Dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and Hertwig's epithelial root sheath/epithelial rests of Malassez cells through the Fas-Fas ligand pathway

Hertwig's epithelial root sheath (HERS), epithelial rests of Malassez (ERM) cells, and reduced ameloblasts undergo apoptosis during tooth development. This study examined the effects of dental follicle cells and cementoblasts on the apoptosis of ameloblast-lineage and HERS/ERM cells derived from the enamel organ. We also elucidated the induction pathways and identified the apoptotic pathway involved in this process. Here, we showed terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL)-positive HERS cells and reduced ameloblasts near dental follicle cells during tooth development. Co-culturing ameloblast-lineage cell line (ALC) ameloblasts and HERS/ERM cells with either dental follicle cells or OCCM-30 cementoblasts markedly enhanced the apoptosis of ameloblasts and HERS/ERM cells compared with cells cultured alone. However, dental follicle cells and cementoblasts did not modulate the apoptotic responses of co-cultured non-odontogenic MCF10A or KB cells. When ameloblasts + HERS and cementoblasts + dental follicle cells were co-cultured, the expression of Fas ligand (FasL) increased in cementoblasts + dental follicle cells, while the expression of Fas increased in ameloblasts + HERS. Interestingly, recombinant FasL induced ameloblast apoptosis while the cementoblast-induced ameloblast apoptosis was suppressed by the Fas/FasL antagonist Kp7-6. These results suggest that during tooth development, dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and HERS/ERM cells through the Fas-FasL pathway, but do not induce the apoptosis of non-odontogenic epithelial cells.     

大鼠磨牙上皮根鞘发育的组织学观察

目的 观察SD大鼠磨牙上皮根鞘的发育。方法 选择出生后0、5、10、15、25 d的SD大鼠,制备大鼠下颌组织切片,抗角蛋白14免疫组化染色。结果 SD大鼠磨牙上皮根鞘在其出生后5 d时开始形成,出生后15 d时开始断裂,出生后25 d时大部分发生断裂,根尖处部分保持完整。结论 大鼠磨牙上皮根鞘有序发生、生长和断裂,可作为研究人类牙根发育的动物模型。     

Elevated expression of calcineurin subunits during active mineralization of developing mouse molar teeth

Calcineurin is a Ca²⁺/calmodulin‐dependent protein phosphatase consisting of two subunits – catalytic subunit A (CnA) and regulatory subunit B (CnB) – and plays a critical role in transducing Ca²⁺ signals into cellular responses. In this study, we investigated the expression of calcineurin in the mouse developing tooth. In‐situ hybridization detected mRNAs for the CnAα and CnAβ isoforms of CnA and for the CnB1 isoform of CnB in the upper molar tooth germ at embryonic day 15. Immunohistochemistry with antibodies specific for CnAα, CnAβ, and CnB1 showed strong immunoreactivity of these proteins in secretory‐stage ameloblasts and in odontoblasts during dentin formation. CnAβ and CnB1 were strongly immunoreactive in ruffle‐ended ameloblasts at the enamel‐maturation stage. In ameloblasts and odontoblasts, we also noted different subcellular distributions of CnAα and CnAβ. From these data, temporal profiles of calcineurin expression appear to correlate with active mineralization in tooth development. Furthermore, the distinct subcellular distribution of the two CnA subunits may reflect their distinct substrates or responsive sites within single cells, thus contributing to the diversity of calcineurin‐dependent cellular responses during active tooth mineralization.     

Damage of the Hertwig's epithelial root sheath: effect upon root growth after autotransplantation of teeth in monkeys

Abstract The effect of damage to the Hertwig's epithelial root sheath (HERS) during autotransplantation was examined in 12 green vervet monkeys. During the transplantation of permanent incisor tooth germs, either one-half or the entire root sheath was amputated or a contusion injury made to the root sheath. Transplanted incisors served as controls, where attempts were made not to injure the HERS. After an observation period of 9 months, a histologic examination of the transplanted teeth was performed. This examination showed variations in root growth among the various experimental groups. Thus, arrested root formation with ingrowth of bone from the base of the socket was the normal finding in the groups where either the entire HERS was amputated or contused. The group with partial amputation of HERS showed formation of a new but diminutive root. The control group showed teeth with normal root formation. The development of pulp necrosis usually led to complete or almost complete arrest of root formation irrespective of group. The present study indicated that the HERS can function normally subsequent to trauma elicited by surgical separation of the follicle and the post-transplantation disturbances in nutrition. Furthermore, a regenerative potential appeared to exist in this structure, even if just a fraction of it was left intact.     

Effect of the antineoplastic agent busulfan on rat molar root development

Objective

The antineoplastic bifunctional-alkylating agent busulfan (Bu) induces developmental anomalies. We examined histopathological changes in the molar roots of rats that received Bu at different stages of root formation.

Design

At different developmental stages, i.e., on postnatal days (P) 13, 15, and 19, rats were administered 7.5 mg/kg of Bu dissolved in dimethyl sulfoxide (DMSO) and then killed on P 30. After micro-computed tomography analysis, the maxillary first molars underwent immunohistochemical analysis for cytokeratin 14 (CK14), nestin, and dentin sialoprotein (Dsp). This was followed by histomorphometric analysis.

Results

The rats receiving Bu at an early stage (i.e., P 13 and P 15) showed osteodentin formation and complete destruction of the Hertwig's epithelial root sheath (HERS). Cells around osteodentin showed nestin and Dsp immunoreactivity. The root lengths in rats treated with Bu at P 13 (1228.44 ± 62.17 μm) and P 15 (1536.08 ± 109.71 μm) were lower than that in the control rats (1674.10 ± 40 μm). A narrowed apical foramen and an increased amount of osteodentin were also present, depending on the rat's age at the time of treatment (P < 0.05).

Conclusion

Busulfan treatment in juvenile rats resulted in abnormal root development, depending on the stage at which Bu was administered. This abnormal development may result from the destruction of the HERS. The administration of Bu caused a shortage of HERS cells, which are required for normal root development. This disturbs root formation, resulting in osteodentin formation and a narrowed apex foramen.     

大鼠Hertwig''''s上皮根鞘细胞的分离培养

目的:建立大鼠Hertwig's上皮根鞘细胞分离、培养和纯化的方法.方法:分离出生后7d SD大鼠上下颌第一、二磨牙牙胚,切取牙冠颈部组织,酶消化法原代培养,利用差速传代纯化上皮细胞.用角蛋白14和波形丝蛋白染色确定细胞来源.结果:原代培养细胞为上皮和间充质混合细胞,经2～3次差速传代后可获得纯化的上皮根鞘细胞.免疫组化染色角蛋白14阳性,波形丝蛋白阴性.结论:利用酶消化法及差速传代法培养并获得纯化的上皮根鞘细胞.     

Hepatocyte growth factor (HGF) in periodontal disease: detection of HGF in gingival crevicular fluid

Hepatocyte growth factor (HGF) acts as a mitogen, motogen, morphogen, anti-apoptotic factor, and scatter factor for various kinds of epithelial cells. It is a protein secreted by mesenchymal cells such as fibroblasts, and promotes motility and matrix invasion of epithelial cells. To clarify whether HGF is involved in periodontal disease, this study was conducted to determine whether HGF is present in gingival crevicular fluid (GCF) and to investigate the relationship between levels of HGF and the clinical parameters of periodontal disease, probing depth (PD), gingival index (GI) and bleeding on probing (BOP). We examined and collected GCF samples from 80 sites in 38 subjects with periodontal or other oral diseases. The concentrations of HGF, IL-1beta and PGE2 were determined by ELISA, and active collagenase activity was determined by functional assay. The HGF level correlated positively with PD and GI, and was significantly higher in specimens from BOP-positive sites and those where PD exceeded 4 mm compared with those from sites that were BOP-negative or with a PD less than 3 mm. There was a significant positive correlation between the concentrations of HGF and IL-1beta. These results indicate that the HGF level in GCF correlates well with clinical parameters of periodontal disease, and suggest that HGF may be involved in epithelial invasion through its role as a scatter factor.     

上皮根鞘细胞表面超微结构及牙根发育中上皮根鞘增殖力的变化

目的:观察体外培养的上皮根鞘细胞超微结构及牙根发育中上皮根鞘增殖力的变化。方法:体外培养大鼠上皮根鞘细胞,扫描电镜观察。制备出生后5 d、7~8 d和15 d的SD大鼠下颌磨牙石蜡切片,增殖细胞核抗原,检测免疫染色。结果:上皮根鞘细胞表面有许多微绒毛,细胞间连接紧密。牙根发育中上皮根鞘增殖力有序变化。结论:上皮根鞘细胞具有典型的上皮细胞表面结构特性。牙根发育中上皮根鞘有序断裂与其增殖力变化有关。     

Three independent canals in the mesial root of a mandibular first molar

Abstract Endodontic therapy was performed in a mandibular first molar with three canals in the mesial root. The mesiobuccal and the mesiolingual canals were found in their normal locations. The third was located in the middle of the distance between the other two. Radiographically, it ended in its own distinct foramen. Many reports deal with three orifices in the mesial root, but very few describe three independent canals, indicating a rare anatomical configuration. To locale the third possible intermediate canal it was suggested to reduce the mesial dentinal wall of the pulp chamber after instrumenting the main two canals.     

肝细胞生长因子受体在大鼠牙胚发育中的表达及其意义

目的：研究肝细胞生长因子受体(c—Met)在大鼠牙胚发育过程中的时空表达模式。方法：用免疫组织化学方法，检测c—Met在大鼠牙胚发育不同阶段表达的位置。结果：c—Met在牙胚发育的不同阶段表达于牙胚中特定的部位：帽状期(胚胎15d)，内釉上皮阳性表达，中间层及部分星网状层细胞弱阳性表达；钟状期(胚胎19d)，内、外釉上皮阳性表达，但内釉上皮较外釉上皮表达弱，中间层及星网状层细胞弱阳性表达；矿化期(生后7d)，成牙本质细胞阳性表达。结论：c—Met在大鼠牙胚发育过程中的表达具有时空特异性，在牙齿形态发生的过程中具有重要的作用，还可能与牙齿的矿化有关。     

Differences in the confluence of mesial canals in mandibular molar teeth with three or four root canals

Aim  To investigate the correlation between number of root canals and their confluence in mandibular molar teeth.
Methodology  A total of 553 first and 383 second mandibular molars were root filled between the beginning of 1997 and the end of 2006. Access cavities were prepared and the pulp chamber floors were carefully inspected with an endodontic explorer under magnification of 4×. After negotiating the root canal system, the 'straightest' canal of each root was instrumented. A gutta-percha cone was placed in the canal and a small file (08 or 10) was inserted to working length and then removed in all other canals. The gutta-percha cone was removed and inspected for notches indicating the presence of confluences. If it was not possible to probe completely one or more canals, the canal confluences were classified as 'not determined' (ND) if there was no sign of confluence; if a notch was present canals were considered confluent. For each tooth, the number of root canals and the presence of confluences were recorded. To test whether there was a difference in frequency of confluences between teeth with three and four root canals the chi-squared analysis with Yates' continuity correction was performed.
Results  The frequency of a confluence in mesial canals of first molars was 56% in teeth with three root canals and 34% in teeth with four canals. In second molars, it was 67 and 41%, respectively. Differences in the frequency of confluences in teeth with three and four root canals were statistically significant both in first and in second molars.
Conclusions  A greater incidence of confluent canals occurred in teeth with three rather than with four root canals both in first and second mandibular molars.     

Hepatocyte growth factor secreted by periodontal ligament and gingival fibroblasts is a major chemoattractant for gingival epithelial cells

To ascertain whether periodontal fibroblasts could be involved in the pathogenesis of periodontal pocket formation, the chemotactic activity of periodontal ligament fibroblast-conditioned medium (PLF-CM) and gingival fibroblast-conditioned medium (GF-CM) for gingival epithelial cells was examined using a modified Boyden chamber assay. Both PLF-CM and GF-CM possessed significant chemotactic activity, which was decreased markedly by treatment with anti-human hepatocyte growth factor (HGF) neutralizing antibody. Furthermore, the chemotactic activity of PLF-CM and GF-CM was well correlated with HGF content. These results show that PLF and GF secrete an HGF-like factor, and suggest that such a factor derived from periodontal fibroblasts might play a role in epithelial apical migration in periodontitis.     

Unusual root development following surgical repositioning of horizontally developing central incisor

Dilaceration is one of the causes of maxillary central incisor eruption failure. If the dilacerated maxillary permanent central incisor is in a horizontal or vertical position and root formation is in early stages, surgical repositioning is frequently the treatment of choice. In this article, the case of a horizontally impacted and dilacerated maxillary central incisor is presented which was treated by surgical repositioning. The tooth developed an unusual form of the root with discontinuity at the middle third region with the coronal and apical parts growing separately. Radiographic and clinical monitoring of the case was regularly performed. The tooth completely erupted in 2 years after the repositioning and even after 6 years of surgery is currently successfully functional in the arch with the malformed root. This article highlights the ability of Hertwig's epithelial root sheath to withstand trauma and its ability to recover.     

Exogenous hepatocyte growth factor inhibits myoblast differentiation by inducing myf5 expression and suppressing myoD expression in an organ culture system of embryonic mouse tongue

We examined the effects of exogenous hepatocyte growth factor (HGF) on the differentiation and proliferation of tongue myoblasts by using an organ culture system of tongue obtained from mouse embryos at embryonic day (E) 13. Exogenous HGF induced reductions in the quantities of muscle creatine kinase and myogenin mRNAs and in the number of fast myosin heavy chain-positive myoblasts and myotubes, suggesting that HGF suppressed the differentiation of myoblasts in the cultured E13 tongues. Exogenous HGF induced no significant changes in the percentage of proliferating cell nuclear antigen (PCNA)-positive cell nuclei to total cell nuclei (labeling index) in the muscle portion of the cultured E13 tongue, suggesting that HGF did not affect the proliferation of myoblasts. Exogenous HGF induced the expression of myf5 mRNA but inhibited the expression of myoD mRNA. Since mouse tongue myoblasts are reported to complete proliferation by E13, it appears that exogenous HGF arrests myoblasts in the cell cycle and does not allow them to enter the differentiation process. This is achieved by controlling the expression of myf5 and myoD mRNAs, thus inhibiting the differentiation of tongue myoblasts.     

神经生长因子在大鼠牙髓炎症中的作用的实验研究

目的 探讨神经生长因子（NGF）在牙髓炎症中的作用。方法 建立大鼠磨牙内毒素炎症模型后,进行NGF的免疫组化染色。结果 经开髓和内毒素处理后12h－3d,冠髓内牙髓细胞表达NGF明显增加,5d反恢复正常。结论 NGF参与牙髓炎症过程,可能与牙髓炎的激发痛症状相关。     

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目的利用锥形柬计算机断层扫描（CBCT）研究上颌第一磨牙近中颊根第二根管（MB2）发生率、根管解剖形态以及根管口位置分布。方法随机选取2011-2013年航天中心医院口腔科20～30岁患者CBCT影像160张（男80张、女80张）。平行近中颊根长轴平面观察其根管形态,并按照Vertucci分类统计。水平面观察根管口的位置,记录MB、MB2及腭根根管口间距离以及MB、MB2根管口分别到腭根根管口连线夹角,并比较不同性别间差异。结果本研究观察了279颗上颌第一磨牙,其中存在MB2牙齿233颗,检出率为83．5％,不同性别间差异无统计学意义（P〉0．05）。不同类型的根管出现概率不同,各类型出现率男女间无差异。MB与MB2根管口间距男性为（2．195±0．504）mm,女性为（1．966±0．419）mm,差异有统计学意义（P〈0．05）。结论上颌第一磨牙MB2发生率高,不同类型MB2发生概率不同,男性与女性之间无差异。MB与MB2根管口间距在两性之间存在统计学差异。