ORIGINAL ARTICLE

Novel evaluation method of dentin repair by direct pulp cappingusing high-resolution micro-computed tomography

Motoki Okamoto1& Yusuke Takahashi1 & Shungo Komichi1 & Manahil Ali1 & Naomichi Yoneda1 & Takuya Ishimoto2

&

Takayoshi Nakano2& Mikako Hayashi1

Received: 31 August 2017 /Accepted: 29 January 2018# Springer-Verlag GmbH Germany, part of Springer Nature 2018

AbstractObjectives We evaluated a novel micro-computed tomography (micro-CT) assessment for quality and quantity of dentin repair,which is difficult to visualize by histological analysis, after direct pulp capping under standardized cavity preparation.Materials and methods Standardized cavities were prepared on Wistar rats and direct pulp capping was performed using twocommercial bioceramics, ProRoot MTA, and iRoot BP Plus. After 2 or 4 weeks, quality and quantity of tertiary dentin formationwere evaluated using high-resolution micro-CT analyses including dentin mineral density, dentin mineral contents, compactnessand integrity of tertiary dentin, and dentin volume with/without void space. Reproducibility of micro-CTanalyses was confirmedby histological evaluation of the same specimen.Results The exposed pulp area sizes were similar between iRoot BP Plus and ProRoot MTA. Micro-CT analysis of 2-weeksamples showing compactness of tertiary dentin was significantly higher in iRoot BP Plus than ProRootMTA (p < 0.05). Tertiarydentin volume without void space, dentin mineral contents, and density were not significantly different between the groups. In 4-week samples, a significant increase was observed in dentin mineral density, compactness, and dentin volume with/without voidspace induced by iRoot BP Plus (p < 0.05). Micro-CT analysis of tertiary dentin integrity demonstrated that some ProRoot MTAspecimens had small defects and lacked continuity (6/512 images). No defects were observed with iRoot BP Plus.Conclusions Micro-CT analysis was confirmed as an accurate, objective, and inclusive approach for evaluating quality andquantity of dentin repair.Clinical relevance These multifaceted approaches to evaluate pulp capping materials may accelerate review processes, ultimatelyimproving vital pulp therapy.

Keywords Micro-computed tomography evaluation . Direct pulp capping . iRoot BP Plus . Mineral trioxide aggregate

Introduction

As one of themost important endodontic treatments, direct pulpcapping has been widely used to maintain dental pulp vitality.

This treatment involves covering the pulpal wound surface withdental material to facilitate reparative dentin formation [1].

Evaluation of direct pulp capping materials in vivo hasbeen primarily performed by histopathological analysis [2].However, sectioned histological specimens are relativelythin, do not reflect the entire structure, and can be vulnerableto subjective evaluation. Additionally, other factors such asangle or direction of the specimen during sectioning mayproduce different results. It is crucial to measure qualityand quantity of regenerated dentin with precise, objective,and all-inclusive methods for accurate evaluation of pulpcapping agents. In the field of bone measurement, panopticimage analysis has been recently introduced to objectivelyevaluate bone-filling materials and other regenerative proce-dures [3, 4]. In the field of dental materials, several reportsalso evaluated tertiary dentin formation using micro-

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s00784-018-2374-5) contains supplementarymaterial, which is available to authorized users.

* Yusuke Takahashitakahasi@dent.osaka-u.ac.jp

1 Department of Restorative Dentistry and Endodontology, OsakaUniversity Graduate School of Dentistry, 1-8, Yamadaoka, Suita,Osaka 565-0871, Japan

2 Division of Materials and Manufacturing Science, Osaka UniversityGraduate School of Engineering, Suita, Osaka 565-0871, Japan

Clinical Oral Investigationshttps://doi.org/10.1007/s00784-018-2374-5

computed tomography (micro-CT) images [5–7]. However,these reports used relatively low-resolution images that couldnot be used to analyze detailed characteristics such as densityor integrity. Additionally, most previous reports measureddentin thickness or evaluated dentin morphology from onlyone or a few slices of all images. Such evaluations could besimilar to histological methods using thin sections. Thus, wesuggest that more comprehensive methods are necessary foraccurate and objective evaluation such as three-dimensionalreconstructed images from micro-CT, which contains thewhole structure of the specimen. However, to our knowl-edge, no study has investigated the complete structure ofspecimens at the same resolution as the thickness of a histo-logical tissue slice.

One type of direct pulp capping material, mineral trioxideaggregate (MTA), has been widely accepted because of itshigh potential to promote wound healing of the dentin-pulpcomplex [8–10]. Previous studies demonstrated that MTA in-duced greater dentin bridge formation compared with conven-tional calcium hydroxide materials [11, 12]. However, con-cerns regarding MTA—including possible release of hazard-ous substances, tooth discoloration, delayed setting time, mix-ture inconsistency, lack of adhesion to dentin, and especiallyhandling inconvenience—limit its application in dental proce-dures [13].

Thus, nano-sized bioceramic materials have been con-sidered to play crucial roles in pulpal repair and regener-ation by facilitating biological function and/or improvinghandling convenience of conventional bioceramics [14].iRoot BP Plus (Innovative BioCeramix Inc., Vancouver,Canada), a ready-to-use, laboratory-synthesized, calciumsilicate-based nanoparticulate bioceramic putty, has beensuggested to offer improved performance in dental prac-tice compared with hand-mixed MTA [15]. It is insoluble,radiopaque, and aluminum-free, contains zirconium oxide,and was developed for dental surgery applications [16].iRoot BP Plus is speculated to be a promising MTA alter-native because of its nano-modified nature and handlingconvenience [17, 18]. A previous report compared thesematerials; however, evaluation methods used for tertiarydentin formation were conventional histology and micro-CT from only several sliced sections [19]. Additionally,another report evaluating pulp capping materials did notmeasure or standardized the size of exposed pulp area, animportant factor of the pulpal healing process [20].

Therefore, the aim of this study was to establish an all-inclusive method for evaluating the pulpal repair process.We used high-resolution micro-CT to evaluate quantity andquality of tertiary dentin formation induced by two com-mercial pulp capping materials. We also confirmed micro-CT findings with those of conventional histologicalmethods under the condition of standardized size of ex-posed pulp for a fair evaluation.

Materials and methods

Pulp capping procedure

TenmaleWistar rats (weighing 180–200 g; CLEA Japan, Inc.,Tokyo, Japan) were used for direct pulp capping experiments.Twenty maxillary first molars were randomly divided into twogroups by pulp capping material used iRoot BP Plus (Lot1403BPP) or ProRoot MTA (Dentsply, Tulsa, OK, USA;Lot 108824). Rats were anesthetized with medetomidine hy-drochloride (Domitol; Meiji Seika Pharma Co., Ltd., Tokyo,Japan) at a dose of 0.3 mg/kg, midazolam (Dormicum;Astellas Pharma Inc., Tokyo, Japan) at a dose of 4.0 mg/kgand butorphanol (Vetorphale;Meiji Seika Pharma Co., Ltd.) ata dose of 5.0 mg/kg. Then, local infiltrative anesthesia wasgiven as a 0.5-mL injection of 2% lidocaine with 1:100,000epinephrine (Xylocaine; Dentsply Pharmaceuticals Inc., York,PA, USA). After cleaning and disinfecting teeth with cottonsoaked in 75% ethanol, a rubber dam sheet (Heraeus Kulzer,South Bend, IN, USA) with a custom-made rubber dam clamp(YDM, Tokyo, Japan) was placed on the maxillary first molarto prevent contamination (Fig. 1). A class I cavity was pre-pared on the occlusal surface with pulp exposure using a steel#1 round bur (diameter of 0.8 mm; Dentsply Maillefer,Ballaigues, Switzerland) with a low-speed electric engine(VIVAMATE G5, NSK, Tochigi, Japan) under sterile salinespray by one experienced operator to establish a stable andstandardized sized cavity (approximately 1-mm diameter anddepth) with pulp exposure. Drilling time was around 10 s foreach sample. Bleeding was controlled with light pressureusing sterile cotton pellets. Then, the exposed pulp was direct-ly capped with iRoot BP Plus or ProRoot MTA using anendodontic explorer (Hu-Friedy, Chicago, IL, USA) withoutpressure following the manufacturer’s protocol. After direct

�Fig. 1 Pulp capping and micro-CT evaluation methods. Direct pulpcapping was performed towards the mesial pulpal horn under rubberdam application (a). Micro-CT images from three directions (sagittal,axial and coronal) were obtained (b–d, representative micro-CT imagesof pulp capped with ProRoot MTA). After determining the exposed pulparea from the deepest area of the pulp capping material in the sagittalplane (black dotted line, e), the area enclosed by the black dotted linewas measured as the exposed pulp area (f). The area enclosed by the blackdotted line indicates tertiary dentin formation induced by ProRoot MTA(g, h). Images taken in coronal and sagittal planes were used to measureand evaluate the quality of tertiary dentin. A representativephotomicrograph of H-E staining illustrates the methodology used tomeasure the total area of tissue volume (TV; black dashed line) (i). Theyellow area beneath the pulp capping material in the sagittal planeindicates TV of induced tertiary dentin area containing no hard tissuespace (j) and dentin volume (DV) of tertiary dentin without any voidspace (k). E = enamel, GIC = glass ionomer cement, P = pulp, PCM =pulp capping material, PD = primary dentin, TD = tertiary dentin, M =mesial, D = distal, P = palatal, B = buccal, O = occlusal, A = apex. Thecolor scale indicates the mineral density of different tissue (1 mg/cm3

(blue) to 2550 mg/cm3 (red)) (b–h)

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pulp capping, the cavity was restored with glass ionomer ce-ment (Fuji IX; GC International Corp, Tokyo, Japan) accord-ing to the manufacturer’s instructions. Cusp tips of the oppos-ing mandibular tooth were cut to minimize occlusal forces.

Three-dimensional micro-CT analyses

Rats were sacrificed at 2 or 4 weeks after direct pulp capping.Induced tertiary dentin (n = 5/group and time point) was ana-lyzed using a micro-CT scanner (SMX100CT; Shimadzu,Kyoto, Japan) with a scanning resolution of 7.1-μm pixel sizeunder the following settings: voltage, 50 kV; current, 150 μA;and exposure time, 20 min in a vacuum environment. Afterscanning, 512 consecutive image slices were obtained. In eachspecimen, a calibration curve was prepared from mineral ref-erence phantoms (Ratoc System Engineering, Tokyo, Japan)to convert the CT value to the calcification degree.Representative micro-CT images for microstructure of tertiarydentin are shown in Fig. 1b–d. After scanning, image datawere reconstructed and quantitatively analyzed using three-dimensional reconstruction imaging software (TRI/3D-BON;Ratoc System Engineering).

The size of exposed pulp area was evaluated in 2 and4 weeks samples by measuring the area at the pulpal edge ofthe applied capping material in the axial plane with guiding ofthe sagittal plane (Fig. 1e, f; indicated by black dotted lines).The region of tertiary dentin for structural morphometry wasdefined from the mesial pulp horn in sagittal plane imageswith guiding of the coronal plane (Fig. 1g, h). Finally, theregion of tertiary dentin was confirmed by histological sec-tion. A representative photomicrograph of hematoxylin-eosin(H-E) staining to illustrate the methodology used to measurethe total area of tissue volume (TV) is shown in Fig. 1i. Dentinmineral density (DMD; mg/mm3) of tertiary dentin in sagittalplane images was standardized using reference phantoms: thelower-calcified region beneath the cavity compared with pri-mary dentin was assumed to be the area of induced tertiarydentin. The volume of each region was measured as TV,which was defined as the volume of tertiary dentin includingdefects or cellular contents (Fig. 1j). Dentin volume (DV) wasdefined as the tertiary dentin area without any necrotic area,defect, or cellular content (Fig. 1k), and DVwas automaticallycalculated from TV based on the mineral density using thesame software. Dentin mineral contents (DMC) were calcu-lated by multiplying DMD by DV; compactness of tertiarydentin was defined by the DV/TV ratio; and DMD, presentedas DMC/DV, was evaluated using TRI/3D-BON.

Histological evaluation

After micro-CT analysis, the same specimens were used forhistological evaluation to confirm micro-CT findings for faircomparison. After perfusion, specimens including

experimental teeth were dissected and fixed in 4% paraformal-dehyde for 24 h at 4 °C. Specimens were subsequentlydemineralized in Kalkitox solution (Wako Pure Chemicals,Osaka, Japan) for 1 day at 4 °C following the manufacturer’sprotocol and then embedded in paraffin. Then, sagittal sec-tions (7-μm thick) were stained with H-E. Histological criteria[21] were adopted for histologic evaluation according to thecriteria presented in Supplemental Table 1. For the histologi-cal evaluation, two observers were trained to evaluate histo-logical features according to the criteria with 95% consistency.In cases of disagreement, the two observers discussed until aconsensus was reached.

Statistical analysis

All values are expressed as mean ± standard deviation.Differences between evaluation methods were determinedby Student’s t test. P values < 0.05 were considered statistical-ly significant.

Results

Size of standardized cavity preparation measurement

The size of exposed pulp area was not significantly differentbetween the two pulp capping materials (Table 1).

Micro-CT analysis of tertiary dentin formation

In 2-week samples, micro-CT images of both materialsshowed similar characteristics of induced tertiary dentin for-mation according to DV (Fig. 2a), TV (Fig. 2b), DMD(Fig. 2c), and DMC (Fig. 2d). For compactness of tertiarydentin (Fig. 2e), iRoot BP Plus showed significantly superiorresults compared with ProRoot MTA (p < 0.05).

In 4-week samples, iRoot BP Plus demonstrated signifi-cantly greater compactness of tertiary dentin compared withProRoot MTA (p < 0.05) (Fig. 2e). Furthermore, iRoot BPPlus induced greater density (Fig. 2c). DV, TV, and DMCvalues were similar between both groups (Fig. 2a, b, d).

Table 1 The size of exposed pulp area capped with iRoot BP Plus orProRoot MTA at 2 or 4 weeks. Data are expressed as mean ± standarddeviation. The areas were not significantly different between the two pulpcapping materials (p > 0.05, Student’s t test; n = 5/group)

The size of exposed area (× 10−2 mm2)

iRoot BP Plus ProRoot MTA

2 weeks 4.89 ± 0.679 5.20 ± 0.865

4 weeks 4.96 ± 0.779 4.80 ± 0.912

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Three-dimensional micro-CT analysis of tertiarydentin integrity

In 4-week samples, tertiary dentin formation induced by bothmaterials was investigated using three planes of micro-CTimages and three-dimensional reconstructed images (Fig. 3a,b). Some ProRoot MTA-treated specimens had small defectsaround the center of tertiary dentin with decreased integrity(3/5 samples). Additionally, these defects (42 μm in size) werevisible only in 6 of 512 images (slices 199–204) (Fig. 3c).Conversely, tertiary dentin formation induced by iRoot BPPlus had no defects.

Histological evaluation

In H-E-stained histological sections, no significant differencein tertiary dentin formation induced by iRoot BP Plus orProRoot MTA was observed in both 2- and 4-week samples

based on histological criteria (Supplemental Fig. 1 andSupplemental Table 2).

A continuous dentin bridge was induced by both ProRootMTA and iRoot BP Plus at each time point. Additionally, nodefects in tertiary dentin were observed in both ProRoot MTAand iRoot BP Plus specimens (Supplemental Fig. 1).

Discussion

Direct pulp capping plays a critical role in vital pulp therapy.Many pulp capping methods under different conditions havebeen investigated; consequently, many pulp capping materialshave been developed and sold on the market. Histologicalanalysis has mainly been used to evaluate pulp capping mate-rials [22, 23]. However, histological methods cannot revealthe entire healing process, even when using serial sections. Itis difficult for histological evaluation to reveal all repair reac-tions of pulp tissue after damage. Additionally, this method is

Fig. 2 Quantitative comparisonof each parameter obtained fromhigh-resolution micro-CTanalysis after direct pulp cappingusing iRoot BP Plus and ProRootMTA. In 2-week samples, tertiarydentin formation induced by eachmaterial showed similarcharacteristics according to dentinvolume (DV) (a), tissue volume(TV) (b), dentin mineral density(DMD) (c), and dentin mineralcontents (DMC) (d) (p > 0.05).However, the DV/TV (e) value,which indicates compactness oftertiary dentin, was significantlyhigher in iRoot BP Plus samplescompared with ProRoot MTAsamples (p < 0.05). For 4-weeksamples, tertiary dentin formationinduced by each material wassimilar according to DV (a), TV(b), and DMC (d). However,iRoot BP Plus samples showedsignificantly higher DMD (c) andDV/TV (e) values compared withProRoot MTA (p < 0.05). n = 5/group

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not suitable for quantitative evaluation because thin sectionsare used, which can lead to subjective bias.

Panoptic image analysis has been recently introduced in thefield of bone measurement to evaluate quality and quantity ofbone regeneration [3, 4]. In the field of pulp capping, previousstudies used both histological and micro-CT analyses to eval-uate tertiary dentin formation; however, most studies usedimage slices and reported only the observation of inductedtertiary dentin [5, 6]. To our knowledge, no study has investi-gated the complete structure of specimens at the same resolu-tion as the thickness of a histological tissue slice. In a previousstudy, the scanning resolution of micro-CTwas approximately17–37-μm pixel size [5–7], which is considered to be lowerthan that in the present study (7.1 μm) and this resolution was

considered to be sufficient to evaluate quality and quantity oftertiary dentin using rat teeth from the results. Additionally, noreports evaluated size of the exposed area of pulp tissue, al-though this affects the pulpal healing process. Stanley empha-sized that control of bleeding from exposed pulp tissue andconfirmation that the pulp capping material directly contactsvital pulp tissue must be considered to improve the successrate of pulp capping [20]. The standardized size of exposedpulp in the present study made it easier to control hemostasis.

Table 1 shows the cavity sizes of the samples, which werenot significantly different. Thus, tertiary dentin formation, in-cluding reparative and reactionary dentin formation, was af-fected only by stimulation of preparation or pulp capping ma-terials. Additionally, we also emphasize the standardized size

Fig. 3 Representative three-dimensional reconstructed micro-CT imagesof tertiary dentin formation induced by iRoot BP Plus (a) and ProRootMTA (b). Tertiary dentin volume appeared to be abundant in ProRootMTA specimens but contained defects. Tertiary dentin induced by iRootBP Plus had less volume but appeared to have greater density than thatinduced by ProRoot MTA. Some ProRoot MTA samples (3/5 samples)had defective structures. Representative images of a ProRoot MTAspecimen are shown from consecutive micro-CT data in axial (upper)

and sagittal (lower) planes (c). A void space was observed in someimage slices (182/512, 192/512 and 212/512), but only 6 of 512 images(slices 199–204) showed a tunnel-like defect in tertiary dentin (blackarrow). GIC = glass ionomer cement, PCM = pulp capping material,PD = primary dentin, TD = tertiary dentin. M = mesial, D = distal, P =palatal, B = buccal, O = occlusal, A = apex. The color scale indicates themineral density of different tissue [1mg/cm3 (blue) to 2550mg/cm3 (red)](c)

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of exposed pulp might be an important factor to evaluate theeffect of direct pulp capping materials susceptible to bleeding[20]. Therefore, the present study evaluated pulpal woundhealing ability under fair comparison using standardized cav-ity preparation followed by application of iRoot BP Plus orProRoot MTA as pulp capping materials and assessed qualityand quantity of tertiary dentin using high-resolution micro-CTin combination with the conventional histological method.

Previous reports showed the effect of MTA on pulpalwound healing and this material has been regarded as a goldstandard for vital pulp therapy [24, 25]. However, disadvan-tages of MTA have also been reported, which may limit itsapplication in dental surgery [11]. iRoot BP Plus, a ready-to-use, laboratory-synthesized, calcium silicate-basednanoparticulate bioceramic putty, has been suggested to offerimproved performance in dental practice comparedwith hand-mixed MTA [15]. iRoot BP Plus is speculated to be a prom-isingMTA alternative because of its nano-modified nature andhandling convenience [17, 18] As a potential alternative,iRoot BP Plus has been demonstrated to have an importantrole in tissue repair and regeneration because of its uniquenanostructure, which facilitates bioactivity, biological func-tion and/or handling convenience of bioceramics [12].Additionally, iRoot BP Plus could induce odontoblastic dif-ferentiation [26]. Recently, newly developed bioceramicshave improved handling convenience, setting time, and toothdiscoloration [27]. Regarding biologically appropriateness forapplication in acidic environments such as wound area, iRootBP plus shows favorable results compared with MTA [28].However, limited information is available as few studies havecompared these materials.

The three-dimensional radiographic assessment used in thisstudymade it possible to observe the entire structure of tertiarydentin in a panoptic view with a high resolution of 7.1 μm onindividual images. We also evaluated quality and quantity oftertiary dentin by calculating micro-CT parameters using astandardized phantom. A tunnel-like defect in tertiary dentin,which is considered to be a negative factor for protecting pulpvitality, is difficult to evaluate using only histological imagesbecause of limited information acquired from thin sections[29]. Thus, we used a more reliable evaluation method bycalculating the above parameters for both quantitative andqualitative analyses under standardized cavity preparation.Use of high-resolution micro-CT images allowed detectionof small defects, and we found decreased integrity fromProRoot MTA specimens (3/5 samples). Conversely, no de-fect in tertiary dentin was observed in iRoot BP Plus samples.One ProRoot MTA specimen exhibited a complete tunnel-shaped defect on micro-CT analysis (Fig. 3c). However, thisdefect was not detected in histological analysis; histologicalresults of the two direct pulp capping materials were the sameconcerning tertiary dentin formation (Supplemental Table 2).Histological findings in this study were similar to those

previously reported [19]. However, defects may have beenmissed in studies using only histological sections(Supplemental Fig. 1).

The volume of tertiary dentin (DV and TV, Fig. 2a, b)induced by iRoot BP Plus or ProRoot MTA was not signifi-cantly different. iRoot BP Plus tended to induce early tertiarydentin formation with fewer defects compared with ProRootMTA (Fig. 2a). However, DV/TV values, indicating compact-ness of tertiary dentin, were significantly higher in iRoot BPPlus than in ProRoot MTA at both 2- and 4-week time points(Fig. 2e).

From the result of DMD, tertiary dentin formation inducedby iRoot BP Plus had less porosity than tertiary dentin inducedby ProRoot MTA (Fig. 2c). Additionally, iRoot BP Plus couldinduce tertiary dentin formation quicker than ProRoot MTA(Fig. 2a). This may be attributed in part to apatite-formingability of iRoot BP Plus [30] and its suitability in acidic con-ditions [28]. Higher DMD values observed in iRoot BP Plusspecimens might be because of earlier tertiary dentin forma-tion, and later, tertiary dentin formation induced by ProRootMTA could result in greater mineralized crystal growth at4 weeks after pulp capping (Fig. 2c). From our results, iRootBP Plus has the potential to induce higher quality tertiarydentin than ProRoot MTA, suggesting that iRoot BP Plus ismore suitable for clinical use as a pulp capping material.Additionally, the reasons for these results may be related toconcerns about ProRootMTA characteristics, such as possiblerelease of hazardous substances and delayed setting time [31].

To our knowledge, this study is the first report to evaluatequality and quantity of tertiary dentin using high-resolutionmicro-CT analysis at the same resolution as the thickness ofa histological tissue slice under the standardized size of ex-posed pulp. It should be noted that this study was performedunder ideal conditions with artificially exposed healthy pulptissue in vivo. Therefore, further investigation is required toconfirm our results using caries or pulpitis models.

Additionally, micro-CT can only evaluate hard tissues.Thus, micro-CT evaluation cannot evaluate pulp status andcompletely replace histological assessment. Moreover,micro-CT observation must be performed under strong vacu-um conditions to obtain high-resolution images, which re-quires a highly dried condition compared with typical histo-logical specimens. As a result, pulp tissue shrinkage and de-creased dye staining may have occurred. Thus, our analysesfocused on tertiary dentin formation, which is not affected bydrying, rather than pulp inflammatory response which mightnot be the optimal condition for evaluation. Nevertheless, thecombined use of high-resolution micro-CT and conventionalhistological methods may overcome the limitations of eachtechnique. Furthermore, development of contrast agents andmagnetic resonance imaging technology to evaluate soft tis-sues may improve diagnostic imaging methods, which willfacilitate the evaluation of pulpal wound healing ability of

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bioceramics more accurately. Moreover, such techniques willreduce the number of experimental animals needed, therebyimproving animal welfare.

Although many kinds of bioceramic materials have beennewly developed, evaluation of the clinical effectiveness andapplication of such products remains time-consuming. Thus, amultifaceted approach to evaluate new materials may acceler-ate review processes, ultimately improving vital pulp therapy.

Conclusion

We confirmed micro-CT analysis is a powerful and objectiveapproach to evaluate induced tertiary dentin formation afterpulp capping. Additionally, iRoot BP Plus has a greater po-tential to induce tertiary dentin formation than ProRoot MTAunder standardized cavity preparation.

Funding This work was supported by JSPS KAKENHI (grant numbersJP16K20453, 17H06848, and 17K11704).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflicts ofinterest.

Ethical approval The animal experimental protocol in this study wasapproved by the Ethical Guidelines Committee for Animal Care ofOsaka University Graduate School of Dentistry (no. 23-005-1). All sur-geries were performed under general anesthesia and efforts were made tominimize suffering.

Informed consent For this type of study, formal consent is not required.

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