Revascularization after Pulpectomy of Immature Molars in Rats

272019年 2月

Revascularization after Pulpectomy of Immature Molars in Rats

MINATO Hanae1, KITAJIMA Kayoko2,ARAI Kyoko2 and IGARASHI Masaru1,2

1The Nippon Dental University, Graduate School of Life Dentistry at Niigata, Course of Clinical Science, Field of Advanced Conservative Dentistry and Periodontology, Endodontics

2Department of Endodontics, The Nippon Dental University School of Life Dentistry at Niigata

Abstract　Purpose: The purpose of this study was to histologically examine the amount of regenerated tissue and the healing of periapical tissue after revascularization following the pulpectomy of rat immature teeth by using mineral trioxide aggregate（MTA）compared with calcium hydroxide（CH）.　Materials and Methods: Six-week-old Wistar male rats were used. The pulp was removed and the root canals were shaped with a Ni-Ti rotary file under rubber dam isolation. An H-file was inserted to induce bleeding over 1 mm from the apical foramen. Blood clot formation in the entire root canal was confirmed and MTA or CH was applied on the canal orifice. The experimental periods were one, two and four weeks after the operation. The specimens were observed histologically by HE staining and immunohistochemically by DMP1 and nestin expression.　Results: The formation of regenerated tissue was greater in the MTA groups than in the CH groups. Sig-nificantly more regenerated tissue was seen in the MTA groups compared with the CH groups at one week（p＝0.041）and four weeks（p＝0.015）after the operation. Significantly more hard tissue was formed at the cervical third of the MTA group at four weeks compared with the CH group（p＝0.004）. Each postoperative period of the MTA groups（p＝0.004）and each postoperative period of the CH groups（p＝0.041）showed a gradually increasing tendency in the formation of hard tissue toward four weeks after the operation. Each postoperative period of the MTA groups（p＝0.015）showed a gradually increasing tendency in the formation of bone-like tissue four weeks after the operation. All of the hard tissue formed in the root canal was bone-like tissue. No inflammatory cell infiltration was observed. Expression of DMP1, a regulator of mineralization, was observed underneath the MTA, in the granulation-like tissue under the MTA and around the lumpy structure of the apex side at one week. Expression of nestin, an odontoblast-like cell marker, gradually increased in the apex toward four weeks after the operation.　Conclusion: This study shows that revascularization using MTA is more effective compared to CH in the formation of regenerated tissue and bone-like mineralized tissue in the root canal following the pulpectomy of vital immature teeth.

Key words: revascularization, immature molars, MTA

　Corresponding author: Dr. MINATO, The Nippon Dental University, Graduate School of Life Dentistry at Niigata, Course of Clinical Science, Field of Advanced Conservative Dentistry and Periodontology, Endodontics, 1‒8, Hamaura-cho, Chuo-ku, Niigata 951‒8580, Japan　TEL: ＋81‒25‒211‒8171, FAX: ＋81‒25‒267‒1795, E-mail: [email protected]　Received for Publication: November 15, 2018/Accepted for Publication: January 9, 2019　DOI:10.11471/shikahozon.62.27

日歯保存誌 62（1）：27～38，2019Original Article

28 日　本　歯　科　保　存　学　雑　誌第 62巻　第 1号

Introduction

　Dental root development progresses through the for-mation of dentin by odontoblasts, achieved by signal-induced differentiation of dental papilla inside the Her-twig epithelial sheath derived from the inner and outer enamel epithelium. When dentin grows in the apical direction, the Hertwig epithelial sheath on the cervical side becomes a net-l ike epithelial funiculus, and cemento-blasts are differentiated and induced on the outer surface of the exposed dentin and form cemen-tum. When the root length is determined, the formation of the tooth root is completed by forming a narrowed portion at the apical end of the root. Permanent teeth that are in the progress of this root formation are referred to as having immature root formation and are classified into 1/4, 1/2 or 3/4 tooth root formation, tooth root length completion, apical foramen completion, etc. based on the degree of the root formation. A tooth prior to the phase of root length completion is charac-terized by a blunderbuss shaped apical foramen and a thin root canal wall.　When examining the X-ray of a tooth with immature root formation, it is difficult to clearly understand the condition of the root apex because of its insufficient cal-cification due to the enlarged apical foramen. Regular root canal treatment is challenging partly because the root canal cannot be properly formed, and it is impossi-ble to apply an apical seat and/or apical collar, resulting in the failure to tightly close the apical foramen. Hence, apexogenesis is used to promote the physiological growth of vital teeth with immature root formation; in nonvital teeth, apexification is initially used to induce the closure of the apical foramen to enable conventional root canal filling. That is, the aim of apexogenesis is to preserve the vital dental pulp in the root canal and keep it alive so that the root formation induced in the Hertwig epithelium sheath continues and reaches the completion of the root apex formation.　In apexification, a chemical agent that promotes hard tissue formation is applied inside the root canal, after removing its contents, to seal the apical foramen with hard tissue or fibrous tissue, and then root canal filling is conducted. However, thickening of the dentin on the root canal wall cannot be expected. Instead, there is a thick root canal with a thin wall. The risk of dental

fracture following long-term occlusal force, etc. has been pointed out; hence, applying hard tissue to the root canal wall would be appropriate1）. Regenerative end-odontic therapies have recently been reported, includ-ing attempts to add hard tissue to the root canal wall2,3）. Regenerative endodontic therapies aim to fill the root canal with stem cells and scaffolds after removing the root canal contents of the nonvital tooth, regenerate the pulp, form hard tissue on the root canal wall, and complete the tooth root formation; one of these therapies is revascularization2,3）. The procedure involves inducing bleeding from the apical periodontal tissue of the inferior tooth root after removing the root canal contents, filling the root canal with blood and forming a blood clot, on which mineral trioxide aggre-gate（MTA）or calcium hydroxide（CH）is applied. Initially, revascularization treatment involved the use of CH paste in the root canal4）. Later, MTA was used instead of CH in the revascularization procedure, with MTA paste applied on a clot in the root canal5）. Using the clot itself as a scaffold, the regeneration of pulp blood flow, induction of differentiation into odontoblasts and formation of regenerative tissue at the root canal wall are expected2,3,6,7）.　In the case of irreversible pulpitis where acute inflammation has spread to the root canal pulp due to caries progression, a pulpectomy would be necessary; it would be required even for teeth with immature root formation. In some cases of a pulpectomy conducted near the root apex because the position of the apex cannot be determined, CH paste may be applied follow-ing apexification8）. In this case, continuous growth of the apex may be expected after treatment because there is still living tissue around the root apex, but the root canal where CH was applied remains thick7）. On the other hand, case studies5,9‒14） and animal experi-ments15‒20） have reported that root growth and tissue formation in the root canal occurred after revascular-ization therapy. In a case of revascularization therapy for a dog tooth, an increase was reported in the thick-ness of the root and the vertical root length due to the ingrowth of cementum-like tissue15）. We selected rat teeth in this study because the maxillary first molar of the rat has five roots, each with a root canal. Especially for the mesial root, the root canal is large and of a form that makes it easier to conduct root canal therapy.　In this study, we developed a model of nonvital teeth

292019年 2月 Revascularization after Pulpectomy of Immature Molars in Rats

with immature root formation treated with pulpectomy due to irreversible pulpitis using rat teeth and applied MTA and CH on the blood clots after conducting a revascularization procedure. The purpose of this study was to histologically observe and compare the differ-ences between MTA and CH application in the wound areas in the root canal and the apical periodontal tissue.

Materials and Methods

1 ．Animal experiments　Six-week-old Wistar male rats（Japan SLC, Shizuoka, Japan）were used. The first molar of the maxillary right side served as the test tooth, and the mesial root was used. The animals were given chow diet and water and were managed under an appropriate environment. This study was conducted with the approval of the Animal Experiment Ethics Committee at The Nippon Dental University School of Life Dentistry at Niigata（Approval Number NDUN-189）.

2 ．Operative method　General anesthesia was administered intraperitone-ally using a mixed anesthetic of medetomidine hydro-chloride（0.15 mg/kg）, midazolam（2 mg/kg）and butorphanol tartrate（2.5 mg/kg）. A rat was strapped to the operation table, moisture control was conducted using a rubber dam clamp for the rat（YDM, Tokyo, Japan）and a 5.0×3.5 cm sized rubber dam sheet, and the operative field was disinfected（Fig.　1）. A maxillary molar endodontic access opening was created with φ 0.5 mm round-type diamond point MI-1L（HORICO DENTAL Hopf, Berlin, Germany）under a surgical microscope（Leica M 650; LEICA Microsystems, Wetz-lar, Germany）. Dental pulp removal at the crown of the

tooth and cutting at the root canal were performed with a microexcavator（SUNDENTAL Co., Osaka, Japan）, and the pulp chamber was alternately cleaned with 6％ sodium hypochlorite（SIOE PHARMACEUTI-CAL Co., Hyogo, Japan）and 3％ hydrogen peroxide water（Daiichi Sankyo Company, Tokyo, Japan）. After confirming the mesial root canal, the root canal pulp was removed using H-files #10, #15, #20（MANI, Inc., Tochigi, Japan）with a working length of 3.5 mm and pulling needle #00（MANI）, after which a Ni-Ti file（PROTAPER F1; DENTSPLY Maillefer, Ballaigues, Swi tzer l and）was a t tached to the endo motor（X-SMART; DENTSPLY Maillefer）for enlargement of the root canal（Fig.　2）. Any remaining pulp tissue in the root canal was completely removed using an H-file and a pulling needle, and the inside of the root canal was irrigated with physiological saline（Otsuka Pharmaceuti-cal Co., Tokyo, Japan）. The teeth from which the tissue in the root canal was removed were regarded as a non-vital model. After drying the root canal with a #20 paper point（Morita Corporation, Osaka, Japan）and confirming hemostasis of the mesial root canal, the root canal was filled with blood by inserting a #10 H-file about 1 mm past the apical foramen. After confirming clot formation, MTA（PROROOT MTA; DENTSPLY Maillefer）or CH（Wako Pure Chemical Industries, Osaka, Japan）was affixed to the top of the blood clot. In root canals other than the mesial root canal, the pulpec-tomy was performed by a conventional method. The cavity was sealed with resin lining material（Cavios; Neo Dental Chemical Products Co., Tokyo, Japan）, bonding（G-Bond Plus; GC, Tokyo, Japan）and composite resin（Unifil Flow; GC）to prevent microleakage and maintain aseptic conditions. The experimental periods

Fig.　1　 Rubber dam using spe-cial clamp for rat Fig.　2　 Confirmation of cleaned

root canal


were one, two and four weeks after the operation. 3 ．Preparation of specimen, histological observa-

tion　After the completion of the experimental periods in each group, the animals were again placed under gen-eral anesthesia, and whole-body perfusion fixation was performed with 4％ paraformaldehyde phosphate buffer solution（pH 7.4）. The test teeth including the periapical tissue were extracted and fixation was achieved by immersion at room temperature for one week. Samples were decalcified with 10％ EDTA-2Na solution（pH 7.4）at room temperature for four weeks, dehydrated, pene-trated and embedded in paraffin according to a conven-tional method, and a continuous section of 6μm in thickness was prepared in the buccolingual direction. After deparaffinization, hematoxylin-eosin（HE）staining was performed and histological observation was per-formed under an optical microscope. Observations were made on the formation of regenerated tissue, hard tis-sue, bone-like structures and dentin, and inflammation of periapical tissue.

4 ．Immunohistological observation　Tissues were embedded in paraffin, continuously sec-tioned at a thickness of 6μm, and deparaffinized according to a conventional method. DMP1（dilution ratio 1：1,000; TaKaRa Bio, Shiga, Japan）and nestin（dilution ratio 1：1,000; NES; Merck Millipore, Darm-stadt, Germany）were used as the primary antibodies. DMP1 is a dentin noncollagenous extracellular matrix protein that has been implicated in the regulation of mineralization21,22）. Nestin is used as a marker of differ-entiation or differentiated odontoblast-like cells21,23）. The sections were incubated with DMP1 antibodies at room temperature for 5 min using Hysto/Zyme（Diagnostic BioSystems, CA, USA）. For NES, the slides were immersed in Immunosaver buffer diluted 200 times, microwaved at 95℃ continuously for 15 min using a microwave processor and then reacted at room tem-perature for 30 min. After each antigen activation, the cells were washed with Tris-buffered saline（TBS）（TBS Tablets, pH 7.6; TaKaRa Bio）and cultured in 2.5％ horse serum（VECTOR LABORATORIES, CA, USA）, followed by blocking by incubating for 30 min. Next, reaction with the primary antibody was carried out overnight at 4℃ and then washed with TBS. Endogenous peroxidase inactivation was performed using 3％ H2O2（Dako REAL Peroxidase-Blocking Solu-

tion; Dako, CA, USA）, and then DAB（Liquid DAB Plus, Dako）was inoculated using a polymerized secondary antibody（EnVision Plus Dual Link System-HRP, Dako）to develop color. After counterstaining with hematoxy-lin solution, histological observation similar to that with HE staining was performed using an optical micro-scope.

5 ．Statistical analysis　A total of 36 cases, 6 cases in each group, were examined. The results for the MTA group at one week after the operation are referred to as MTA1, two weeks after the operation as MTA2, and four weeks after the operation as MTA4; the results for the CH group one week after the operation are referred to as CH1, two weeks after the operation as CH2, and four weeks after the operation as CH4. Based on the obser-vation with HE staining, the root of each tooth, from the apical end to the stenotic portion of the cervical canal, was divided into three parts: the root apex（apex 1/3）, the middle third of the root（central 1/3）and the root cervix（cervical 1/3）for evaluation. Evaluation criteria were set using each observation item and the numeri-cal values for statistical processing（Table 1）, and histo-logical observation and analysis were performed. The difference between the MTA group and the CH group in each postoperative period was compared by the Mann-Whitney U test（Table 2）. In addition, the differ-ence between each postoperative period within the MTA group and the CH group was compared using the Kruskal-Wallis test（Table 3）（IBM SPSS Statistics ver.　24; IBM Japan, Tokyo, Japan）.

Results

1 ．Formation of regenerated tissue in the root canal

　Formation of regenerated vital tissue in the root canal was observed in all cases in both groups. Com-pared to the CH group, the MTA group showed signifi-cant increases in the amount of tissue formation across a wide range of areas up to the cervical 1/3 at one week（p＝0.041）and four weeks（p＝0.015）after the operation（Fig.　3, Table 2）.　In the MTA group, an eosinophilic layer was found underneath the MTA paste, and granulation-like tissue was observed directly under the layer. In addition, mas-sive structures with heavy hematoxylin concentration


were found in the root canal（Fig.　3, MTA1, MTA2 and MTA4）. In the CH group, the formation of granulation tissue was observed only up to the central 1/3 in all the test weeks, which was smaller than the area of tissue formation in the MTA group, and hematoxylin-concen-trated mass structures were found from the first week after the operation in the CH group（Fig.　3, CH1, CH2, CH4）, similarly to the MTA group. There was also an eosinophilic nonstructural region from the cervical 1/3 underneath the CH paste to the central 1/3（Fig.　3, CH1 and CH2）. Revascularization was observed up to the cervical 1/3 in the MTA group and up to the center 1/3 in the CH group（Fig.　3B）.

2 ．Formation of hard tissue　In the MTA group, the extent of hard tissue forma-tion increased to the cervical 1/3 over time（Fig.　3B, Table 1）. In most of the CH group, the formation of

hard tissue was observed only up to the apex 1/3, which was the same region where the granulation tis-sue had formed（Fig.　3C, Table 2）. In both groups, the formation of hard tissue increased from one to four weeks after the operation. In a comparison between the MTA group and the CH group, a significant difference was observed at four weeks after the operation（p＝0.004, Table 2）. The MTA group（p＝0.004）and the CH group（p＝0.041）showed an increasing tendency in the formation of hard tissue（Table 3）. Furthermore, cementum formation occurred in the apical end of the root canal in all the cases, and stenosis of the apical foramen was observed at four weeks after the opera-tion（Fig.　3C, MTA4, CH4）.

3 ．Formation of bone-like hard tissue　Bone-like hard tissue was observed in the root canal in all cases（Fig.　3, Table 1）. The MTA group showed

Table　1　Numerical scales in each experimental group

Scales for each parameterExperimental groups（n＝6）

MTA1 CH1 MTA2 CH2 MTA4 CH4

Formation of regenerated tissue　0＝No formation 0 0 0 0 0 0　1＝Formation at apex 1/3 1 2 2 1 0 0　2＝Formation up to central 1/3 0 4 2 5 1 6　3＝Formation up to cervical 1/3 5 0 2 0 5 0

Formation of hard tissue　0＝No formation 5 4 2 2 0 0　1＝Formation at apex 1/3 1 2 0 4 0 5　2＝Formation up to central 1/3 0 0 3 0 2 1　3＝Formation up to cervical 1/3 0 0 1 0 4 0

Formation of bone-like tissue　0＝No formation 5 4 2 2 0 0　1＝Formation 1 2 4 4 6 6

Inflammation of periapical tissue　0＝No infiltration of inflammatory cells 6 6 6 6 6 6　1＝Infiltration of inflammatory cells 0 0 0 0 0 0

Table　2　 Differences between the MTA and the CH groups at each postoperative period

Parameter Week 1 Week 2 Week 4

Formation of regenerated tissue 0.041a 0.818 0.015a

Formation of hard tissue　Formation of bone-like tissue

0.6990.699

0.2401.000

0.004b

1.000

Inflammation of periapical tissue 1.000 1.000 1.000

Significant difference in a：α＜0.05；b：α＜0.01.

Table　3　 Differences between each postoperative period of MTA and CH

Parameter MTA CH

Formation of regenerated tissue 0.128 0.322

Formation of hard tissue　Formation of bone-like tissue

0.004b

0.015a0.041a

0.059

Inflammation of periapical tissue 1.000 1.000

Significant difference in a：α＜0.05；b：α＜0.01.


Fig.　3　HE staining for each group（bars＝400μm）　（A）Magnified views of central 1/3 of MTA4, （B）cervical 1/3 of MTA4 and（C）apex 1/3 of CH4 are shown（bars＝100μm）. Hematoxylin-positive lumpy structures（black arrows）, bone-like tissue（＊）, and blood vessels（▲）are observed.


an increasing tendency in bone-like hard tissue（p＝0.15, Table 3）. In the enlarged images of the hard tissue found in MTA4 and CH4, bone-like tissue containing some cells and tissues were observed in massive struc-tures concentrated in the hematoxylin（Fig.　3A, B, C）.

4 ．Inflammation of apical periodontal tissue　No inflammatory cell infiltration was observed in any of the cases（Fig.　3, Table 1）.

5 ．Immunostaining 1 ）Directly under medical application

　In MTA1, cell-dense granulation tissue was observed directly under the medical application（Fig.　4A）. There was a DMP1-positive reaction directly at the interface with MTA and the area directly underneath（Fig.　4B, C）, and the positive reaction was weak in MTA2 and MTA4. Moreover, no NES-positive reaction was observed in any of the cases（Fig.　4D）.

2 ）Apical foramen　In MTA1, the cementum extended from the apex, and hematoxylin-stained massive structures were observed in the apex 1/3（Fig.　4A）. DMP1-positive reac-tion was found around a massive structure in the same area（Fig.　5B, C）.　MTA4 showed a greater increase in the thickness of cementum extending from around the apex compared to that in MTA1（Fig.　5,　6A）. NES-positive reaction was observed at the apical end of the same area（Fig.　6B）.

Discussion

　Apexification has been considered an appropriate procedure for treating a nonvital tooth with immature root formation caused by a pulpectomy for treating irreversible pulpitis of the pulp of an immature perma-nent tooth; however, revascularization has recently drawn attention as an alternative therapy2‒4）. While conventional apexification halts the growth of the tooth root and thickening of the root canal wall, studies have reported that revascularization retained these abili-ties15‒17）. Some studies suggest that the growth of the tooth root length and the thickening of the root canal wall are induced by the formation of cementum instead of dentin because the formation of dentin was not found. Other studies report the formation of cementum or bone-like hard tissue in the root canal15‒20）. Details on tissue formation in the root canal following revascular-ization are still unclear.

　Dogs and/or monkeys have usually been used in ani-mal experiments of endodontic treatment, but the recent development of clamps for rats has enabled their experimental use. The present study used Wistar SPF rats, which are considered suitable for animal experi-ments because the structure of their molars is similar to that of humans, having one apical foramen in one main root canal, individual differences are minimal, and their short life cycle allows prompt completion of the observation24）. However, based on a report that cemen-tum builds up in the root canal as the rats age25）, it is important to understand the normal structural changes of the molar with age. This experiment used untreated symmetric teeth as a control for comparison. In addi-tion, it was confirmed that the root length growth due to dentin formation occurred at six to ten weeks of age, and that cementum had not formed in the root canal. In this experiment, mechanical enlargement was used in order to develop a model of nonvital teeth with imma-ture root formation, and the working length was deter-mined by referring to the average value at six weeks of age. It was considered that a procedure for aseptic treatment could be established because the application of the photopolymerized cementum to the top of the spinal cord material did not cause infection26）.　The results showed a clear quantitative difference between the MTA and the CH groups in tissue forma-tion in the root canal. The formation of regenerated tis-sue and hard tissue, and the regeneration of blood ves-sels were confirmed up to 1/3 of the tooth cervix in the MTA group, whereas in the CH group, the formation of regenerated tissue was limited from 1/3 of the central area of the root to 1/3 of the apex of the root. Both MTA and CH have antibacterial properties and cause cell necrosis due to their alkalinity at the time of appli-cation27）; furthermore, CH exhibits more extensive necrosis layer formation and inflammatory cell infiltra-tion compared to MTA8,28）. Similarly, this experiment also found an unstructured layer from 1/3 of the cervi-cal part to 1/3 of the central part of the root in the CH group, which indicates the necrotic layer because there was no tissue formation in this part. It is reported that MTA shows faster neutralization and apatite formation compared with CH. From this, there is a difference in the amount of hard tissue formation after surgery between MTA and CH. Based on this finding, it may be expected that hard tissue is more likely to be formed


Fig.　4　Magnified view of cervical 1/3 in MTA1　（A）HE,（B, C）DMP1, and（D）NES staining. （A）Eosin-positive layer（▲）underneath the MTA paste layer（＊）. Granu-lation-like tissue containing dense cells（black arrows）on the apex side over the eosin-positive layer（bar＝100μm）. （B）and（C）DMP1 expression was observed underneath the MTA paste layer（＊）and granulation-like tissue（black arrows）under the MTA（B：bar＝100μm；C：bar＝50μm）. （D）NES expression was not observed underneath the MTA paste layer（＊）（bar＝100μm）.

Fig.　5　Magnified view of apex 1/3 in MTA1　（A）HE and（B）, （C）DMP1 staining. （A）Hematoxylin-positive lumpy structures（black arrows）and cementum in apex 1/3（white arrows）were observed（bar＝100μm）. （B）and（C）DMP1 expression was observed around a lumpy structure（▲）（B：bar＝100μm；C：bar＝50μm）.

Fig.　6　Magnified view of the apex in MTA4　（A）HE and（B）NES staining. （A）Amount of cementum in apex 1/3 in MTA4 is greater than that in MTA1（Fig.　5）. （B）NES expression was observed in the apex（bars＝100μm）.


over the entire root canal when treated with revascu-larization using MTA compared to CH. Moreover, DMP1 shows a positive reaction at the interface, whereas NES does not show a clear positive reaction. This suggests the occurrence of ectopic calcification rather than hard tissue formation by odontoblasts dur-ing this experiment29,30）.　Cementum was formed at the apical end of the den-tin, and the amount of formation increased from one to four weeks after the operation, indicating that the tooth roots grew due to cementogenesis. DMP1 showed a positive reaction on the surface of the root canal in which a massive structure was observed, indicating that the structure may be associated with calcification. In addition, because cementum formation extended from the root surface on the inner wall surface of the root canal, cementum at the root surface may have invaded through the apical foramen. A regular cell sequence was not found on the dentin-exposed surface of the root canal wall, which was also negative by immunostaining, and hard tissue formation by odonto-blasts was not clearly observed during this experiment period; however, bone- and cementum-like hard tissue formation starting from the dentin-exposed root canal wall was observed. For the root canal irrigation in this experiment, a 6％ sodium hypochlorite solution follow-ing clinical practice was used for a short period of time. EDTA was not used in this experiment because it was not used in the past protocol for revascularization announced by the American Association of Endodontics（AAE）. Yet, it is necessary to compare and investigate the hard tissue formation when EDTA is used because the current AAE protocol includes EDTA for cleansing the root canal, and it is also reported that the use of EDTA promotes the adhesion of stem cells to the den-tin31,32）.　Based on the findings in this experiment, namely the small growth of the tooth root length and the thicken-ing of the root canal wall, we intend to conduct a longi-tudinal experiment to compare and investigate the application of MTA and CH in detail.

Conclusion

　Histological observation was conducted on the root canal of nonvital rat teeth with immature root forma-tion, which was filled with blood clots to which MTA or

CH paste was applied. The results are presented below.　1．At one and four weeks after the operation, the amount of regenerated tissue formation was signifi-cantly larger in the MTA group than in the CH group. Moreover, there was a significant difference in the for-mation of hard tissue at four weeks after the operation.　2．At one, two and four weeks after the operation, the formation of regenerated tissue and hard tissue was found up to the cervical 1/3 in the MTA group, whereas the tissue formation was observed only from the central 1/3 to the apex 1/3 in the CH group.　3．There was an unstructured layer from the cervi-cal 1/3 to the central 1/3 of the root in the CH group, and no regeneration of tissue was found.　4．All the formed hard tissue was bone-like hard tis-sue.　5．Inflammatory cell infiltration was not observed in any samples.　Taken together, we conclude that revascularization therapy is effective for adding hard tissue in the root canal when conducting a pulpectomy on a vital tooth with immature root formation. In addition, the forma-tion of hard tissue in the entire root canal is more likely to occur with MTA than CH, suggesting that the formed hard tissue would be bone-like hard tissue.

　The authors declare that there is no conflict of interest with regard to this study.

Acknowledgement　We thank Professor Yuichiro Noiri at Niigata University Graduate School of Medical and Dental Sciences, Depart-ment of Oral Life Science, Oral Health Science; and Dr. Rie Yamada at Nippon Dental University, School of Life Den-tistry at Niigata.

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　責任著者連絡先：湊　華絵　〒 951‒8580：新潟市中央区浜浦町 1‒8　日本歯科大学大学院新潟生命歯学研究科生命歯学専攻臨床科学系硬組織機能治療学分野硬組織機能治療学　TEL：025‒211‒8171，FAX：025‒267‒1795，E-mail：[email protected]　受付：2018年 11月 15日/受理：2019年 1月 9日

ラットの歯根未完成臼歯の抜髄後に行ったリバスクラリゼーション

湊　　　華　絵1 　北　島　佳代子2

新　井　恭　子2 　五　十　嵐　　勝1,2

1日本歯科大学大学院新潟生命歯学研究科　生命歯学専攻　臨床科学系　硬組織機能治療学分野　硬組織機能治療学2日本歯科大学新潟生命歯学部歯科保存学第 1講座

抄録　目的：本研究は，ラットの歯根未完成歯を抜髄した後の失活歯にリバスクラリゼーションを施し，血餅上部にMTA（mineral trioxide aggregate）と calcium hydroxide（CH）を用いた場合の新生組織の形成および根尖歯周組織の創傷部を組織学的に比較観察する目的で施された．　材料と方法：6週齢のWistar系雄性ラットを使用した．ラバーダム防湿下で根管歯髄の除去を行った．Hファイルで根尖孔外に約 1 mm突き出して出血させた．血餅の形成を確認し，MTAまたは CHを根管口部に貼付した．実験期間は術後 1，2，4週とした．これらの標本は HE染色で組織学的，DMP-1，nestinで免疫組織学的に観察した．　成績：MTA群は CH群に比べ新生組織の形成が根管全域にみられた．新生組織の形成は，術後 1週（p＝0.041）と術後 4週（p＝0.015）においてMTA群が CH群と比較して形成量が多く，有意に異なっていた．硬組織の形成は術後 4週においてMTA群が CH群と比較して歯頸部 1/3までの形成量が多く，有意に異なっていた（p＝0.004）．また硬組織の形成は，MTA群間（p＝0.004）と CH群間（p＝0.041）において術後 4週にかけて徐々に増加する傾向があった．骨様硬組織の形成は術後 4週にかけて徐々に増加する傾向がみられ，MTA群間において形成量は有意に異なっていた（p＝0.015）．硬組織の形成はすべての症例で骨様硬組織であった．すべての症例で，炎症性細胞浸潤はみられなかった．石灰化の制御に関与する DMP-1の発現は，術後 1週においてMTA直下，MTA下の顆粒様組織，根尖側の塊状構造の周囲に観察された．象牙芽細胞の分化マーカーの nestinの発現は，術後 4週において根尖で観察された．　結論：この研究により歯根未完成歯に抜髄をし，リバスクラリゼーションを行った場合，CHよりMTAが根管内の組織再生と硬組織形成に有効であることが示された．

キーワード：リバスクラリゼーション，根未完成歯，MTA

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