group) based on the capping material (MTA or CGF). Two subgroups per group (12 teeth/group) were formed based on the time following pulpotomy (two weeks and four weeks). Compared to the control group, MTA and CGF applications decreased the formation of inflammation and edematous areas. We also observed that it decreased Nuclear Factor kappa B/P65 (NF-kB/P65) and Tumor Necrosis Factor (TNF)-α positivities. We determined fewer cells showing NF-kβ/p65 positivity in the MTA treatment group compared to the CGF group. Moreover, we determined fewer cells showing NF-kβ/p65 positivity on day 28 after the MTA application compared to day 14 after the MTA application. In the CGF group, both histological damage scores and TNF positivity decreased. This study has established that, second-generation autologous growth factors have a protective effect in treating inflammation after pulpotomy. Concentrated growth factor Inflammation Mineral trioxide aggregate Pulpotomy NF-kB/p65 TNF-α Figures Figure 1 Figure 2 Figure 3 Introduction Caries is known to be a preventable chronic health problem; however, comprehensive global reports [ 1 ] estimate that there were 573 million children with untreated caries in 2015 [ 2 ]. Caries most commonly affect young children; however, it is a lifelong condition that is also encountered during adolescence and adulthood. Therefore; the treatment of caries, especially deciduous caries, holds great importance [ 3 ]. The pulp must be preserved during the treatment processes of deciduous molar teeth' complex root canal systems [ 4 , 5 ] Exposure of pulp tissue may be caused by caries, as well as iatrogenic causes, or traumatic injuries. Pulpotomy is widely used on deciduous teeth as a successful treatment in pedodontics [ 6 , 7 ]. It is an important pulp treatment that includes the removal of a part of the coronal pulp tissue and the capping of the radicular tissue by materials that prevent further injury to the pulp as well as allowing and promoting healing [ 8 ]. Several materials have been used so far to stimulate the formation of a dentin bridge through the dentinogenic potential of pulp cells [ 9 ]. The increasing focus on biocompatible materials has generated a wider variety of Mineral Trioxide Aggregate (MTA) choices in pulpotomy. MTA, which shields periradicular or dental pulp tissues may preserve pulp vitality and stimulate its recovery [ 10 ]. Despite its high success rates, MTA has disadvantages such as pulp chamber obliteration, change of tooth color, pulp degeneration, and a high cost [ 11 ]. Thus, biocompatible treatments aimed at protecting pulp vitality and increasing tooth survival must be developed [ 7 ]. Concentrated growth factor (CGF), which is among second-generation autologous growth factors, is a recently discovered platelet concentrate that is usually obtained from autologous venous blood and produces a larger and more dense fibrin matrix at levels greater than previously known platelet concentrates [ 12 ]. Serving as a scaffold material, CGF also produces many growth factors where it is applied [ 13 ]. Although previous research includes a multitude of studies on CGF, its direct use as a pulpotomy material has not been examined [ 12 , 14 – 18 ]. Our study aims to shed light on the alternative materials that may be used in the pulpotomy treatment soon of endodontic procedures by investigating the potential effects of the inflammation that develops after the use of MTA, which is a bioactive endodontic cement, and of CGF, which is a second generation autologous growth factor, as pulpotomy materials on the TNF-α and Nf-kB/p65 pathways. Materials and Methods Ethics Approval The required ethics committee decision for the study was granted by Recep Tayyip Erdogan University, Faculty of Medicine, Animal Research Ethics Committee (approval number: 2020/47). I confirmed that all methods are reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Experimental Design The study used the maxillary anterior central teeth of thirty-six young male rats (Sprague Dawley, weighing between 250 and 350 g). In total, 72 maxillary central teeth were included. We randomly assigned 48 teeth to two groups (12 rats/group; 24 teeth/group) based on the capping material (MTA or CGF). Two subgroups per group were formed (12 teeth/group) based on the time following pulpotomy (two weeks and four weeks). The central teeth of the 12 animals assigned to the control group were not manipulated in any way, both in the two-week group and in the four-week group; thus, these teeth served as the control group for histological identification. Throughout the experiment, we housed the animals in polypropylene cages (with four animals per cage) in an experimental animal unit with a 12:12 hour light-dark cycle, 55–60% humidity and room temperature, as well as unlimited availability of water and food pellets (Nutrilabor; Guabi, Campinas, SP, Brazil). The cage bedding was renewed three times per week at minimum. Operational procedure The animals received anesthesia through the intramuscular injection of a mixture of 50 mg/kg (i.p) ketamine hydrochloride (Ketalar ®, Eczacıbaşı Parke-Davis, Istanbul, Turkey) and 10 mg/kg (i.p) Xylazine HCl (Alfazyne ®, Alfasan International B.V. Woerden, The Netherlands) and immobilized on a surgical board. For pulpotomy, the conventional technique was followed. The permanent maxiller right and left central incisors were isolated according to the method previously described [ 17 ] and adapted for this experiment. In summary, a round diamond bur (#1011, Dentsply Indústria e Comércio Ltda, Petropólis, RJ, Brazil) was used together with a high-speed rotary handpiece to create an opening for pulpotomy and a 1 mm cavity, equal to the diameter of the diamond wheel used, was opened in the middle third of the crown in the palatal part of the incisors. The coronal pulp tissue was removed using an excavator (#5, SS White, Dental Objects Ltda., Juiz de Fora, MG, Brazil). The bleeding in the pulp chamber was controlled using saline-soaked cotton pellets. During the performance of this technique, loupes with illumination ( Loupes, YDL-EW, 2.5x) were used to increase the field of view. Prior to the pulpotomy procedure, the mucosa of the central teeth were subjected to infiltrative local anesthesia with articaine (ULTRAVER D-S), 80 mg articaine hydrochloride, and 0,01819 mg epinephrine bitartrate. Animals in the control group (CG) were also divided into two subgroups in the same way as in the experimental groups (two weeks and four weeks). In consideration of the standardization of the study, animals in this group underwent immobilization with appropriate anesthetics and local anesthesia even though no dental procedures were performed. In the experimental groups, either MTA or CGF was used as the pulpotomy material. In the MTA (PROROOT, WHITE, Dentsply, Konstanz, Germany) group (MTAG), a 1:1 mixture of MTA powder and distilled water was placed in the pulp chamber where bleeding control was performed, using an MTA carrier (MAP One, Produits Dentaires SA, Switzerland). In the CGF group (CGFG), 4 ml of blood was collected from the jugular vein of each rat. The blood was placed in CGF kit tubes with 0.025M calcium chloride (S6M, STR Biotechnologies, Çorum, Turkey). The tubes were placed in a centrifuge device designed specifically for CGF (Labfuge, STR Biotechnologies, Çorum, Turkey). This device was operated on a standard procedure of centrifugation that automatically increased the speed and then slowed down for 45 seconds as follows: 2700 rpm (735 g) for 2 minutes, 2400 rpm (580 g) for 4 min, 2700 rpm (735 g) for 4 min. and 3000 rpm (905 g) for 3 min. As a result of centrifugation, four layers formed within the tubes. The topmost fourth layer consisted of serum, the second layer (buffy coat) and the third layer (unipotent stem cell) composed of the CGF layer of a gel-like consistency, and the bottom layer was the first layer consisting of red blood cells. After the serum layer was removed, the CGF layer of gel-like consistency was extracted from the tube using forceps, cut off from the red blood cell layer underneath, and placed onto the exposed pulp tissue using a sterile handpiece (Medisana double-ended dental spatula). Cavities in both groups were restored and capped using conventional glass ionomer cement (Ketac-molar, ESPE Platz, Seefeld, Germany). The animals were kept under observation during healing. Potential changes in the feeding and drinking cycles as well as the behavioral profile were evaluated. After the experimental period (2nd and 4th weeks after pulpotomy), the rats were sacrificed by the administration of sevoflurane (sevorane 100%, Abbott, United States of America). Histopathological analysis procedure Maxillary tissue specimens obtained from the rats were fixed by being kept in 10% phosphate-buffered neutral formalin (Sigma-Aldrich, Germany, Ph:7.4) for 24 hours. Subsequent to fixation, these specimens were decalcified by being kept in Morse’s solution for 24 hours. In the following stages, the specimens were subjected to a dehydration procedure with ethanol series of gradually increasing concentration (50%, 70%, 80%, 90%, 96%, and 100%, Merck GmbH, Germany) using an autotechnicon device (Citadel 2000, ThermoScientefic, Germany), clearing, and paraffin inclusion (Merck GmbH, Germany) procedures. Maxillary tissue specimens subjected to paraffin inclusion were embedded in hard paraffin (Merck GmbH, Germany) and sections of 4–5 µm were cut using a rotary microtome (RM2525, Leica Biosystems, Germany). The sections were stored in an incubator and passed through two series of xylol (Merck GmbH, Germany). Following the deparaffinization stage, they were stained with Harris hematoxylin (Merck, Germany) and Eosin G (Merck, Germany) stains with the use of a staining device. Stained sections were mounted with entellan (Merck GmbH, Germany) and photographed under a light microscope with an attached digital camera (BX51, Olympus Corp., Japan). Immunohistochemistry (IHC) analysis procedure In order to examine the maxillary inflammation induced by the invasive procedure, TNF-Alpha (ab220210, Abcam, UK) and NF-kß/P65 (ab16502, Abcam, UK) primary antibody kits and corresponding secondary antibody kits (Goat Anti-Rabbit IgG H&L (HRP)) and (ab205718, Abcam, UK), respectively) were used. Sections of 1–2 µm thickness were obtained from maxillary tissue sections using a rotary microtome (Leica Biosystem, RM2525, Germany). The obtained maxillary tissue sections were incubated with primary and secondary antibodies for one hour according to the manufacturer’s guidelines in the catalog and stained using the immunoperoxidase method with an IHC staining device (Bond Max III, Leica Systems, Germany). Finally, they were counterstained with Harris hematoxylin (Merck, Germany). Semi-Quantitative analysis Harris hematoxylin (Merck GmbH, Germany) and Eosin G (Merck GmbH, Germany) -stained maxillary tissue sections were given a histopathological damage score (HDS) in accordance with the studies that evaluated the indications caused by invasive procedures of the maxilla concerning edema, inflammation, fibrosis, cartilage and bone callus formation as shown in Table 1 . From each preparation, 20 different randomly selected sites were scored by a histopathologist under a light microscope using x21 and x20 objectives. Specimens of maxillary tissue incubated with TNF-α and NF-kB/p65 primary antibodies were scored as shown in Table 2 . From each preparation, 20 different randomly selected sites were scored by a histopathologist under a light microscope using x40 objectives. The histopathologists were blind to the experimental groups. Table 2 Semi-Quantative Analysis Scoring Methods. Findings Score 0 Less than 5% 1 Between 6%-25% 2 Between 26%-50% 3 More than 50% Statistical analysis Data obtained from the semi-quantitative analysis were evaluated for normality of distribution using the Skewness-Kurtosis values, Q-Q plot, Levene’s test, and the Shapiro-Wilk test on the SPSS 20 (IBM, USA) statistics software. For non-parametric data, median-25% and 75% interquartile range were calculated and analyzed with the Kruskal Wallis test followed by Tamhane’s T2 test. P value < 0.05 was accepted as significant. Results Histopathological Findings In the evaluation of the maxillary tissue sections stained with Harris hemotoxylin and Eosin G, odontoblasts, dentin, pulp, alveolar bone and periodontal ligaments of the normal structure were present in maxillary tissue sections obtained from the two-week CG and the four-week CG (Fig. 1 a-b, respectively; HDS: 0.5(0–1)), 1(0–1)). In contrast, sections from the two-week CGF treatment group showed extensive inflammation accompanied by edematous areas (Fig. 1 c, respectively; HDS: 5(4–5)). In maxillary tissue sections from the four-week CGFG, a decrease was found in the extensive edematous areas and infiltrative areas along with fibrous callus formation (Fig. 1 d, respectively; HDS: 4(3–4)). In the two-week MTAG, a decrease in infiltrative and edematous areas was observed along with the formation of cartilage callus at places ((Fig. 1 e, HDS: 2.5(3–4)). In the four-week MTAG, a decrease in infiltrative and edematous areas was observed along with typical odontoblasts, dentin, periodontal ligaments, and alveolar bone structures (Fig. 1 e, HDS: 2(1–2)). The two-week and four-week subgroups of the control group were not significantly different concerning HDS scores (Fig. 1 a-b, Respectively; HDS 0.5(0–1), 1(0–1)). In contrast, we determined a higher HDS score in the two-week CGFG (HDS:5(4–5) compared to the two-week (HDS 0.5(0–1)) and four-week control groups (1(0–1)) (Table 3 ). Similarly, we determined a higher HDS score in the four-week CGFG (HDS:4(3–4) compared to the two-week (HDS 0.5(0–1)) and four-week (1(0–1)) CG groups (Table 3 ). In contrast, we determined lower HDS scores in the two-week (HDS:2.5(3–4)) and four-week (HDS: 2(1–2)) MTAG compared to the two-week (HDS:5(4–5) and four-week (HDS:4(3–4) CGFG (Table 3 ). Table 3 Histopathological Damage Score Results HDS, (median-25%-75% interquartile range) Group Oedematous Area Inflammation HDS CG 2W 0(0–1) 0(0-0.5) 0.5(0–1) CG 4W 0.5(0–1) 0(0-0.5) 1(0–1) CGFG 2W 2(2–3) a,c 2(2–3) a,b 5(4–5) a,b CGFG 4W 1.5(1–2) a,d,e 3(2-2.5) a,b,i 4(3–4) a,b,i MTAG 2W 2(2–2) a,c,f 2(1–2) a,b,j 2.5(3–4) a,b,g MTAG 4W 1(1–1) b,c,g,h,i 1(0–1) a,b,g,k 2(1–2) a,m,g,k,i a p =0.000; versus to CG 2W, b p =0.038; versus to CG 4W, c p =0.000; versus to CG 4W, d p =0.001; versus to CG 4W, e p =0.044; versus to CGFG 2W, f p =0.011; versus to CGFG 2W, g p =0.000; versus to CGFG 2W, h p =0.046; versus to CGFG 4W, i p =0.000; versus to MTAG 2W, j p =0.001; versus to CGFG 2W, k p =0.000; ; versus to CGFG 4W, j p =0.002; versus to CGFG 2W, m p =0.012;versus to CGFG 2W, Kruskal Wallis/Tamhane T2 Test Immunohistochemical Analysis On evaluation of the maxillary tissue sections incubated with the TNF-α primary antibody under a light microscope, we observed that immune-negative cells were common in the maxillary tissues of the two-week and four-week control groups (Fig. 2 a and 2 b, respectively; TNF-α score: 0(0-0.5), 0(0–1)). In contrast, we determined an increase in the number of cells showing TNF-alpha positivity in sections from the two-week CGFG and the four-week MTAG compared to those from the two-week and four-week control groups (Fig. 2 c, respectively; 3 TNF-α score : (2–3), 2(2–2) p = 0.000, p = 0.000). On the other hand, we determined a decrease in the number of cells showing TNF-α positivity in the two-week MTAG compared to the two-week CGFG (Fig. 2 b, d, respectively; TNF-α score: 3(2–3), 1.5(1–2), p = 0.000). Similarly, we observed fewer cells showing TNF-alpha positivity in the maxillary tissue sections from the four-week MTAG compared to the four-week CGFG (Fig. 2 c,e, respectively; TNF-α score: 2(2–2), 1(1–1), p = 0.001). When the two-week and four-week MTAG subgroups were compared to each other about the number of cells showing TNF-α positivity, we found a significant decrease in the number of cells showing TNF-α positivity in the two-week MTAG subgroup compared to the four-week MTAG subgroup (Fig. 2 d,e, respectively; TNF-α score: 1.5(1–2), 1(1–1), p = 0.015). In sections of maxillary tissue incubated with TNF-alpha primary antibody, a decrease in cells showing immune positivity is observed (Fig. 2 f; TNF-alpha positivity score: 1(1–1)) (Table 4 ). Table 4 Semi-Quantitative Analysis Results (median-25%-75% interquartile range) Group TNF-α Positivity Score NF-kβ/p65 Positivity Score CG 2W 0(0-0.5) 0(0–1) CG 4W 0(0–1) 0(0–1) CGFG 2W 3(2–3) a,b 2(2–3) a,b CGFG 4W 2(2–2) a,b 2(2–2) a,b MTAG 2W 2(1.5-2) a,b,c 1.5(1–2) a,b,c MTAG 4W 1(1–1) a,b,c,d,e 1(1–2) b,c,e,g a p =0.000; versus to CG 2W, b p =0.000; versus to CG 4W, c p =0.000; versus to CGFG 2W, d p =0.001; versus to CGFG 4W, e p =0.015; versus to MTAG 2W, f p =0.008; versus to CGFG 2W, g p =0.014; versus to MTAG 2W, Kruskal Wallis/Tamhane T2 Test On light microscopic examination of the maxillary tissue sections incubated with NF-kB/p65 primary antibody, we observed that immune-negative cells were common in the maxillary tissues of the two-week and four-week CG (Fig. 3 a-b, respectively; NF-kB/p65 score: 0(0–1), 0(0–1)). In contrast, we determined an increase in the number of cells showing NF-kB/p65 positivity in the sections from the two-week and four-week MTAG compared to those from the two-week and four-week CG (Fig. 3 c, respectively; NF-kB/p65 score: 2(2–3), 2(2–2) p = 0.000, p = 0.000). On the other hand, we determined a decrease in the number of cells showing NF-kB/p65 positivity in the two-week MTAG compared to the two-week CGFG (Fig. 3 b,d, respectively; NF-kB/p65 score: 2(2–3), 1.5(1–2), p = 0.000). Similarly, we observed fewer cells showing NF-kB/p65 positivity in the maxillary tissue sections of the four-week CGFG compared with those of the four-week MTAG (Fig. 3 c,e, respectively; NF-kB/p65 score: 2(2–2), 1(1–2), p = 0.015). When the two-week and four-week subgroups of CGFG were compared to each other concerning the number of cells showing NF-kB/p65 positivity, there was a significant decrease in the number of cells showing NF-kB/p65 positivity in the two-week CGFG compared to the four-week CGFG (Fig. 3 d,e respectively; NF-kB/p65 score: 1.5(1–2), 1(1–2), p = 0.014) (Table 4 ). Discussion As there is no data in the literature regarding the effects of second-generation autologous growth factors on the tissue damage induced by pulpotomy, the present study is the first that has investigated the effects of CGF, which is a second-generation autologous growth factor, on the TNF-α and NF-kB/p65 pathways at a molecular level and different intervals, in comparison to MTA, which is a bioactive endodontic cement. Although studies that have investigated the tissue injuries that develop due to pulpotomy in clinics are experimental animal models, many researchers have stated that human and rat teeth have morphometric similarities [ 19 , 20 ]. Rat incisors are different from human teeth in the sense that they show continuous growth, they were proven to represent a model for the evaluation of the reactions of the human dental pulp to bioactive molecules [ 21 , 22 ]. Vital pulp treatments use an appropriate pulp capping material and this material is directly placed onto the pulp tissue; therefore, biocompatibility, the anti-inflammatory property, and cytotoxicity need to be completely evaluated before a new material is used as a pulp capping material [ 23 ]. In a study by Minic et al. [ 24 ] that investigated pulpotomy-induced pulpitis using a rat model, findings of inflammation and cellular degeneration were reported. Similarly, in the present study, we determined extensive edematous areas accompanying inflammation. In a study by Nguyen [ 25 ] on pulpotomy-induced pulpitis, the inflammation and edematous areas observed in the second week were reported to be reduced in the fourth week. On the other hand, we did not determine any difference in terms of inflammation and edematous areas in the present study. Minic et al. [ 24 ] observed an increase in TNF-α levels after pulpotomy. Although no scientific studies examine NF-kB/p65 levels in pulpitis that develops after pulpotomy. We determined elevated TNF-α and NF-kB/p65 levels in pulpitis developed after the present study's pulpotomy. Biological scaffolds and CGF, which is known to be a cytokine reservoir, are used for bone regeneration in the current clinical implantology practice [ 14 , 26 ]. As a latest generation platelet concentrate product, CGF has a simpler modified production process that requires the repeated alteration of the centrifugation speed [ 5 ]. Consequently, its relatively dense structure is more comparable to that of natural fibrin and involves ample amounts of growth factors and proteins that are derived from autologous platelets and leukocytes. No studies are addressing the effect of pulp damage induced by CGF pulpotomy. In addition, studies are reporting that CGF increases periodontal regeneration in clinical studies [ 27 ]. Öngöz Dede et al. [ 28 ] demonstrated that CGF contributed to gingival regeneration by increasing the gingival thickness. In addition, Firouzi et al. (2022) [ 29 ] reported that CGF enhanced bone regeneration induced by lipopolysaccharide in cell culture. Similarly, in our study, we observed that CGF regenerated pulpotomy-induced damage. In current studies, although the effects of CGF on tissue regeneration are available, the information about its effect on tissue damage mechanisms is not clear. In addition, studies conducted today have reported that CGF suppresses TNF-α and NF-kB/p65 expressions [ 30 ]. Similarly, Xu et al. described in their in vitro and in vivo studies that CGF TNF-α expression mediated inflammation by suppressing pulp regeneration [ 18 ]. Although there are no studies on the effects of CGF on NF-kB/p65 and any studies on its impact on odontogenesis. Yuan et al. demonstrated that, NF-kB/p65 in lipopolysaccharide-induced dental pulp damage. They reported that increasing (NF-kB/p65) expression, triggers inflammation together with TNF-α [ 31 ]. Again, Kamal et al. [ 32 ] reported that CGF application reduced inflammation and pain induced by low-dose laser application. Similarly, our study found that CGF decreased NF-kB/p65 positivity together with TNF-α. MTA has been adopted as an endodontic material, and especially a pulp capping material for longer than a decade; however, its certain biological effects on pulp tissue regeneration remain unknown [ 33 ]. Silva et al. [ 6 ], reported that the cytokines IL-1, IFN-, and the chemokine CCL5 in the pulp tissue of rat incisors were modified when exposed to MTA. Inflammatory cytokines and chemokines play a role in the aggregation of leukocytes towards tissues that are injured or infected; however, may exacerbate tissue damage as well [ 34 ]. Tohma et al. [ 35 ] employed the MTA pulpotomy model for a period of 14 days. The results of the study revealed a scar-healing pattern that was generally consistent with previous studies. Similarly, Prabhakar et al. [ 36 ] reported that the inflammation and edema caused by pulpotomy-related pulpitis in rats decreased after MTA application. They reported an improvement in tissue structure on days 14 and 30 after the pulpotomy. Salako et al. [ 37 ] reported that MTA reduced the inflammation resulting from pulpotomy-induced pulpitis in 14 days. Moreover, they determined that inflammation was further reduced by MTA on day 30 compared to day 14. Similarly, we observed that the MTA treatment reduced the inflammation encountered in pulpotomy-induced pulpitis in 14 days and also that this reduction was more pronounced on day 30. A study by Benetti et al. [ 38 ] on dental root canal treatment reported MTA to exert an anti-inflammatory effect by causing a decrease in TNF-α expression. Similarly, in the present study, we found that the MTA treatment reduced the pulpotomy-induced increase in TNF-α positivity. Moreover, we demonstrated a further decline in cells showing TNF-α positivity on day 30 compared to day 14. However, although the effects of MTA on the nuclear factor-kappa B signaling system have not been previously researched, MTA can cause the initial inflammation of rat dental pulp cells through its effects on the NF-kB/p65 signaling system, while a subsequent anti-inflammatory effect is also observed due to the regulation of the expression of certain inflammatory mediators. Minic et al. [ 24 ] revealed that it stimulates the toll-like receptor 4 found on dental pulp cells and produces inflammatory cytokines such as IL-1β and IL-6 by activating the NF-kB pathway in pulpotomy-induced pulpitis. First, our study is a pilot study considering the effects of second-generation autologous growth factors on pulpotomy-induced inflammation by tissue levels of NF-kB/p65 and TNF-α. Additionally, there are some limitations to our study. The present study should be supported by studies dealing with other inflammatory cytokines and cell signaling molecules. Similarly, our study needs to be corroborated by research on intracellular and mitochondrial calcium levels that address cell damage mechanisms. Conclusion In conclusion, in the present study, we determined a reduction in cells showing NF-kβ/p65 positivity in the MTA treatment group compared to the CGF group. Moreover, we determined fewer cells showing NF-kβ/p65 positivity on day 30 after the MTA application compared to day 14 after the MTA application. Declarations Author contribution: A. K. contributed to conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing (original draft preparation), visualization, supervision, project administration, funding acquisition; A. Ş. Ç. contributed to conceptualization, methodology; E. B. contributed to conceptualization, methodology; Z. G. contributed to conceptualization, methodology, T. M. contributed to conceptualization, methodology, validation, formal analysis, data curation; L. T. contributed to conceptualization, methodology, validation, formal analysis, data curation; M. K. contributed to conceptualization, methodology. Funding: This study was financed in part by the Recep Tayyip Erdogan University Research Foundation (Grant No: TDH-2021-1242). The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Availability of data and materials: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Competing interests: The authors declare no competing interests. Ethical Approval : The required ethics committee decision for the study was obtained from Recep Tayyip Erdogan University, Faculty of Medicine, Animal Research Ethics Committee (approval number: 2020/47). Consent to participate: None. Conflict of interest: There is no conflicts of interest. 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Tabatabaei F, Aghamohammadi Z, Tayebi L. In vitro and in vivo effects of concentrated growth factor on cells and tissues. J Biomed Mater Res A. 2020;108:1338-1350. Xu F, Qiao L, Zhao Y, et al. The potential application of concentrated growth factor in pulp regeneration: an in vitro and in vivo study. Stem Cell Res Ther. 2019; 10:134. Pinzon RD, Kozlov M, Burch WP. Histology of rat molar pulp at different ages. Journal of Dental Research. 1967; 46:202-208. Schour I, Van Dyke H. Changes in Teeth Following Hypophysectomy: II. Changes in the Molar of the White Rat. Journal of Dental Research. 1934; 14:69-91. Hu Y, Wilson GS. A temporary local energy pool coupled to neuronal activity: fluctuations of extracellular lactate levels in rat brain monitored with rapid‐response enzyme‐based sensor. Journal of neurochemistry. 1997; 69:1484-1490. Sloan A, Smith A. Stimulation of the dentine–pulp complex of rat incisor teeth by transforming growth factor-β isoforms 1–3 in vitro. Archives of oral biology. 1999; 44:149-156. Qureshi A, Soujanya E. Recent advances in pulp capping materials: an overview. Journal of clinical and diagnostic research. 2014; 8:316. Minic S, Florimond M, Sadoine J, et al. Evaluation of Pulp Repair after BiodentineTM Full Pulpotomy in a Rat Molar Model of Pulpitis. Biomedicines . 2021; 9:784. Nguyen TD. Mineral Trioxide Aggregate/Ferric Sulfate Pulpotomy for Vital Primary Incisors: A Randomized Controlled Trial: University of Toronto (Canada); 2014. Palermo A, Giannotti L, Di Chiara Stanca B, Ferrante F, Gnoni A, Nitti P, Calabriso N, Demitri C, Damiano F, Batani T, Lungherini M, Carluccio MA, Rapone B, Qorri E, Scarano A, Siculella L, Stanca E, Rochira A. Use of CGF in Oral and Implant Surgery: From Laboratory Evidence to Clinical Evaluation. Int J Mol Sci. 2022;23:15164. Mijiritsky E, Assaf HD, Peleg O, Shacham M, Cerroni L, Mangani L. Use of PRP, PRF and CGF in Periodontal Regeneration and Facial Rejuvenation-A Narrative Review. Biology (Basel). 2021; 10. Öngöz Dede F, Bozkurt Doğan Ş, Çelen K, Çelen S, Deveci ET, Seyhan Cezairli N. Comparison of the clinical efficacy of concentrated growth factor and advanced platelet-rich fibrin in the treatment of type I multiple gingival recessions: a controlled randomized clinical trial. Clin Oral Investig. Feb 2023;27(2):645-657. Firouzi N, Yavari HR, Rahimi S, Roshangar L, Chitsazha R, Amini M. Concentrated Growth Factors Combined with Lipopolysaccharide Stimulate the In Vitro Regenerative and Osteogenic Activities of Human Dental Pulp Stem Cells by Balancing Inflammation. 2022. doi:10.1155/2022/2316666. Murakami-Malaquias-Silva F, Rosa EP, Almeida PA, et al. Evaluation of the effects of photobiomodulation on orthodontic movement of molar verticalization with mini-implant: A randomized double-blind protocol study. Medicine (Baltimore) . 2020; 99:e19430. Yuan H, Zhang H, Hong L, et al. MicroRNA let-7c-5p suppressed lipopolysaccharide-induced dental pulp inflammation by inhibiting dentin matrix protein-1-mediated nuclear factor kappa B (NF-κB) pathway in vitro and in vivo. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research. 2018; 24:6656. Kamal A, Salman B, Abdul Razak NH, Qabbani AA, Samsudin AR. The Efficacy of Concentrated Growth Factor in the Healing of Alveolar Osteitis: A Clinical Study. 2020. doi:10.1155/2020/9038629. Modena KCdS, Casas-Apayco LC, Atta MT, et al. Cytotoxicity and biocompatibility of direct and indirect pulp capping materials. Journal of Applied Oral Science. 2009;17:544-554. Silva MJB, Vieira LQ, Sobrinho APR. The effects of mineral trioxide aggregates on cytokine production by mouse pulp tissue. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2008;105(5):e70-e76. Tohma A, Ohkura N, Yoshiba K, et al. Glucose transporter 2 and 4 are involved in glucose supply during pulpal wound healing after pulpotomy with mineral trioxide aggregate in rat molars. Journal of Endodontics. 2020;46(1):81-88. Prabhakar A, Mandroli PS, Bhat K. Pulpotomy with curcumin: Histological comparison with mineral trioxide aggregate in rats. Indian Journal of Dental Research. 2019;30(1):31. Salako N, Joseph B, Ritwik P, Salonen J, John P, Junaid T. Comparison of bioactive glass, mineral trioxide aggregate, ferric sulfate, and formocresol as pulpotomy agents in rat molar. Dental traumatology. 2003;19(6):314-320. Benetti F, Mello PB, Bonini JS, Monteiro S, Cammarota M, Izquierdo I. Early postnatal maternal deprivation in rats induces memory deficits in adult life that can be reversed by donepezil and galantamine. International Journal of Developmental Neuroscience. 2009;27(1):59-64. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 03 Aug, 2024 Read the published version in BMC Oral Health → Version 1 posted Editorial decision: Revision requested 14 May, 2024 Reviews received at journal 13 May, 2024 Reviews received at journal 13 May, 2024 Reviewers agreed at journal 04 May, 2024 Reviews received at journal 02 May, 2024 Reviews received at journal 02 May, 2024 Reviewers agreed at journal 22 Apr, 2024 Reviewers agreed at journal 22 Apr, 2024 Reviewers invited by journal 21 Apr, 2024 Editor assigned by journal 16 Apr, 2024 Editor invited by journal 04 Apr, 2024 Submission checks completed at journal 04 Apr, 2024 First submitted to journal 25 Mar, 2024 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. 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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-4165836","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288418632,"identity":"e6746341-a70e-400c-aa49-fff9e982944c","order_by":0,"name":"Ayca Kurt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYNCCA0DM3sDAwAPlSxClhYfnAIoWAyK0SCQQqYV/RvLDDwxnDufbSz4+9uFNRa28OQPzwds8DH/ycWmRuJFmLMFw47Blj3Ra8sw5Z44b7mxgS7bmYTCwbMCl50YO0BkfDhvwSOcYM/O2HWPccIDHTBqoBafL5G/kMP8Aa5E8/xmkxX7DAf5veLUY3MhhAznMgEeChxmopSYRaAsbXi2GZ56ZWSScSTfgOZNmzDjnzIHkDYfZjC3nGBjj1CJ3PPnxjQ/HrA3Y2w8/ZnhTUWe74XjzwxtvKuRwh7JAAgNDAoJ7mIGBGexgnBqAEXMAhVuHR+koGAWjYBSMVAAAZrdTMelm1q4AAAAASUVORK5CYII=","orcid":"","institution":"Recep Tayyip Erdogan University","correspondingAuthor":true,"prefix":"","firstName":"Ayca","middleName":"","lastName":"Kurt","suffix":""},{"id":288418635,"identity":"bb01ee44-a5cb-4250-a02b-eed5bc416cbb","order_by":1,"name":"Ahter Sanal Cıkman","email":"","orcid":"","institution":"Recep Tayyip Erdogan University","correspondingAuthor":false,"prefix":"","firstName":"Ahter","middleName":"Sanal","lastName":"Cıkman","suffix":""},{"id":288418636,"identity":"01e13824-4b1b-4bfa-89b9-13da2a552494","order_by":2,"name":"Emre Balaban","email":"","orcid":"","institution":"Recep Tayyip Erdogan University","correspondingAuthor":false,"prefix":"","firstName":"Emre","middleName":"","lastName":"Balaban","suffix":""},{"id":288418637,"identity":"e73d329e-bda0-494c-a755-b12f59c874ca","order_by":3,"name":"Zeynep Gumrukcu","email":"","orcid":"","institution":"Recep Tayyip Erdogan University","correspondingAuthor":false,"prefix":"","firstName":"Zeynep","middleName":"","lastName":"Gumrukcu","suffix":""},{"id":288418638,"identity":"45de366a-3087-4c28-8dd9-a8c4f441c495","order_by":4,"name":"Tolga Mercantepe","email":"","orcid":"","institution":"Recep Tayyip Erdogan University","correspondingAuthor":false,"prefix":"","firstName":"Tolga","middleName":"","lastName":"Mercantepe","suffix":""},{"id":288418639,"identity":"475895f9-7623-4632-bc82-727ce6ec80b5","order_by":5,"name":"Levent Tümkaya","email":"","orcid":"","institution":"Recep Tayyip Erdogan University","correspondingAuthor":false,"prefix":"","firstName":"Levent","middleName":"","lastName":"Tümkaya","suffix":""},{"id":288418640,"identity":"100c97a0-ab65-4e4b-93b6-51732fd61d20","order_by":6,"name":"Mert Karabağ","email":"","orcid":"","institution":"Recep Tayyip Erdogan University","correspondingAuthor":false,"prefix":"","firstName":"Mert","middleName":"","lastName":"Karabağ","suffix":""}],"badges":[],"createdAt":"2024-03-25 22:59:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4165836/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4165836/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12903-024-04577-z","type":"published","date":"2024-08-03T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54336013,"identity":"7fcfa808-4947-4554-8f64-03f7ebc17461","added_by":"auto","created_at":"2024-04-09 03:15:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12425931,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative light microscopic screenshot of maxillary sections stained with H+E.\u003c/p\u003e\n\u003cp\u003eBone (B), dentin (d), periodontal ligament(po), odontoblast(ob), Pulp(p).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA(x20) CG 2W\u003c/strong\u003e: Dentin (d), periodontal ligament (po), alveolar bone (b) and pulp structures formed by normal odontoblasts (ob) are observed (HDS: 0.5(0-1)). \u003cstrong\u003eB(x20) CG 4W\u003c/strong\u003e: The maxilla, which consists of dentin (d), periodontal ligament (po), alveolar bone (b) and pulp (p) structures formed by normal odontoblasts (ob), is observed (HDS:1(0-1)). \u003cstrong\u003eC(x20) CGFG 2W\u003c/strong\u003e: Widespread inflammation (tailed arrow) accompanied by granuloma tissue (g) and widespread edematous areas are observed in the application areas (HDS: 5(4-5)). \u003cstrong\u003eD(x20) CGFG 4W:\u003c/strong\u003e Inflammation (tailed arrow), edematous areas and fibrous callus (fc) are observed in the application areas (HDS:4(3-4)). \u003cstrong\u003eE(x20) MTAG 2W:\u003c/strong\u003e A decrease is observed in infiltrative and edematous areas. In addition, diffuse fibrous callus (fc) and occasional cartilage callus formations (kc) are observed (HHS:2.5(3-4)). \u003cstrong\u003eF(x20) MTAG 4W:\u003c/strong\u003e A decrease is observed in infiltrative and edematous areas. In addition, odontoblasts (o), the pulp (p), periodontal ligament (PO) with typical structures are observed in places. HDS: 2(2(1-2)).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4165836/v1/1bb26c0fc9ea088cbe818ebf.png"},{"id":54336014,"identity":"cba00c36-04ca-4a1c-9c21-a278933b1a04","added_by":"auto","created_at":"2024-04-09 03:15:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15159385,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative light microscopic screenshot of maxillary tissue incubated with TNF-alpha primary antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA(x40) CG 2W:\u003c/strong\u003eImmune-negative cells are commonly observed in sections of maxillary tissue incubated with TNF-alpha primary antibody (TNF-alpha positivity score: 0(0-0.5)). \u003cstrong\u003eB(x40) CG 4W:\u003c/strong\u003e Immune negative cells are commonly observed in sections of maxillary tissue (TNF-alpha positivity score: 0(0-1)). \u003cstrong\u003eC(x40) CGFG 2W: \u003c/strong\u003eA large number of cells showing TNF-alpha positivity are observed in the sections of the maxillary tissue (TNF-alpha positivity score: 3(2-3)). \u003cstrong\u003eD(x40) CGFG 4W:\u003c/strong\u003e Cells showing TNF-alpha positivity are observed in sections of maxillary tissue (TNF-alpha positivity score: 2(2-2)). \u003cstrong\u003eE(x40) MTAG 2W\u003c/strong\u003e: In sections of maxillary tissue incubated with TNF-alpha primary antibody, a decrease in cells showing TNF-alpha positivity is observed (TNF-alpha positivity score: 1.5(1-2)). \u003cstrong\u003eF(x40) MTAG 4W:\u003c/strong\u003e In sections of maxillary tissue incubated with TNF-alpha primary antibody, a decrease in cells showing immune positivity is observed (TNF-alpha positivity score: 1(1-1)).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4165836/v1/0c01268a98a7c59dea1dc605.png"},{"id":54336015,"identity":"b8123948-a8a1-4c79-b27a-2e02f08fdd89","added_by":"auto","created_at":"2024-04-09 03:15:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16228141,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative light microscopic screenshot of maxillary tissue incubated with NF-kß/p65 primary antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA(x40) CG 2W:\u003c/strong\u003eNF-kß/p65 negative cells are commonly observed in sections of maxillary tissue (NF-kß/p65 positivity score: 0(0-1)). \u003cstrong\u003eB(x40) CG 4W:\u003c/strong\u003e Immune-negative cells are commonly observed in sections of maxillary tissue incubated with NF-kß/p65 (NF-kß/p65 positivity score: 0(0-1)). \u003cstrong\u003eC(x40) CGFG 2W:\u003c/strong\u003e A large number of cells showing NF-kß/p65 positivity are observed in the sections of the maxillary tissue (NF-kß/p65 positivity score: 2(2-3)). \u003cstrong\u003eD(x40) CGFG 4W:\u003c/strong\u003e Cells showing NF-kß/p65 positivity are observed in sections of maxillary tissue (NF-kß/p65 positivity score: 2(2-2)). \u003cstrong\u003eE(x40) MTAG 2W:\u003c/strong\u003e In sections of maxillary tissue incubated with NF-kß/p65 primary antibody, a decrease in cells showing TNF-alpha positivity is observed (NF-kß/p65 positivity score: 1.5(1-2)). \u003cstrong\u003eF(x40) MTAG 4W:\u003c/strong\u003e In sections of maxillary tissue incubated with NF-kß/p65 primary antibody, a decrease in cells showing immune positivity is observed (NF-kß/p65 positivity score: 1(1-2)).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4165836/v1/6ec03f98290b1028a339522b.png"},{"id":61793397,"identity":"2d807fd1-dacc-43c4-b066-ab8715ee0e60","added_by":"auto","created_at":"2024-08-05 16:12:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":76533049,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4165836/v1/fea8dcd5-8613-4b94-a7cb-c59bd2a69b5d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Effects of Mineral Trioxide Aggregate and Second-Generation Autologous Growth Factor on Pulpotomy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCaries is known to be a preventable chronic health problem; however, comprehensive global reports [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] estimate that there were 573\u0026nbsp;million children with untreated caries in 2015 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Caries most commonly affect young children; however, it is a lifelong condition that is also encountered during adolescence and adulthood. Therefore; the treatment of caries, especially deciduous caries, holds great importance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pulp must be preserved during the treatment processes of deciduous molar teeth' complex root canal systems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Exposure of pulp tissue may be caused by caries, as well as iatrogenic causes, or traumatic injuries. Pulpotomy is widely used on deciduous teeth as a successful treatment in pedodontics [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It is an important pulp treatment that includes the removal of a part of the coronal pulp tissue and the capping of the radicular tissue by materials that prevent further injury to the pulp as well as allowing and promoting healing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral materials have been used so far to stimulate the formation of a dentin bridge through the dentinogenic potential of pulp cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The increasing focus on biocompatible materials has generated a wider variety of Mineral Trioxide Aggregate (MTA) choices in pulpotomy. MTA, which shields periradicular or dental pulp tissues may preserve pulp vitality and stimulate its recovery [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite its high success rates, MTA has disadvantages such as pulp chamber obliteration, change of tooth color, pulp degeneration, and a high cost [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, biocompatible treatments aimed at protecting pulp vitality and increasing tooth survival must be developed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConcentrated growth factor (CGF), which is among second-generation autologous growth factors, is a recently discovered platelet concentrate that is usually obtained from autologous venous blood and produces a larger and more dense fibrin matrix at levels greater than previously known platelet concentrates [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Serving as a scaffold material, CGF also produces many growth factors where it is applied [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although previous research includes a multitude of studies on CGF, its direct use as a pulpotomy material has not been examined [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study aims to shed light on the alternative materials that may be used in the pulpotomy treatment soon of endodontic procedures by investigating the potential effects of the inflammation that develops after the use of MTA, which is a bioactive endodontic cement, and of CGF, which is a second generation autologous growth factor, as pulpotomy materials on the TNF-α and Nf-kB/p65 pathways.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eEthics Approval\u003c/h2\u003e\n \u003cp\u003eThe required ethics committee decision for the study was granted by Recep Tayyip Erdogan University, Faculty of Medicine, Animal Research Ethics Committee (approval number: 2020/47). I confirmed that all methods are reported in accordance with ARRIVE guidelines (\u003cspan\u003e\u003cspan\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003eExperimental Design\u003c/h2\u003e\n \u003cp\u003eThe study used the maxillary anterior central teeth of thirty-six young male rats (Sprague Dawley, weighing between 250 and 350 g). In total, 72 maxillary central teeth were included. We randomly assigned 48 teeth to two groups (12 rats/group; 24 teeth/group) based on the capping material (MTA or CGF). Two subgroups per group were formed (12 teeth/group) based on the time following pulpotomy (two weeks and four weeks). The central teeth of the 12 animals assigned to the control group were not manipulated in any way, both in the two-week group and in the four-week group; thus, these teeth served as the control group for histological identification. Throughout the experiment, we housed the animals in polypropylene cages (with four animals per cage) in an experimental animal unit with a 12:12 hour light-dark cycle, 55–60% humidity and room temperature, as well as unlimited availability of water and food pellets (Nutrilabor; Guabi, Campinas, SP, Brazil). The cage bedding was renewed three times per week at minimum.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003eOperational procedure\u003c/h2\u003e\n \u003cp\u003eThe animals received anesthesia through the intramuscular injection of a mixture of 50 mg/kg (i.p) ketamine hydrochloride (Ketalar ®, Eczacıbaşı Parke-Davis, Istanbul, Turkey) and 10 mg/kg (i.p) Xylazine HCl (Alfazyne ®, Alfasan International B.V. Woerden, The Netherlands) and immobilized on a surgical board.\u003c/p\u003e\n \u003cp\u003eFor pulpotomy, the conventional technique was followed. The permanent maxiller right and left central incisors were isolated according to the method previously described [\u003cspan\u003e17\u003c/span\u003e] and adapted for this experiment. In summary, a round diamond bur (#1011, Dentsply Indústria e Comércio Ltda, Petropólis, RJ, Brazil) was used together with a high-speed rotary handpiece to create an opening for pulpotomy and a 1 mm cavity, equal to the diameter of the diamond wheel used, was opened in the middle third of the crown in the palatal part of the incisors. The coronal pulp tissue was removed using an excavator (#5, SS White, Dental Objects Ltda., Juiz de Fora, MG, Brazil). The bleeding in the pulp chamber was controlled using saline-soaked cotton pellets. During the performance of this technique, loupes with illumination ( Loupes, YDL-EW, 2.5x) were used to increase the field of view. Prior to the pulpotomy procedure, the mucosa of the central teeth were subjected to infiltrative local anesthesia with articaine (ULTRAVER D-S), 80 mg articaine hydrochloride, and 0,01819 mg epinephrine bitartrate.\u003c/p\u003e\n \u003cp\u003eAnimals in the control group (CG) were also divided into two subgroups in the same way as in the experimental groups (two weeks and four weeks). In consideration of the standardization of the study, animals in this group underwent immobilization with appropriate anesthetics and local anesthesia even though no dental procedures were performed.\u003c/p\u003e\n \u003cp\u003eIn the experimental groups, either MTA or CGF was used as the pulpotomy material. In the MTA (PROROOT, WHITE, Dentsply, Konstanz, Germany) group (MTAG), a 1:1 mixture of MTA powder and distilled water was placed in the pulp chamber where bleeding control was performed, using an MTA carrier (MAP One, Produits Dentaires SA, Switzerland). In the CGF group (CGFG), 4 ml of blood was collected from the jugular vein of each rat. The blood was placed in CGF kit tubes with 0.025M calcium chloride (S6M, STR Biotechnologies, Çorum, Turkey). The tubes were placed in a centrifuge device designed specifically for CGF (Labfuge, STR Biotechnologies, Çorum, Turkey). This device was operated on a standard procedure of centrifugation that automatically increased the speed and then slowed down for 45 seconds as follows: 2700 rpm (735 g) for 2 minutes, 2400 rpm (580 g) for 4 min, 2700 rpm (735 g) for 4 min. and 3000 rpm (905 g) for 3 min. As a result of centrifugation, four layers formed within the tubes. The topmost fourth layer consisted of serum, the second layer (buffy coat) and the third layer (unipotent stem cell) composed of the CGF layer of a gel-like consistency, and the bottom layer was the first layer consisting of red blood cells. After the serum layer was removed, the CGF layer of gel-like consistency was extracted from the tube using forceps, cut off from the red blood cell layer underneath, and placed onto the exposed pulp tissue using a sterile handpiece (Medisana double-ended dental spatula). Cavities in both groups were restored and capped using conventional glass ionomer cement (Ketac-molar, ESPE Platz, Seefeld, Germany). The animals were kept under observation during healing. Potential changes in the feeding and drinking cycles as well as the behavioral profile were evaluated. After the experimental period (2nd and 4th weeks after pulpotomy), the rats were sacrificed by the administration of sevoflurane (sevorane 100%, Abbott, United States of America).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003eHistopathological analysis procedure\u003c/h2\u003e\n \u003cp\u003eMaxillary tissue specimens obtained from the rats were fixed by being kept in 10% phosphate-buffered neutral formalin (Sigma-Aldrich, Germany, Ph:7.4) for 24 hours. Subsequent to fixation, these specimens were decalcified by being kept in Morse’s solution for 24 hours. In the following stages, the specimens were subjected to a dehydration procedure with ethanol series of gradually increasing concentration (50%, 70%, 80%, 90%, 96%, and 100%, Merck GmbH, Germany) using an autotechnicon device (Citadel 2000, ThermoScientefic, Germany), clearing, and paraffin inclusion (Merck GmbH, Germany) procedures. Maxillary tissue specimens subjected to paraffin inclusion were embedded in hard paraffin (Merck GmbH, Germany) and sections of 4–5 µm were cut using a rotary microtome (RM2525, Leica Biosystems, Germany). The sections were stored in an incubator and passed through two series of xylol (Merck GmbH, Germany). Following the deparaffinization stage, they were stained with Harris hematoxylin (Merck, Germany) and Eosin G (Merck, Germany) stains with the use of a staining device. Stained sections were mounted with entellan (Merck GmbH, Germany) and photographed under a light microscope with an attached digital camera (BX51, Olympus Corp., Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003eImmunohistochemistry (IHC) analysis procedure\u003c/h2\u003e\n \u003cp\u003eIn order to examine the maxillary inflammation induced by the invasive procedure, TNF-Alpha (ab220210, Abcam, UK) and NF-kß/P65 (ab16502, Abcam, UK) primary antibody kits and corresponding secondary antibody kits (Goat Anti-Rabbit IgG H\u0026amp;L (HRP)) and (ab205718, Abcam, UK), respectively) were used. Sections of 1–2 µm thickness were obtained from maxillary tissue sections using a rotary microtome (Leica Biosystem, RM2525, Germany). The obtained maxillary tissue sections were incubated with primary and secondary antibodies for one hour according to the manufacturer’s guidelines in the catalog and stained using the immunoperoxidase method with an IHC staining device (Bond Max III, Leica Systems, Germany). Finally, they were counterstained with Harris hematoxylin (Merck, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eSemi-Quantitative analysis\u003c/h2\u003e\n \u003cp\u003eHarris hematoxylin (Merck GmbH, Germany) and Eosin G (Merck GmbH, Germany) -stained maxillary tissue sections were given a histopathological damage score (HDS) in accordance with the studies that evaluated the indications caused by invasive procedures of the maxilla concerning edema, inflammation, fibrosis, cartilage and bone callus formation as shown in Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e. From each preparation, 20 different randomly selected sites were scored by a histopathologist under a light microscope using x21 and x20 objectives.\u003c/p\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n \u003cp\u003eSpecimens of maxillary tissue incubated with TNF-α and NF-kB/p65 primary antibodies were scored as shown in Table\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e. From each preparation, 20 different randomly selected sites were scored by a histopathologist under a light microscope using x40 objectives. The histopathologists were blind to the experimental groups.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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BCQgAQlIQAISkMDTCJgMPA2thiUgAQlIQAISkIAEJPDaBEwGXvv+6N0NBH777bdvv/zysUf5X//617d///vff8/2+++/f7f5559//t3nhQQkIAEJSEACEjgagY9FUEej4XpehgABfoL81S/B+6NKbGWOTgYeZVs7EpCABCRwH4G8jOn9/48//viWFzWpLRKQwOMImAw8jqWWnkBgvrFnil9//ZXLh9Rb8zzEuEYkIAEJSOBdBEgE8mKIkn0/yYHJAESsJfAYAiYDj+GolScR+Kwg/bPmeRImzUpAAhI4FAH+qeZcVBICk4FJxbYEPkbAZOBj/NR+MoEZpOdtUQ6JLjkc+m8K8s99opfCXzH3ePp565TxvHma87SNyPPPlnId2egxR/pS2mbGZ4nN9Kf2r7onHdsSkIAEfhAgGch+2WW1d2Zf5TfPB/ZdxrNPU3pfz3jv6ezz6Z820beWwFEI/DNiOcrKXMchCGRD7sMgm3dvzAnys1kT7LPxs5EHQt4iRYa3SQTttDkQmKdtRJ9xDovoYwNfZrt1Yi9y2M91+3OIG+UiJCABCTyYAPv41n7Jvjv38vSnZM/lbKCNrd6jI5N9OfOlIJNr9+vvSPzPwQmYDBz8Bu99eX0YZIPOL5tzl2zkqw2/ZVovsgTmyGSe7ss1B0NkODg4ZNKX8fSncGB8b/z1n54z4+1j9DjAWsdrCUhAAhL4QSB7MXt/6t43M9b7aicHff3D2n8DfXTY19tm9uo+C6KbeWdf2/RaAnsnYDKw9zt4cP9nkJ6NOr8u2djZ3NOfTbsD+fRlMydwz/W0MeeZNjg0et7Wib3Y3UoWOJgi0762Pa8lIAEJSGBNIPtm9s/ev9O3FaSv9uRY7r19ta9nnHm6dt9e3xd7j0HAZOAY9/Gwq+iAO4tMUD0D+WzSvVH3Zg+YbOrPTAYyT3xljrxp6kMLP+InB0y/jWLcWgISkIAE/vu3ras9Mvtn9tqU7KfPSAa2bHpfJHBUAiYDR72zB1nXTAZWy3pvMrCyOftmQrF6gzR1eBNFsE9iEJ8z1gfb9Hm1LvskIAEJnJVA9sxVUJ59NXtvytynYRUZXsj0PpzxTiAylv26S9vv/pUvPe61BPZM4Oc/BXteib4fksAMuFeLnIH16oDIhs+hwAHA3zBwaEQmtlKmDXR6/vYtNtBtGa5JFGjHvocLNKwlIAEJ/EyAPXPuk72X888vWyZ6vHhJf+Rps9fzzzlX+3q8iE7bjB7nx89e2pLAMQiYDBzjPh5uFWzS2ZT5saH3YhOQM57rBOS0U3NY0McGzyGR/uh0YD9ttOzKZuTnPMwXuykcULO/1+K1BCQgAQn8lwB7JkkBe+cMygnwGWePh+Pcv7f6SRAYx17q7PEWCRyZgMnAke+ua/tUAvOQyuS+UfrUW+BkEpCABCQgAQm8k4DJwDuBKS6BFQH+dmCO5W1T3mxZJCABCUhAAhKQwCsSMBl4xbuiT7sjsPVXyfwzod0tSIclIAEJSEACEjgFAZOBU9xmF/kZBBL4978zzbVFAhKQgAQkIAEJvDKBq9HK29vbN38y8BnwGfAZ8BnwGfAZ8BnwGfAZeN1n4N6E42oycK9h9SQgAQlIQAISkIAEJCCB1yZgMvDa90fvJCABCUhAAhKQgAQk8DQCJgNPQ6thCUhAAhKQgAQkIAEJvDYBk4HXvj96JwEJSOAuAvnYkv9vVnehU0kCEpDAqQiYDJzqdrtYCUhgLwTml1Xn/1NV2vNrq6yNr66aDEDEWgISkIAEtgiYDGyRsV8CEpDACxC49IY/H7vbKpf0tnTsl4AEJCCB8xEwGTjfPXfFEpDAjgjcG9Tfq7cjNLoqAQlIQAIPIGAy8ACImpCABCTwLAKroP7PP//89ttvv/1jSj58l78xWOmln39u1Pq55iN52OCfGP3+++9LnUyOrdTI/8MpOyQgAQlI4KUJmAy89O3ROQlI4OwEVkF9AvQO5sMowTh9/O8NOkDPdfRSGMcOQX1kkmjklz6Siuh0wsB88W3a+97hfyQgAQlIYDcETAZ2c6t0VAISOCOBBNwE610T+IdJrjvwT18nEQn+5/++IPL0EegnCaBknGA/ffwNATIZbx/iW7exYy0BCUhAAq9NwGTgte+P3klAAicn0EE9KHijT7vf4NPXegT7nUzkmgSCcXRTX0sGkCVJMBmAiLUEJCCBfREwGdjX/dJbCUjgZAQ6qGfpeTvfb+FvSQb4WwBsdH1PMkASwN8emAw0Ua8lIAEJ7IeAycB+7pWeSkACJySwSgbAkH/+k0B+JdN9BO7oURPIvzcZ4H9TkPkpJgOQsJaABCSwLwImA/u6X3orAQmcjEAH9b10AvLU/A+CCe4jl+CcX9r5Zz/9twPRQ/69yQDzJclIoR07FglIQAIS2BcBk4F93S+9lYAETkKAAJuAflV3cM/bf+RWSUQSAsb53wtEjr7UJBn0ZQ6SBfriW/pp5xrb7dNJbpXLlIAEJLBrAiYDu759Oi8BCUhAAhKQgAQkIIH7CZgM3M9OTQlIQAISkIAEJCABCeyagMnArm+fzktAAhKQgAQkIAEJSOB+AiYD97NTUwISkIAEJCABCUhAArsmcDUZeHt7++ZPBj4DPgM+Az4DPgM+Az4DPgM+A6/7DNybkVxNBu41rJ4EJCABCUhAAhKQgAQk8NoETAZe+/7onQQkIAEJSEACEpCABJ5GwGTgaWg1LAEJSEACEpCABCQggdcmYDLw2vdH7yQgAQlIQAISuJFAPoTnl7BvhKWYBP4iYDLgo3B6Av3FVb7KenooApCABCQwCPReydenv3rPzBe0Kfj02cnA/Fp4M+kvg2/5xVe+WYe1BD6bgMnAZxN3vpciwOGWzTwlm3Vv5C/lrM5IQAISeAEC2ScTeL9CmX6kvRV0P8vfBPOcIT1Hny8kDFMuvn62v+2j1xIIgdf40+y9kMAXEchbpX6z9P0PxS+/fMvbHIsEJCABCfyTwKskA9m7vzoZSHCfZGBV+FsBxmaickkXHWsJfAYBk4HPoOwcL0sgm/MM/FcJwssuQMckIAEJfDKB9yQDCZSzz85AmLfm6c91v5RBh7fmGZ8FGWwTkDMPgXjavcf3vBlDL/bRiUzbn3N3u+XmW3/sIY9v3ebaWgJfScBk4CvpO/eXEuBQmBt4DiX/qdCX3honl4AEXpjArclA9lEC8eyzHZhnjCA/1wTlJABZPvMgN5Ew3v2ZI/ZILuZ+3gE5wXp84xp9zoWW73m4Rg5fWEfG+4xh/chHjmtsWUvgqwiYDHwVeef9cgK9Ubcz8/DoMa8lIAEJnJ0Age8lDgl0OzCObAf9ue5gGNnYJpCPTq7fmwy0PkE+via4J0HhDKCN7PSr7WFnq57JAza7P2vMLwUfMg6DLdv2S+BZBEwGnkVWuy9PgE24N/44nY0/B5VFAhKQgAT+SeCWZACZBLn9Y2/NPkt/78G5pv9aEM4c7WF0CbTTTzA+Ewp0Iz+TgZZNgP6eID22LslnfT0eHvg7fe91eS2BZxIwGXgmXW2/PIE+CHA2G/W1QwhZawlIQAJnI0AgvVp3gt38ItNB75Zs9uD8pmyCZMY6OG87Kz+iQ3Ad2ZkMoJOaF0KPTAZmsN/+5jr+UZg/OilhMDkgay2BZxL48VQ+cxZtS+BFCSTon4F/NmsOhxd1W7ckIAEJfBkBAuqVAwSzBOFThv2WOuMJhrPvkkR08J+koGXb3sqPS8kA82CfYJz9Hp8Zz1zvDdBjI36tSmwR+Gec+el771yrOeyTwD0ETAbuoabOYQjMzTibOIfZYRbpQiQgAQk8kABBeAfNMZ9+/hlQ2rnu/TTyBPYZI2hmH049bUQfubmEDt6xeykZQJ7gm3bqFNq9rvcE6LHb629/s4bVOprD9L31vZbAMwmYDDyTrrZ3QSAbeDbh/Prg2oXzOikBCUjgkwgQtLNfrmqCclxKsItcB8qRS3DMGAF5+t6zJ6PfttI37aePYB2dyHA96ynbvrO29jP6W+dHfNsaa6ZbMsxnLYFnETAZeBZZ7UpAAhKQgAQkIAEJSODFCZgMvPgN0j0JSEACEpCABCQgAQk8i4DJwLPIalcCEpCABCQgAQlIQAIvTuBqMvD29vbNnwx8BnwGfAZ8BnwGfAZ8BnwGfAZe9xm4N+e4mgzca1g9CUhAAhKQgAQkIAEJSOC1CZgMvPb90TsJSEACEpCABCQgAQk8jYDJwNPQalgCEpCABCQgAQlIQAKvTcBk4LXvj95JQAISkIAEJCABCUjgaQRMBp6GVsN7IsAHa/IRGYsEJCCBoxJgr5sf2aLNx79uXT9fDb5V/pXk+CBaf1AsH/6CRX+JGL/5SNhqDBlqZLE3P8iGHH4g1+cQfe0jeqlzv7bGWs5rCVwiYDJwiY5jpyDQG3FvwqdYvIuUgAROSSD73gxO+SLv7L8EKMlFfnsqCaATZM8v/mYdBNZhwHWvLX23JALRaX2+VjzZxpfZx3zxj7HY4prx+NFz0G8tgfcSMBl4LzHlD0mAjdpk4JC310VJQAKDwCq4jAhvxof4ssmb7z0lAyQCqWfp4Bu5DvwTjK/0pp20V3LRn8F72j1H20rCgp336rYdryVwjYDJwDVCjp+CgMnAKW6zi5SABP4i8J5kgP2Rf7ISE7OPN+3I8BabduoObJGPrfQjN4PljDOWul/YJHjPj6Qk48z71zJ/qpDbSl4uJQPx8ZLtnybaaGTezEHpda9sZz3NrNlEnjHsWUvgXgImA/eSU+9QBDjY+qA51AJdjAQkIIEisEoGCE47ME1fB6EJZrudgLWDa/bSftsde60TN5hjBsiRI2AmeMdWZDNf2pHJdeSxjf/I13K/X2ZOAuzU6CMX+9hqn2OPfmTvqeNzB/CcN7GPP+175OGU+bmODa7v8UMdCUwCJgOTiO1TEuAAY3M+JQQXLQEJnIZAgksC0K7nHpiAtPsIuOmLbicDAZi+DnoJ4tFJzXX86AAY2fTlum0TNNMX3zpIZxzb82ZGNj8CaeRjh5JreGQ8pX3Ev7lG9Lfq2Op5VnLxq9cTGXyhP3a4zvjK35Vt+yRwiYDJwCU6jp2GQA6PbLpbh8hpQLhQCUjgFAQSUBIUs2AC/eyFFILRWRPsp5/gHJ3Y7cCXIJf5qAnGp+20sxd3oNsy6Ge858EevuEPddY8fSW4j+6qZC7scU5EFlZbetNW+znHup11XrKZNTAe30kM4ifXbc9rCdxC4Mef+FuklZHAQQmwyZsMHPQGuywJSOAnAgkcCap7gCCXMQLzlunrjM8Am/2UoDntDlaxnfFLwW8C6Gm7535EMsB6CbDbfsbwNf0dfOM7+lkHv/R1iY1bz5YO9ttGrmOnbWf9+Icfq3VMO7YlMAmYDEwitk9JgMPr1g37lJBctAQkcBgCW8kAeyFBZuRmQB4ZgtJVMhBI6Y8MdrAbW+i2XIPNeOSju3qjjs33JgPRy3q6ZK7Zl/EE1bN/Kxloe/N6rneOz/ack/FmSZ/JACSsP0rAZOCjBNU/BAEOqtQWCUhAAkcnkKCToJq18rY7gTx7YYLQbkc2eryBxk7anTQQeHdfZGML3djK+OwjAcCfthF/8kt5bzKwspe5sffd6F//ia/tZ7o5J9IPlynTNuJ3+46N2YdO5oQ7fakzR8ZmiR364T1lbEvgFgImA7dQUubQBHKg5EDgNw/IQy/exUlAAqcikACSvW6rngEugS/yHTxjj6AUmOjQTt3B6+zHduoOiONLj7E/Zz76c02gTl/mWpVpr9eCfOyt+jPOejPPlkzk4ie+zLrn6bHJPXL4uxrLeJ9fWzLMZy2BLQImA1tk7JeABCQgAQlIQAISkMDBCZgMHPwGuzwJSEACEpCABCQgAQlsETAZ2CJjvwQkIAEJSEACEpCABA5OwGTg4DfY5UlAAhKQgAQkIAEJSGCLwNVk4O3t7Zs/GfgM+Az4DPgM+Az4DPgM+Az4DLzuM7AV7F/rv5oMXDPguAQkIAEJSEACEpCABCSwTwImA6UHMfsAACAASURBVPu8b3otAQlIQAISkIAEJCCBDxMwGfgwQg1IQAISkIAEJCABCUhgnwRMBvZ53/RaAhKQgAQkIAEJSEACHyZgMvBhhBrYO4H5Jcu9r0f/JSABCUDg0pdw8/Xb/tovOpfqyF/68u4l3a8Y6y/0Zr2XSssi1/y2WL2XY391ONfzy8E9jh/UfV7R1zVfYu4+ryVwjcDlPxnXtB2XwM4JZPPvjTibcDZbiwQkIIEjEcje9ttvv/20JILf2f+T0GgkON5LMhBfO4AnkB5L+i6z2vuzzvSnhBHXrR+GPUePra5j5xLvzAHfnj+2sp7Ml5Ka655n5WOPey2BFQGTgRUV+05BIEnA3MTZfDtBOAUMFykBCRyaQILEVRCaAPnWFyC8dSZYfXVg00/8732fvhWbDr6Ra91rgf2Kz6VgPfZmgJ97g299nb5pK7rt32p++ySwImAysKJi32kJsOGfFoALl4AEDkngPckAL0WiQ6LQfenPL8En1wlO8xKFdmoCU+QSXKcQyGKjgU8b/WImvvC2n3kIlNvG1jW222bs4NfUu5QMZG0zcJ/6s93rXvndwT66kWOeHsdWy61sMm4tgUsETAYu0XHsdASymW4dDKeD4YIlIIHDEEjQO4NFAsruzzXBZxafAJQ2wXS/cSdJaFCRRyf90WOO7K+9x3YwPl/GRC7j+JHr9gf/e+5L1/G1/UKfNcQ+88UO/blGFvstR9+1muSIdU4baWfOLj1veOB/M74nMek5vJaAyYDPgASKQA4aiwQkIIGjEUigufr1W/KsOXtg9xGMpi+/GbDSR6AbGwTx2EmAy3X0uyCLXgfDBM30dSIQecax3XZX1wmgWzbt+BMfKGn3OYB/6WeN0eM6vmUsv7B6T+mAPnqxwVqxA3/a8Y356EubsvKXMWsJbBH48QRtSdgvgZMQyKbLBn+SJbtMCUjgJARWwSqBJsFvAmUCzVlnb2R8BqwJajugJiAlOGYsNqZd2rHdgS79qbGTcWzltmHvln07Pl/zOzYjkzm3bMYX/IFHZN/jC48c+rQz7/Qxc6V/q6wSk8he09uyZ/85CWw/Yefk4apPSiAbMBv8SRG4bAlI4MAEOqjuZRI0piY4Tb0qjM+AlQA6Otjpt94E8ATMK9vpS7A/bbfsvclA5sWHtjeTmIzhY+pZ0hcdSq87fTCeSc2ls6UD/ehN2fjdczJ36si2fMteWkfb8FoCIWAy4HNwegLZNFcHxenBCEACEjgMAQLVuSACWoLKyM2APO3sk1vJAP29l7bd9Ke0XPuRuTO2Cs7Tj2/3JAPY7fm4jt0OxtMfX2cf8rOfNfY4vtJ3qZ6+RXcG/qsEITbj55Q1GbhE27FLBEwGLtFx7PAEVhtqFj032cODcIESkMChCSSQnYEqgW/GEpimECDTTl/vh9iJbicNkUng2n2Rza9LAtbZh338aRvxJ/0p700GsobozMJ86Y8v/TJoK/iODn5gL/ajn358nzLIruroNmf8Yf2pV/4jN21GHrbcxyljWwIrAj//KV1J2CeBgxJg48zmOX/v2dAPisdlSUACByBA8D33uG7PZRJIItP7IfY6oI5+dGbgGtkpF1lsYL8DYoJqxmI3hXbq2Jz7d9pdpp3Wn7I9xnxtK/Ot+iPTfmzJYKvnyfWqkGBkfPJEPmN9T+hP3Wy3ZFreawmEwPpplI0EJCABCUhAAhKQgAQkcHgCJgOHv8UuUAISkIAEJCABCUhAAmsCJgNrLvZKQAISkIAEJCABCUjg8ARMBg5/i12gBCQgAQlIQAISkIAE1gSuJgNvb2/f/MnAZ8BnwGfAZ8BnwGfAZ8BnwGfgdZ+Bdah/vfdqMnDdhBISkIAEJCABCUhAAhKQwB4JmAzs8a7pswQkIAEJSEACEpCABB5AwGTgARA1IQEJSEACEpCABCQggT0SMBnY413TZwlIQAISkIAE/ibAx7/6A2Z/D3ohAQlcJGAycBGPg2cgkK9L8mXI1dcyz8DANUpAAhKAAPth6nzR9lLpL95ufTH3kv5HxvCNRCD+fnYy0OvP/PMrxD0+v3wcWVjPMbiE6dYYMtYS+CgBk4GPElR/1wQS/PPJdj4DPzfzXS9Q5yUgAQncQaAD7C119swEtOyjW7KP7o9/JAOxjb+fmQxkrksJUPzrF0zhRGAfXmnHxpbv0Uf+0fy0J4EmYDLQNLw+HYF5cGTz7QPmdEBcsAQkIIG/gmv+1nTrBQn75VckA5mz9+qtgPqZNzPzbwXrOVsI9vEhHEke+hrZtpXrXh82rCXwDAImA8+gqs3dEsjhNxOE3S5GxyUgAQncSSDBaAJWEoKVmQS2vOGefzOQQJhfvx2PnegRSEeGZANb6K3mJHBGJnV8zS/XGcfntLtknqnHeHTya/uXgvGWI8DHVuoO9ulHJ3WP0581pKS9sokdawk8msDPf1IebV17EtgRgRwE80Dbkfu6KgEJSOBhBEgGCNAJ2Jkg7fwY772TAB3ZtAluU6ed/TbBdurYyXzIRC/93cYWdcY6WCcZSD++ZB78ZjyB9rSfufCROac881IzR9rRiX50KKyNduoO+uGWvjlX7OFn63stgWcRMBl4Flnt7oYAG3E287mh72YROioBCUjggQSyLxJIEyy3+eyVKQS1BMfRIaBGnj02dUrGp0zmwEZk0Ok+7GFjlQy0fGwigz1sxE/WkL7Itk8E7m0P3VWNfYL4a8lAbOBDnzvRg1Pmzlh+rGM1t30S+CgBk4GPElT/MAR64z3MolyIBCQggTsIJCAlGWBvpJ2aa8ZSpySozm+WBLToJOiewS1B76wJjKe9aWMG45d8iS7zYHf6TTKwNT96XccG8rckA62b6+g2l/iYvnt8mbZtS+ASAZOBS3QcOx2BbLzZgC0SkIAEzkwgeyHBezgk0GVvTDDNG/BHJgMkFLdwvycZIAmI71kb62F9ncTcE4AnkA+3lNjPfF1gBbseS1/LT9m53tb1WgIfJWDU81GC6h+KQDbg3pAPtTgXIwEJSOBGAjMZIDidQSn9qVMSECfI7oCXwBqZaSN66evkI32RJ7j+brz+M21Ebs7bb/vnm/pnJAOZj3WzZtpxfZUgsKSsp2XhSt9cL3rWEngEAZOBR1DUxmEIZMPdOnwOs0gXIgEJSOAKgZkMRJy/HSBATR9BK4F++hKUZy+lzEB8FdgSzLed6PVc2Es9A330W75lct0+sRZstmz6YifruPU8iK8zmZnrjr1eH3NHbjUP87/XF+xaS+BWAiYDt5JS7nAE2GCz4fLrg+RwC3ZBEpCABG4gwH5Izb6YQDaBKyXBNTKpO9ju/tbp/gTgXQjokVkFyMi3LG/50Yuf7Vuu08d46vjUba5XsjPIjw9Tf8tXko7Y30oEmg/rS90+b8m0vNcSuJeAycC95NSTgAQkIAEJSEACEpDAzgmYDOz8Buq+BCQgAQlIQAISkIAE7iVgMnAvOfUkIAEJSEACEpCABCSwcwImAzu/gbovAQlIQAISkIAEJCCBewlcTQbe3t6++ZOBz4DPgM+Az4DPgM+Az4DPgM/A6z4DT0sG7jWsngQkIAEJSEACEpCABCTw2gSu/s3Aa7uvdxKQgAQkIAEJSEACEpDAvQRMBu4lp54EJCABCUhAAhKQgAR2TsBkYOc3UPclIIH9E5gfwOODRntfWdbRH6La+3r0XwISkMARCZgMHPGuuiYJSODTCMxAnq+b3uoA+sj3l1Pp20Odday+1LoH3/VRAhKQwJkJmAyc+e67dglI4GEEEggnEXhvid58e36vrffO/Uj5+JyfRQISkIAE9kXg/SfXvtantxKQgAQ+hcC9Afzqn9Lca+tTFrqYhL/dMBlYwLFLAhKQwIsTMBl48RukexKQwD4IrAL4X3/99Vt+BMv9T4i6L/358TcEW7aQSx19StrR+f3337/bSTvXKat/dsTckWGu6Pzxxx/fdXKdH/4wD/9bBsYjnx9tauxEPzpdpo1eB75EHr+nD23LawlIQAIS+DgBk4GPM9SCBCQggb+DalAkCSCgJqAlWO8A+Ja/GZgysUuQTQDegTfy6WOuyCXYJhHAt/SlkLikPwU5kooO1DMe29GhYJ92xtOHn+nPdeukHZkkD9hPG7+nD9i2loAEJCCBxxEwGXgcSy1JQAInJkAw2wgS+CawpRDc8uY8/QTuyKSetmYQTeCOzgy6V0lH/IidFPzIPJQ5Z/pjF5nUvZbpd8ticzVnr505SBDwIf5RYgMf6LOWgAQkIIHHETAZeBxLLUlAAicmQCDbCGbQThDO2/bIzqA6fStb6Uc/gTcBdPpnIH5rMtB+rOacdjNXSvrz6+RgJdvJwMqn2Or1r3xoG/+d3f9KQAISkMAjCZgMPJKmtiQggdMSWAWyj0oGSAIIvqfdGYivAu8OqrH33mSAJCA3uYP4tKcP6es5Vz5NOyuGbSPyFglIQAISeCwBk4HH8tSaBCRwUgKrQHYG7asgfAbVwTdtJSBOH2XanYH4KvDuoHrlx5wzc7XdzBlfKdPvlkWm58w/D1rJtN2VD20Du9YSkIAEJPA4AiYDj2OpJQlI4MQEVoHsDNpXQfgMqoNw2koQ3YF42rFNmUH2M5KBBOU9Z9r5UQjas8b4n0IfMllDfOV/N0CCEJ2Uue6VDWxZS0ACEpDAYwiYDDyGo1YkIIGTEiDAT5DLjwCfdoJiAl/6/vd///dvefqiR8BMH8E17dRThrH0E1DTN+elnzry097UyTgJBnqt0z6SICCXupOI1ssYZfZPrm0DHWsJSEACEvg4gR878cdtaUECEpCABCQgAQlIQAIS2BEBk4Ed3SxdlYAEJCABCUhAAhKQwCMJmAw8kqa2JCABCUhAAhKQgAQksCMCJgM7ulm6KgEJSEACEpCABCQggUcSuJoMvL29ffMnA58BnwGfAZ8BnwGfAZ8BnwGfgdd9Bu5NEK4mA/caVk8CEpCABCQgAQlIQAISeG0CJgOvfX/0TgISkIAEJCABCUhAAk8jYDLwNLQaloAEJCABCUhAAhKQwGsTMBl47fujdxKQgAQkIAEJ3EggH7LjC9g3qigmgdMTMBk4/SMggP7SKV9PlYoEJCABCfxMoPdKvjD91XtmvlxNwafPTgbmF7ubSX+5e8uvfF17a4y1WUvgmQRMBp5JV9svT4DDLZt5Sjbk3shffgE6KAEJSOCTCWSfTOD9CmX6kfZnB9YJ5jlDmkmfLyQMUy6+fra/7aPXEgiB1/jT7L2QwBcRyFulfrP0/Q/FL798y9sciwQkIAEJ/JPAqyQD2bu/OhlIcJ9kYFX4WwHGZqJySRcdawl8BgGTgc+g7BwvSyCb8wz8VwnCyy5AxyQgAQl8MoH3JAMJlLPPzkCYt+bpz3W/lEGHt+YZnwUZbBOQMw+BeNq9x/e8GUMv9tGJTNufc3e75eZbf+whj2/d5tpaAl9JwGTgK+k795cS4FCYG3gOJf+p0JfeGieXgARemMCtyUD2UQLx7LMdmGeMID/XBOUkAFk+8yA3kTDe/Zkj9kgu5n7eATnBenzjGn3OhZbvebhGDl9YR8b7jGH9yEeOa2xZS+CrCJgMfBV55/1yAr1RtzPz8OgxryUgAQmcnQCB7yUOCXQ7MI5sB/257mAY2dgmkI9Ort+bDLQ+QT6+JrgnQeEMoI3s9KvtYWernskDNrs/a8wvBR8yDoMt2/ZL4FkETAaeRVa7L0+ATbg3/jidjT8HlUUCEpCABP5J4JZkAJkEuf1jb80+S3/vwbmm/1oQzhztYXQJtNNPMD4TCnQjP5OBlk2A/p4gPbYuyWd9PR4e+Dt973V5LYFnEjAZeCZdbb88gT4IcDYb9bVDCFlrCUhAAmcjQCC9WneC3fwi00Hvlmz24PymbIJkxjo4bzsrP6JDcB3ZmQygk5oXQo9MBmaw3/7mOv5RmD86KWEwOSBrLYFnEvjxVD5zFm1L4EUJJOifgX82aw6HF3VbtyQgAQl8GQEC6pUDBLME4VOG/ZY64wmGs++SRHTwn6SgZdveyo9LyQDzYJ9gnP0enxnPXO8N0GMjfq1KbBH4Z5z56XvvXKs57JPAPQRMBu6hps5hCMzNOJs4h9lhFulCJCABCTyQAEF4B80xn37+GVDaue79NPIE9hkjaGYfTj1tRB+5uYQO3rF7KRlAnuCbduoU2r2u9wTosdvrb3+zhtU6msP0vfW9lsAzCZgMPJOutndBIBt4NuH8+uDahfM6KQEJSOCTCBC0s1+uaoJyXEqwi1wHypFLcMwYAXn63rMno9+20jftp49gHZ3IcD3rKdu+s7b2M/pb50d82xprplsyzGctgWcRMBl4FlntSkACEpCABCQgAQlI4MUJmAy8+A3SPQlIQAISkIAEJCABCTyLgMnAs8hqVwISkIAEJCABCUhAAi9OwGTgxW+Q7klAAhKQgAQkIAEJSOBZBK4mA29vb9/8ycBnwGfAZ8BnwGfAZ8BnwGfAZ+B1n4F7k4WrycC9htWTgAQkIAEJSEACEpCABF6bgMnAa98fvZOABCQgAQlIQAISkMDTCJgMPA2thiUgAQlIQAISkIAEJPDaBEwGXvv+6J0EJCABCUhAAhKQgASeRsBk4GloNbwnAny9Ml+UtEhAAhI4KgH2uvnFXdp8CfjW9ecLurG5x8LXkfvrwvkKMCyytln4YvBqbEsWe/PrzMjjB3J9DtHXPqKXOvdra6zlvJbAJQImA5foOHYKAr0R9yZ8isW7SAlI4JQEsu/N4DTtBJ+z/xKgJAJ7SwYSQGedCfy7ZB0E1mHAdcuk75ZEIDqtn7NlxTa+bPGOf4zFFtf4Ez96DvqtJfBeAiYD7yWm/CEJsFGbDBzy9rooCUhgEFgFlxHhzfgQXzZ5S76nZIBEIPUsHXwj14F/gvGV3rST9kou+jN4T7vnaFtJHrDzXt2247UErhEwGbhGyPFTEDAZOMVtdpESkMBfBN6TDLA/JjjNL2X2pZ9EIte8xUYndQe2yMdW+pGbwXLGGUvdL2wyX34kJRln3u9Ojv8gt5W8XEoG4uMl22OqZTPzZg5Kr3tlO+tpZs0m8oxhz1oC9xIwGbiXnHqHIsDB1gfNoRboYiQgAQkUgVUyQHDagWn6OghNMNvtBKwdXLOX9tvu2GuduMEcM0COHAEzwTu2Ipv50o5MriOPbfxHvpb7/TJzEmCnRh+52MdW+xx79CN7Tx2fO4DnvIl9/GnfIw+nzM91bHB9jx/qSGASMBmYRGyfkgAHGJvzKSG4aAlI4DQEElwSgHY998AEpN1HwE1fdDsZCMD0ddBLEI9Oaq7jRwfAyKYv122boJm++NZBOuPYnjczsvkRSCMfO5RcwyPjKe0j/s01or9Vx1bPs5KLX72eyOAL/bHDdcZX/q5s2yeBSwRMBi7Rcew0BHJ4ZNPdOkROA8KFSkACpyCQgJKgmAUT6GcvpBCMzppgP/0E5+jEbge+BLnMR00wPm2nnb24A92WQT/jPQ/28A1/qLPm6SvBfXRXJXNhj3MisrDa0pu22s851u2s85LNrIHx+E5iED+5bnteS+AWAj/+xN8irYwEDkqATd5k4KA32GVJQAI/EUjgSFDdAwS5jBGYt0xfZ3wG2OynBM1pd7CK7YxfCn4TQE/bPfcjkgHWS4Dd9jOGr+nv4Bvf0c86+KWvS2zcerZ0sN82ch07bTvrxz/8WK1j2rEtgUnAZGASsX1KAhxet27Yp4TkoiUggcMQ2EoG2AsJMiM3A/LIEJSukoFASn9ksIPd2EK35RpsxiMf3dUbdWy+NxmIXtbTJXPNvownqJ79W8lA25vXc71zfLbnnIw3S/pMBiBh/VECJgMfJaj+IQhwUKW2SEACEjg6gQSdBNWslbfdCeTZCxOEdjuy0eMNNHbS7qSBwLv7Ihtb6MZWxmcfCQD+tI34k1/Ke5OBlb3Mjb3vRv/6T3xtP9PNOZF+uEyZthG/23dszD50Mifc6UudOTI2S+zQD+8pY1sCtxAwGbiFkjKHJpADJQcCv3lAHnrxLk4CEjgVgQSQ7HVb9QxwCXyR7+AZewSlwESHduoOXmc/tlN3QBxfeoz9OfPRn2sCdfoy16pMe70W5GNv1Z9x1pt5tmQiFz/xZdY9T49N7pHD39VYxvv82pJhPmsJbBEwGdgiY78EJCABCUhAAhKQgAQOTsBk4OA32OVJQAISkIAEJCABCUhgi4DJwBYZ+yUgAQlIQAISkIAEJHBwAleTgbe3t2/+ZOAz4DPgM+Az4DPgM+Az4DPgM/C6z8C9OcvVZOBew+pJQAISkIAEJCABCUhAAq9NwGTgte+P3klAAhKQgAQkIAEJSOBpBEwGnoZWwxKQgAQkIAEJSEACEnhtAiYDr31/9E4CEpCABCQgAQlIQAJPI2Ay8DS0Gt4Lgfnxmr34rZ8SkIAErhG49PGrfPCqP/B1zVbGI3/pY1u32PhMmf4oV9Z7qbQscs1vi9V7OfaHxnI9PxbW4/hB3ecVfV3z8bXu81oC1whc/pNxTdtxCeycQDb/3oizCWeztUhAAhI4EoHsbfOrvAS/s//SuhMc7yUZiK8dwBNIz/URQM+9P+sMtxS+PDx1w7DnmOOzHTuXeGc++Pb8sZP1ZL6U1Fz3HPjbfV5L4BoBk4FrhBw/LIEkAXMTZ/PtBOGwAFyYBCRwGgKrZCCLTwA8g+AtKATNBKtbcq/SP/3E/9736VsF6B18I9e61wL7FYdLwXrszQA/9wbf+jp901Z027/V/PZJYEXAZGBFxb7TEmDDPy0AFy4BCRySwHuSAV6KRIdEofvSn1+CT64TnOYlCu3UBKbIJbhOIZDFRgOfNvrFTHzhbT/zECi3ja1rbLfN2MGvqXcpGcjaZuA+9We7173yu4N9dCPHPD2OrZZb2WTcWgKXCJgMXKLj2OkIZDPdOhhOB8MFS0AChyGQoHcGiwSU3Z9rgs8sPgEobYLpfuNOktCgIo9O+qPHHNlfe4/tYHy+jIlcxvEj1+0P/vfcl67ja/uFPmuIfeaLHfpzjSz2W46+azXJEeucNtLOnF163vDA/2Z8T2LSc3gtAZMBnwEJFIEcNBYJSEACRyOQQHP167fkWXP2wO4jGE1ffjNgpY9ANzYI4rGTAJfr6HdBFr0Ohgma6etEIPKMY7vtrq4TQLds2vEnPlDS7nMA/9LPGqPHdXzLWH5h9Z7SAX30YoO1Ygf+tOMb89GXNmXlL2PWEtgi8OMJ2pKwXwInIZBNlw3+JEt2mRKQwEkIrIJVAk2C3wTKBJqzzt7I+AxYE9R2QE1ASnDMWGxMu7RjuwNd+lNjJ+PYym3D3i37dny+5ndsRiZzbtmML/gDj8i+xxceOfRpZ97pY+ZK/1ZZJSaRvaa3Zc/+cxLYfsLOycNVn5RANmA2+JMicNkSkMCBCXRQ3cskaExNcJp6VRifASsBdHSw02+9CeAJmFe205dgf9pu2XuTgcyLD21vJjEZw8fUs6QvOpRed/pgPJOaS2dLB/rRm7Lxu+dk7tSRbfmWvbSOtuG1BELAZMDn4PQEsmmuDorTgxGABCRwGAIEqnNBBLQElZGbAXna2Se3kgH6ey9tu+lPabn2I3NnbBWcpx/f7kkGsNvzcR27HYynP77OPuRnP2vscXyl71I9fYvuDPxXCUJsxs8pazJwibZjlwiYDFyi49jhCaw21Cx6brKHB+ECJSCBQxNIIDsDVQLfjCUwTSFApp2+3g+xE91OGiKTwLX7IptflwSssw/7+NM24k/6U96bDGQN0ZmF+dIfX/pl0FbwHR38wF7sRz/9+D5lkF3V0W3O+MP6U6/8R27ajDxsuY9TxrYEVgR+/lO6krBPAgclwMaZzXP+3rOhHxSPy5KABA5AgOB77nHdnsskkESm90PsdUAd/ejMwDWyUy6y2MB+B8QE1YzFbgrt1LE59++0u0w7rT9le4z52lbmW/VHpv3YksFWz5PrVSHByPjkiXzG+p7Qn7rZbsm0vNcSCIH10ygbCUhAAhKQgAQkIAEJSODwBEwGDn+LXaAEJCABCUhAAhKQgATWBEwG1lzslYAEJCABCUhAAhKQwOEJmAwc/ha7QAlIQAISkIAEJCABCawJXE0G3t7evvmTgc+Az4DPgM+Az4DPgM+Az4DPwOs+A+tQ/3rv1WTgugklJCABCUhAAhKQgAQkIIE9EjAZ2ONd02cJSEACEpCABCQgAQk8gIDJwAMgakICEpCABCQgAQlIQAJ7JGAysMe7ps8SkIAEJCABCfxNgI9/9QfM/h70QgISuEjAZOAiHgfPQCBfl+TLkKuvZZ6BgWuUgAQkAAH2w9T5ou2l0l+83fpi7iX9j4zhG4lA/P3sZKDXn/nnV4h7fH75OLKwnmNwCdOtMWSsJfBRAiYDHyWo/q4JJPjnk+18Bn5u5rteoM5LQAISuINAB9hb6uyZCWjZR7dkH90f/0gGYht/PzMZyFyXEqD41y+YwonAPrzSjo0t36OP/KP5aU8CTcBkoGl4fToC8+DI5tsHzOmAuGAJSEACfwXX/K3p1gsS9suvSAYyZ+/VWwH1M29m5t8K1nO2EOzjQziSPPQ1sm0r170+bFhL4BkETAaeQVWbuyWQw28mCLtdjI5LQAISuJNAgtEErCQEKzMJbHnDPf9mIIEwv347HjvRI5CODMkGttBbzUngjEzq+JpfrjOOz2l3yTxTj/Ho5Nf2LwXjLUeAj63UHezTj07qHqc/a0hJe2UTO9YSeDSBn/+kPNq69iSwIwI5COaBtiP3dVUCEpDAwwiQDBCgE7AzQdr5Md57JwE6smkT3KZOO/ttgu3UsZP5kIle+ruNLeqMdbBOMpB+fMk8+M14Au1pP3PhI3NOeealZo60oxP96FBYG+3UHfTDLX1zrtjDz9b3WgLPImAy8Cyy2t0NATbibOZzQ9/NInRUAhKQwAMJZF8kkCZYbvPZK1MIagmOo0NAjTx7bOqUjE+ZzIGNolQANwAAAzVJREFUyKDTfdjDxioZaPnYRAZ72IifrCF9kW2fCNzbHrqrGvsE8deSgdjAhz53ogenzJ2x/FjHam77JPBRAiYDHyWo/mEI9MZ7mEW5EAlIQAJ3EEhASjLA3kg7NdeMpU5JUJ3fLAlo0UnQPYNbgt5ZExhPe9PGDMYv+RJd5sHu9JtkYGt+9LqODeRvSQZaN9fRbS7xMX33+DJt25bAJQImA5foOHY6Atl4swFbJCABCZyZQPZCgvdwSKDL3phgmjfgj0wGSChu4X5PMkASEN+zNtbD+jqJuScATyAfbimxn/m6wAp2PZa+lp+yc72t67UEPkrAqOejBNU/FIFswL0hH2pxLkYCEpDAjQRmMkBwOoNS+lOnJCBOkN0BL4E1MtNG9NLXyUf6Ik9w/d14/WfaiNyct9/2zzf1z0gGMh/rZs204/oqQWBJWU/LwpW+uV70rCXwCAImA4+gqI3DEMiGu3X4HGaRLkQCEpDAFQIzGYg4fztAgJo+glYC/fQlKM9eSpmB+CqwJZhvO9HrubCXegb66Ld8y+S6fWIt2GzZ9MVO1nHreRBfZzIz1x17vT7mjtxqHuZ/ry/YtZbArQRMBm4lpdzhCLDBZsPl1wfJ4RbsgiQgAQncQID9kJp9MYFsAldKgmtkUnew3f2t0/0JwLsQ0COzCpCRb1ne8qMXP9u3XKeP8dTxqdtcr2RnkB8fpv6WryQdsb+VCDQf1pe6fd6SaXmvJXAvAZOBe8mpJwEJSEACEpCABCQggZ0TMBnY+Q3UfQlIQAISkIAEJCABCdxLwGTgXnLqSUACEpCABCQgAQlIYOcETAZ2fgN1XwISkIAEJCABCUhAAvcSuJoMvL29ffMnA58BnwGfAZ8BnwGfAZ8BnwGfgdd9Bp6WDNxrWD0JSEACEpCABCQgAQlI4LUJXP2bgdd2X+8kIAEJSEACEpCABCQggXsJmAzcS049CUhAAhKQgAQkIAEJ7JyAycDOb6DuS0ACEpCABCQgAQlI4F4CJgP3klNPAhKQgAQkIAEJSEACOydgMrDzG6j7EpCABCQgAQlIQAISuJeAycC95NSTgAQkIAEJSEACEpDAzgn8P7nqXZeQfUILAAAAAElFTkSuQmCC\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eSemi-Quantative Analysis Scoring Methods.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFindings\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScore\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLess than 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBetween 6%-25%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBetween 26%-50%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMore than 50%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eData obtained from the semi-quantitative analysis were evaluated for normality of distribution using the Skewness-Kurtosis values, Q-Q plot, Levene’s test, and the Shapiro-Wilk test on the SPSS 20 (IBM, USA) statistics software. For non-parametric data, median-25% and 75% interquartile range were calculated and analyzed with the Kruskal Wallis test followed by Tamhane’s T2 test. P value \u0026lt; 0.05 was accepted as significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Findings\u003c/h2\u003e \u003cp\u003eIn the evaluation of the maxillary tissue sections stained with Harris hemotoxylin and Eosin G, odontoblasts, dentin, pulp, alveolar bone and periodontal ligaments of the normal structure were present in maxillary tissue sections obtained from the two-week CG and the four-week CG (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b, respectively; HDS: 0.5(0\u0026ndash;1)), 1(0\u0026ndash;1)). In contrast, sections from the two-week CGF treatment group showed extensive inflammation accompanied by edematous areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, respectively; HDS: 5(4\u0026ndash;5)). In maxillary tissue sections from the four-week CGFG, a decrease was found in the extensive edematous areas and infiltrative areas along with fibrous callus formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, respectively; HDS: 4(3\u0026ndash;4)). In the two-week MTAG, a decrease in infiltrative and edematous areas was observed along with the formation of cartilage callus at places ((Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, HDS: 2.5(3\u0026ndash;4)). In the four-week MTAG, a decrease in infiltrative and edematous areas was observed along with typical odontoblasts, dentin, periodontal ligaments, and alveolar bone structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, HDS: 2(1\u0026ndash;2)).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe two-week and four-week subgroups of the control group were not significantly different concerning HDS scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b, Respectively; HDS 0.5(0\u0026ndash;1), 1(0\u0026ndash;1)). In contrast, we determined a higher HDS score in the two-week CGFG (HDS:5(4\u0026ndash;5) compared to the two-week (HDS 0.5(0\u0026ndash;1)) and four-week control groups (1(0\u0026ndash;1)) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similarly, we determined a higher HDS score in the four-week CGFG (HDS:4(3\u0026ndash;4) compared to the two-week (HDS 0.5(0\u0026ndash;1)) and four-week (1(0\u0026ndash;1)) CG groups (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, we determined lower HDS scores in the two-week (HDS:2.5(3\u0026ndash;4)) and four-week (HDS: 2(1\u0026ndash;2)) MTAG compared to the two-week (HDS:5(4\u0026ndash;5) and four-week (HDS:4(3\u0026ndash;4) CGFG (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistopathological Damage Score Results HDS, (median-25%-75% interquartile range)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOedematous Area\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInflammation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHDS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCG\u003c/p\u003e \u003cp\u003e2W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0(0-0.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5(0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCG\u003c/p\u003e \u003cp\u003e4W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5(0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0(0-0.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGFG\u003c/p\u003e \u003cp\u003e2W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(2\u0026ndash;3)\u003csup\u003e\u003cb\u003ea,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(2\u0026ndash;3)\u003csup\u003e\u003cb\u003ea,b\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5(4\u0026ndash;5)\u003csup\u003e\u003cb\u003ea,b\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGFG\u003c/p\u003e \u003cp\u003e4W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5(1\u0026ndash;2)\u003csup\u003e\u003cb\u003ea,d,e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3(2-2.5)\u003csup\u003e\u003cb\u003ea,b,i\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4(3\u0026ndash;4)\u003csup\u003e\u003cb\u003ea,b,i\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMTAG\u003c/p\u003e \u003cp\u003e2W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(2\u0026ndash;2)\u003csup\u003e\u003cb\u003ea,c,f\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(1\u0026ndash;2)\u003csup\u003e\u003cb\u003ea,b,j\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.5(3\u0026ndash;4)\u003csup\u003e\u003cb\u003ea,b,g\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMTAG\u003c/p\u003e \u003cp\u003e4W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(1\u0026ndash;1)\u003csup\u003eb,c,g,h,i\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1(0\u0026ndash;1)\u003csup\u003e\u003cb\u003ea,b,g,k\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2(1\u0026ndash;2)\u003csup\u003e\u003cb\u003ea,m,g,k,i\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.000; versus to CG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.038; versus to CG 4W,\u003c/p\u003e \u003cp\u003e\u003csup\u003ec\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.000; versus to CG 4W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.001; versus to CG 4W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003ee\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.044; versus to CGFG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.011; versus to CGFG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003eg\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.000; versus to CGFG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003eh\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.046; versus to CGFG 4W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.000; versus to MTAG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003ej\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.001; versus to CGFG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003ek\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.000; ; versus to CGFG 4W,\u003c/p\u003e \u003cp\u003e\u003csup\u003ej\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.002; versus to CGFG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003em\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.012;versus to CGFG 2W,\u003c/p\u003e \u003cp\u003eKruskal Wallis/Tamhane T2 Test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical Analysis\u003c/h2\u003e \u003cp\u003eOn evaluation of the maxillary tissue sections incubated with the TNF-α primary antibody under a light microscope, we observed that immune-negative cells were common in the maxillary tissues of the two-week and four-week control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, respectively; TNF-α score: 0(0-0.5), 0(0\u0026ndash;1)). In contrast, we determined an increase in the number of cells showing TNF-alpha positivity in sections from the two-week CGFG and the four-week MTAG compared to those from the two-week and four-week control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, respectively; 3 TNF-α score : (2\u0026ndash;3), 2(2\u0026ndash;2) p\u0026thinsp;=\u0026thinsp;0.000, p\u0026thinsp;=\u0026thinsp;0.000). On the other hand, we determined a decrease in the number of cells showing TNF-α positivity in the two-week MTAG compared to the two-week CGFG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, d, respectively; TNF-α score: 3(2\u0026ndash;3), 1.5(1\u0026ndash;2), p\u0026thinsp;=\u0026thinsp;0.000). Similarly, we observed fewer cells showing TNF-alpha positivity in the maxillary tissue sections from the four-week MTAG compared to the four-week CGFG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,e, respectively; TNF-α score: 2(2\u0026ndash;2), 1(1\u0026ndash;1), p\u0026thinsp;=\u0026thinsp;0.001). When the two-week and four-week MTAG subgroups were compared to each other about the number of cells showing TNF-α positivity, we found a significant decrease in the number of cells showing TNF-α positivity in the two-week MTAG subgroup compared to the four-week MTAG subgroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed,e, respectively; TNF-α score: 1.5(1\u0026ndash;2), 1(1\u0026ndash;1), p\u0026thinsp;=\u0026thinsp;0.015). In sections of maxillary tissue incubated with TNF-alpha primary antibody, a decrease in cells showing immune positivity is observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef; TNF-alpha positivity score: 1(1\u0026ndash;1)) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSemi-Quantitative Analysis Results (median-25%-75% interquartile range)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003cp\u003ePositivity Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNF-kβ/p65\u003c/p\u003e \u003cp\u003ePositivity Score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCG\u003c/p\u003e \u003cp\u003e2W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(0-0.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0(0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCG\u003c/p\u003e \u003cp\u003e4W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0(0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0(0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGFG\u003c/p\u003e \u003cp\u003e2W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(2\u0026ndash;3)\u003csup\u003e\u003cb\u003ea,b\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(2\u0026ndash;3)\u003csup\u003e\u003cb\u003ea,b\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCGFG\u003c/p\u003e \u003cp\u003e4W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(2\u0026ndash;2)\u003csup\u003e\u003cb\u003ea,b\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(2\u0026ndash;2)\u003csup\u003e\u003cb\u003ea,b\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMTAG\u003c/p\u003e \u003cp\u003e2W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2(1.5-2)\u003csup\u003e\u003cb\u003ea,b,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5(1\u0026ndash;2)\u003csup\u003e\u003cb\u003ea,b,c\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMTAG\u003c/p\u003e \u003cp\u003e4W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(1\u0026ndash;1)\u003csup\u003e\u003cb\u003ea,b,c,d,e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1(1\u0026ndash;2)\u003csup\u003e\u003cb\u003eb,c,e,g\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.000; versus to CG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.000; versus to CG 4W,\u003c/p\u003e \u003cp\u003e\u003csup\u003ec\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.000; versus to CGFG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003ed\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.001; versus to CGFG 4W,\u003c/p\u003e \u003cp\u003e\u003csup\u003ee\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.015; versus to MTAG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003ef\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.008; versus to CGFG 2W,\u003c/p\u003e \u003cp\u003e\u003csup\u003eg\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.014; versus to MTAG 2W,\u003c/p\u003e \u003cp\u003eKruskal Wallis/Tamhane T2 Test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOn light microscopic examination of the maxillary tissue sections incubated with NF-kB/p65 primary antibody, we observed that immune-negative cells were common in the maxillary tissues of the two-week and four-week CG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b, respectively; NF-kB/p65 score: 0(0\u0026ndash;1), 0(0\u0026ndash;1)). In contrast, we determined an increase in the number of cells showing NF-kB/p65 positivity in the sections from the two-week and four-week MTAG compared to those from the two-week and four-week CG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, respectively; NF-kB/p65 score: 2(2\u0026ndash;3), 2(2\u0026ndash;2) p\u0026thinsp;=\u0026thinsp;0.000, p\u0026thinsp;=\u0026thinsp;0.000). On the other hand, we determined a decrease in the number of cells showing NF-kB/p65 positivity in the two-week MTAG compared to the two-week CGFG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,d, respectively; NF-kB/p65 score: 2(2\u0026ndash;3), 1.5(1\u0026ndash;2), p\u0026thinsp;=\u0026thinsp;0.000). Similarly, we observed fewer cells showing NF-kB/p65 positivity in the maxillary tissue sections of the four-week CGFG compared with those of the four-week MTAG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,e, respectively; NF-kB/p65 score: 2(2\u0026ndash;2), 1(1\u0026ndash;2), p\u0026thinsp;=\u0026thinsp;0.015). When the two-week and four-week subgroups of CGFG were compared to each other concerning the number of cells showing NF-kB/p65 positivity, there was a significant decrease in the number of cells showing NF-kB/p65 positivity in the two-week CGFG compared to the four-week CGFG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e respectively; NF-kB/p65 score: 1.5(1\u0026ndash;2), 1(1\u0026ndash;2), p\u0026thinsp;=\u0026thinsp;0.014) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs there is no data in the literature regarding the effects of second-generation autologous growth factors on the tissue damage induced by pulpotomy, the present study is the first that has investigated the effects of CGF, which is a second-generation autologous growth factor, on the TNF-α and NF-kB/p65 pathways at a molecular level and different intervals, in comparison to MTA, which is a bioactive endodontic cement.\u003c/p\u003e \u003cp\u003eAlthough studies that have investigated the tissue injuries that develop due to pulpotomy in clinics are experimental animal models, many researchers have stated that human and rat teeth have morphometric similarities [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Rat incisors are different from human teeth in the sense that they show continuous growth, they were proven to represent a model for the evaluation of the reactions of the human dental pulp to bioactive molecules [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVital pulp treatments use an appropriate pulp capping material and this material is directly placed onto the pulp tissue; therefore, biocompatibility, the anti-inflammatory property, and cytotoxicity need to be completely evaluated before a new material is used as a pulp capping material [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In a study by Minic et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] that investigated pulpotomy-induced pulpitis using a rat model, findings of inflammation and cellular degeneration were reported. Similarly, in the present study, we determined extensive edematous areas accompanying inflammation.\u003c/p\u003e \u003cp\u003eIn a study by Nguyen [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] on pulpotomy-induced pulpitis, the inflammation and edematous areas observed in the second week were reported to be reduced in the fourth week. On the other hand, we did not determine any difference in terms of inflammation and edematous areas in the present study. Minic et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] observed an increase in TNF-α levels after pulpotomy. Although no scientific studies examine NF-kB/p65 levels in pulpitis that develops after pulpotomy. We determined elevated TNF-α and NF-kB/p65 levels in pulpitis developed after the present study's pulpotomy.\u003c/p\u003e \u003cp\u003eBiological scaffolds and CGF, which is known to be a cytokine reservoir, are used for bone regeneration in the current clinical implantology practice [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As a latest generation platelet concentrate product, CGF has a simpler modified production process that requires the repeated alteration of the centrifugation speed [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consequently, its relatively dense structure is more comparable to that of natural fibrin and involves ample amounts of growth factors and proteins that are derived from autologous platelets and leukocytes. No studies are addressing the effect of pulp damage induced by CGF pulpotomy. In addition, studies are reporting that CGF increases periodontal regeneration in clinical studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. \u0026Ouml;ng\u0026ouml;z Dede et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] demonstrated that CGF contributed to gingival regeneration by increasing the gingival thickness. In addition, Firouzi et al. (2022) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] reported that CGF enhanced bone regeneration induced by lipopolysaccharide in cell culture. Similarly, in our study, we observed that CGF regenerated pulpotomy-induced damage. In current studies, although the effects of CGF on tissue regeneration are available, the information about its effect on tissue damage mechanisms is not clear. In addition, studies conducted today have reported that CGF suppresses TNF-α and NF-kB/p65 expressions [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similarly, Xu et al. described in their in vitro and in vivo studies that CGF TNF-α expression mediated inflammation by suppressing pulp regeneration [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although there are no studies on the effects of CGF on NF-kB/p65 and any studies on its impact on odontogenesis. Yuan et al. demonstrated that, NF-kB/p65 in lipopolysaccharide-induced dental pulp damage. They reported that increasing (NF-kB/p65) expression, triggers inflammation together with TNF-α [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Again, Kamal et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] reported that CGF application reduced inflammation and pain induced by low-dose laser application. Similarly, our study found that CGF decreased NF-kB/p65 positivity together with TNF-α.\u003c/p\u003e \u003cp\u003eMTA has been adopted as an endodontic material, and especially a pulp capping material for longer than a decade; however, its certain biological effects on pulp tissue regeneration remain unknown [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Silva et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], reported that the cytokines IL-1, IFN-, and the chemokine CCL5 in the pulp tissue of rat incisors were modified when exposed to MTA. Inflammatory cytokines and chemokines play a role in the aggregation of leukocytes towards tissues that are injured or infected; however, may exacerbate tissue damage as well [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Tohma et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] employed the MTA pulpotomy model for a period of 14 days. The results of the study revealed a scar-healing pattern that was generally consistent with previous studies. Similarly, Prabhakar et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] reported that the inflammation and edema caused by pulpotomy-related pulpitis in rats decreased after MTA application. They reported an improvement in tissue structure on days 14 and 30 after the pulpotomy. Salako et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] reported that MTA reduced the inflammation resulting from pulpotomy-induced pulpitis in 14 days. Moreover, they determined that inflammation was further reduced by MTA on day 30 compared to day 14. Similarly, we observed that the MTA treatment reduced the inflammation encountered in pulpotomy-induced pulpitis in 14 days and also that this reduction was more pronounced on day 30. A study by Benetti et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] on dental root canal treatment reported MTA to exert an anti-inflammatory effect by causing a decrease in TNF-α expression. Similarly, in the present study, we found that the MTA treatment reduced the pulpotomy-induced increase in TNF-α positivity. Moreover, we demonstrated a further decline in cells showing TNF-α positivity on day 30 compared to day 14. However, although the effects of MTA on the nuclear factor-kappa B signaling system have not been previously researched, MTA can cause the initial inflammation of rat dental pulp cells through its effects on the NF-kB/p65 signaling system, while a subsequent anti-inflammatory effect is also observed due to the regulation of the expression of certain inflammatory mediators. Minic et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] revealed that it stimulates the toll-like receptor 4 found on dental pulp cells and produces inflammatory cytokines such as IL-1β and IL-6 by activating the NF-kB pathway in pulpotomy-induced pulpitis.\u003c/p\u003e \u003cp\u003eFirst, our study is a pilot study considering the effects of second-generation autologous growth factors on pulpotomy-induced inflammation by tissue levels of NF-kB/p65 and TNF-α. Additionally, there are some limitations to our study. The present study should be supported by studies dealing with other inflammatory cytokines and cell signaling molecules. Similarly, our study needs to be corroborated by research on intracellular and mitochondrial calcium levels that address cell damage mechanisms.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, in the present study, we determined a reduction in cells showing NF-kβ/p65 positivity in the MTA treatment group compared to the CGF group. Moreover, we determined fewer cells showing NF-kβ/p65 positivity on day 30 after the MTA application compared to day 14 after the MTA application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003eA. K. contributed to conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing (original draft preparation), visualization, supervision, project administration, funding acquisition; A. Ş. \u0026Ccedil;. contributed to conceptualization, methodology; E. B. contributed to conceptualization, methodology; Z. G. contributed to conceptualization, methodology, T. M.\u0026nbsp;contributed to conceptualization, methodology, validation, formal analysis, data curation; \u0026nbsp; L. T. contributed to conceptualization, methodology, validation, formal analysis, data curation; M. K. contributed to conceptualization, methodology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was financed in part by the Recep Tayyip Erdogan University Research Foundation \u0026nbsp;(Grant No: TDH-2021-1242). The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e:\u0026nbsp;The required ethics committee decision for the study was obtained from Recep Tayyip Erdogan University, Faculty of Medicine, Animal Research Ethics Committee (approval number: 2020/47).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThere is \u0026nbsp;no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledge:\u0026nbsp;\u003c/strong\u003eWe thank Recep Tayyip Erdogan University Research Foundation for its support in the study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePeres MA, Macpherson LMD, Weyant RJ, et al. 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Medical Science Monitor: International Medical Journal of Experimental and Clinical Research.\u003cem\u003e \u003c/em\u003e2018; 24:6656.\u003c/li\u003e\n\u003cli\u003eKamal A, Salman B, Abdul Razak NH, Qabbani AA, Samsudin AR. The Efficacy of Concentrated Growth Factor in the Healing of Alveolar Osteitis: A Clinical Study. 2020. doi:10.1155/2020/9038629.\u003c/li\u003e\n\u003cli\u003eModena KCdS, Casas-Apayco LC, Atta MT, et al. Cytotoxicity and biocompatibility of direct and indirect pulp capping materials. Journal of Applied Oral Science.\u003cem\u003e \u003c/em\u003e2009;17:544-554.\u003c/li\u003e\n\u003cli\u003eSilva MJB, Vieira LQ, Sobrinho APR. The effects of mineral trioxide aggregates on cytokine production by mouse pulp tissue. \u003cem\u003eOral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. \u003c/em\u003e2008;105(5):e70-e76.\u003c/li\u003e\n\u003cli\u003eTohma A, Ohkura N, Yoshiba K, et al. Glucose transporter 2 and 4 are involved in glucose supply during pulpal wound healing after pulpotomy with mineral trioxide aggregate in rat molars. \u003cem\u003eJournal of Endodontics. \u003c/em\u003e2020;46(1):81-88.\u003c/li\u003e\n\u003cli\u003ePrabhakar A, Mandroli PS, Bhat K. Pulpotomy with curcumin: Histological comparison with mineral trioxide aggregate in rats. \u003cem\u003eIndian Journal of Dental Research. \u003c/em\u003e2019;30(1):31.\u003c/li\u003e\n\u003cli\u003eSalako N, Joseph B, Ritwik P, Salonen J, John P, Junaid T. Comparison of bioactive glass, mineral trioxide aggregate, ferric sulfate, and formocresol as pulpotomy agents in rat molar. \u003cem\u003eDental traumatology. \u003c/em\u003e2003;19(6):314-320.\u003c/li\u003e\n\u003cli\u003eBenetti F, Mello PB, Bonini JS, Monteiro S, Cammarota M, Izquierdo I. Early postnatal maternal deprivation in rats induces memory deficits in adult life that can be reversed by donepezil and galantamine. \u003cem\u003eInternational Journal of Developmental Neuroscience. \u003c/em\u003e2009;27(1):59-64.\u003c/li\u003e\n\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":"Concentrated growth factor, Inflammation, Mineral trioxide aggregate, Pulpotomy, NF-kB/p65, TNF-α","lastPublishedDoi":"10.21203/rs.3.rs-4165836/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4165836/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of our study is to see the effect of Mineral Trioxide Aggregate (MTA), bioactive endodontic cement, and Concentrated Growth Factor (CGF), second-generation autologous growth factor, on pulpotomy-induced pulp inflammation. The study used the maxillary anterior central teeth of thirty-six young male Sprague Dawley rats. In total, 72 maxillary central teeth were included. We randomly assigned 48 teeth to two groups (12 rats/group; 24 teeth/group) based on the capping material (MTA or CGF). Two subgroups per group (12 teeth/group) were formed based on the time following pulpotomy (two weeks and four weeks). Compared to the control group, MTA and CGF applications decreased the formation of inflammation and edematous areas. We also observed that it decreased Nuclear Factor kappa B/P65 (NF-kB/P65) and Tumor Necrosis Factor (TNF)-α positivities. We determined fewer cells showing NF-kβ/p65 positivity in the MTA treatment group compared to the CGF group. Moreover, we determined fewer cells showing NF-kβ/p65 positivity on day 28 after the MTA application compared to day 14 after the MTA application. In the CGF group, both histological damage scores and TNF positivity decreased. This study has established that, second-generation autologous growth factors have a protective effect in treating inflammation after pulpotomy.\u003c/p\u003e","manuscriptTitle":"The Effects of Mineral Trioxide Aggregate and Second-Generation Autologous Growth Factor on Pulpotomy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-09 03:14:58","doi":"10.21203/rs.3.rs-4165836/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-14T11:16:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-13T13:42:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-13T13:42:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217654974472148549939452940641425137224","date":"2024-05-04T09:26:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-02T12:15:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-02T10:55:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4feb6fe0-6dcf-449d-9a59-9f80d8bcbd31","date":"2024-04-23T02:49:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38845c97-014a-4cc1-ad61-27805f7e17cb","date":"2024-04-22T11:15:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-22T00:49:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-16T12:29:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-04T10:34:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-04T09:40:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2024-03-25T22:56:27+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":"7462b16c-6ee2-4572-9fe7-51d7d96dff5b","owner":[],"postedDate":"April 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T16:00:45+00:00","versionOfRecord":{"articleIdentity":"rs-4165836","link":"https://doi.org/10.1186/s12903-024-04577-z","journal":{"identity":"bmc-oral-health","isVorOnly":false,"title":"BMC Oral Health"},"publishedOn":"2024-08-03 15:57:10","publishedOnDateReadable":"August 3rd, 2024"},"versionCreatedAt":"2024-04-09 03:14:58","video":"","vorDoi":"10.1186/s12903-024-04577-z","vorDoiUrl":"https://doi.org/10.1186/s12903-024-04577-z","workflowStages":[]},"version":"v1","identity":"rs-4165836","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4165836","identity":"rs-4165836","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}