Single cell RNA sequencing reveals mesenchymal heterogeneity and critical functions of Cd271 in tooth development

BACKGROUND Accumulating evidence suggests that the maxillary process,to which cranial crest cells migrate,is essential to tooth development.Emerging studies indicate that Cd271 plays an essential role in odontogenesis.However,the underlying mechanisms have yet to be elucidated.AIM To establish the functionally heterogeneous population in the maxillary process,elucidate the effects of Cd271 deficiency on gene expression differences.METHODS p75NTR knockout(Cd271-/-)mice(from American Jackson laboratory)were used to collect the maxillofacial process tissue of p75NTR knockout mice,and the wildtype maxillofacial process of the same pregnant mouse wild was used as control.After single cell suspension,the cDNA was prepared by loading the single cell suspension into the 10x Genomics Chromium system to be sequenced by NovaSeq6000 sequencing system.Finally,the sequencing data in Fastq format were obtained.The FastQC software is used to evaluate the quality of data and CellRanger analyzed the data.The gene expression matrix is read by R software,and Seurat is used to control and standardize the data,reduce the dimension and cluster.We search for marker genes for subgroup annotation by consulting literature and database;explore the effect of p75NTR knockout on mesenchymal stem cells(MSCs)gene expression and cell proportion by cell subgrouping,differential gene analysis,enrichment analysis and protein-protein interaction network analysis;understand the interaction between MSCs cells and the differentiation trajectory and gene change characteristics of p75NTR knockout MSCs by cell communication analysis and pseudo-time analysis.Last we verified the findings single cell sequencing in vitro.RESULTS We identified 21 cell clusters,and we re-clustered these into three subclusters.Importantly,we revealed the cell–cell communication networks between clusters.We clarified that Cd271 was significantly associated with the regulation of mineralization.CONCLUSION This study provides comprehensive mechanistic insights into the maxillary-process-derived MSCs and demonstrates that Cd271 is significantly associated with the odontogenesis in mesenchymal populations.

Modification of tooth development by heat shock protein 60

several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 （Hsp 60）. To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot （EK） and stratum intermedium （SI）. Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2- deoxyuridine （BrdU） incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.

Bivalent histone modifications during tooth development

Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4trimethylation（H3K4me3） and histone H3 lysine 27 trimethylation（H3K27me3） epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5 B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction（qP CR） analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial–temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5 B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.

Molecular regulatory mechanism of tooth root development

The root is crucial for the physiological function of the tooth, and a healthy root allows an artificial crown to function as required clinically. Tooth crown development has been studied intensively during the last few decades, but root development remains not well understood. Here we review the root development processes, including cell fate determination, induction of odontoblast and cementoblast differentiation, interaction of root epithelium and mesenchyme, and other molecular mechanisms. This review summarizes our current understanding of the signaling cascades and mechanisms involved in root development. It also sets the stage for de novo tooth regeneration.

APPEARANCE PATTERN OF TOOTH GERMS WITH DEVELOPMENTAL PROCESS IN MYLOPHARYNGODON PICEUS

The pharyngeal dental formula of Mylopharyngodon piceus is 4-5 as a rule, and the dentition isasymmetrical. It is difficult to identify each tooth in the larval dentition. In this paper the appearancepattem of tooth germ with developmental process in this fish is described in detail. The formationpattern of the left dentition is contrasted with that of the right one. In the developmental process,the left pharyngeal dentition lacks teeth at position An3. Thus the left dentition is D-type as designatedby Nakajima(1984), while the right one is A-type.

Methods for studying tooth root cementum by light microscopy

The tooth root cementum is a thin, mineralized tissue covering the root dentin that is present primarily as acellular cementum on the cervical root and cellular cementum covering the apical root. While cementum shares many properties in common with bone and dentin, it is a unique mineralized tissue and acellular cementum is critical for attachment of the tooth to the surrounding periodontal ligament （PDL）. Resources for methodologies for hard tissues often overlook cementum and approaches that may be of value for studying this tissue. To address this issue, this report offers detailed methodology, as well as comparisons of several histological and immunohistochemical stains available for imaging the cementum-PDL complex by light microscopy. Notably, the infrequently used Alcian blue stain with nuclear fast red counterstain provided utility in imaging cementum in mouse, porcine and human teeth. While no truly unique extracellular matrix markers have been identified to differentiate cementum from the other hard tissues, immunohistochemistry for detection of bone sialoprotein （BSP）, osteopontin （OPN）, and dentin matrix protein 1 （DMP1） is a reliable approach for studying both acellular and cellular cementum and providing insight into developmental biology of these tissues. Histoloeical and immunohistochemical aooroaches Drovide insight on developmental biology of cementum.

Spatial signalling mediated by the transforming growth factor-β signalling pathway during tooth formation

Tooth development relies on sequential and reciprocal interactions between the epithelial and mesenchymal tissues, and it is continuously regulated by a variety of conserved and specific temporal-spatial signalling pathways. It is well known that suspensions of tooth germ cells can form tooth-like structures after losing the positional information provided by the epithelial and mesenchymal tissues. However, the particular stage in which the tooth germ cells start to form tooth-like structures after losing their positional information remains unclear. In this study, we investigated the reassociation of tooth germ cells suspension from different morphological stages during tooth development and the phosphorylation of Smad2/3 in this process. Four tooth morphological stages were designed in this study. The results showed that tooth germ cells formed odontogenic tissue at embryonic day （E） 14.5, which is referred to as the cap stage, and they formed tooth-like structures at E16.5, which is referred to as the early bell stage, and E18.5, which is referred to as the late bell stage. Moreover, the transforming growth factor-β signalling pathway might play a role in this process.

牙根不同发育状态下闭合式开窗导萌术联合正畸牵引治疗上颌埋伏阻生前牙的临床观察

目的探讨牙根不同发育状态下闭合式开窗导萌术联合正畸牵引治疗对上颌埋伏阻生前牙的临床治疗效果。方法选取2021年1月~2022年1月该院收治并经闭合式开窗导萌术联合正畸牵引治疗的上颌埋伏阻生前牙84例作为研究对象,根据牙根发育状态分为完全组(牙根发育完全)、未完全组(牙根发育不完全),记录两组患者治疗时间,比较两组患者而治疗疗效、恢复情况、并发症发生情况。结果未完全组患者临床治疗总有效率为97.62%高于完全组的85.71%,差异有统计学意义(P<0.05);未完全组患者治疗时间、牙龈指数、出血指数低于完全组,咬合力、咀嚼效率明显高于完全组,差异有统计学意义(P<0.05);未完全组并发生症总发生率为4.76%明显低于完全组的19.05%(χ^(2)=4.086,P=0.043)。结论牙根发育不完全患者采用闭合式开窗导萌术联合正畸牵引治疗效果优于牙根发育完全患者,且并发症少。

Globoside accelerates the differentiation of dental epithelial cells into ameloblasts

Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside （Gb4）, a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins （including neurotrophin-4） and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.

从细胞到器官——牙组织工程任重而道远

Tooth tissue engineering is an emerging biotechnique that will provide replacemental teeth for patients suffering from different diseases causing tooth loss. Although some attempts have been tried to generate whole tooth both in vivo and in vitro, the lack of the knowledge for tooth initiation and development, as well as for tooth shape controlling mechanisms greatly impede the progress of this technique. This article reviewed and discussed some recent findings in tooth tissue engineering related to the cell resource, the concept of reconstruction and regeneration, the application of artificial scaffolds, together with the methods of organ culture and implantation.

学龄前儿童乳牙釉质发育缺陷的患病状况及分布

目的：了解北京地区城乡学龄前儿童乳牙釉质发育缺陷（ＤＤＥ）的患病与分布状况；探讨营养状况与釉质发育缺陷间的关系。方法：采用简化的ＤＤＥ指数进行现况调查。结果：农村儿童ＤＤＥ的患病率（１９．４％）明显高于城市儿童（８．７％）；ＤＤＥ主要发生于上颌切牙；身高、体重测量所反映的儿童营养状况与ＤＤＥ间没有明显的相关性。结论：妊娠的后６个月到出生后６个月为乳牙对ＤＤＥ的敏感时期，因此早期预防非常重要。

乳牙釉质发育缺陷的患病状况及相关因素分析

目的 了解增城市儿童乳牙釉质发育缺陷 (DDE)的患病及分布状况 ,探讨其可能的致病因素。方法 采用世界卫生组织制定的标准进行临床检查及问卷调查。结果 增城市 2～ 4岁儿童乳牙DDE的患病率为 15 .5 % ,主要发生在上颌切牙。母亲在妊娠期患感冒使儿童DDE患病率增加 ,有显著性差别。

Changes of the Unique Odontogenic Properties of Rat Apical Bud Cells under the Developing Apical Complex Microenvironment

Aim To characterize the odontogenic capability of apical bud and phenotypical change of apical bud cells （ABCs） in different microenvironment.Methodology Incisor apical bud tissues from neonatal SD rat were dissected and transplanted into the renal capsules to determine their odontogenic capability. Meanwhile ABCs were cultured and purified by repeated differential trypsinization. Then ABCs were cultured with conditioned medium from developing apical complex cells （DAC-CM）. Immunocytochemistry, reverse transcriptase polymerase chain reaction （RT-PCR） and scanning electron microscope （SEM） were performed to compare the biolo- gical change of ABC treated with or without DAC-CM. Results First we confirmed the ability of apical bud to form crown-like structure ectopically. Equally important, by using the developing apical complex （DAC） condi- tioned medium, we found the microenvironment created by root could abrogate the ＂crown＂ features of ABCs and promote their proliferation and differentiation. Conclusion ABCs possess odontogenic capability to form crown-like tissues and this property can be affected by root-produced microenvironment.

孕期尼古丁暴露对子代小鼠牙釉质形成的影响

目的研究孕期尼古丁暴露对子代小鼠牙釉质形成的影响及其可能的表观遗传学机制。方法通过随机数字表法将10只C57BL/6孕鼠分为对照组(皮下注射生理盐水)及孕期尼古丁暴露(prenatal nicotine exposure, PNE)组(皮下注射尼古丁溶液), 每组5只, 孕鼠生产后分别收集出生后0 d(postnatal day 0, P0;P14、P25含义依此类推)、P14、P25新生小鼠, 根据母代分组分为子代对照组及子代PNE组, 并记录P0、P25子代小鼠体质量。采用显微CT(micro-CT)扫描分析、扫描电镜和维氏显微硬度检测对子代小鼠牙槽骨和下颌切牙进行硬组织相关参数分析。提取P25子代小鼠下颌骨组织及第3代牙上皮干细胞(dental epithelial stem cell, DESC)中的总RNA, 通过实时荧光定量PCR检测成骨向及成釉向分化相关基因、增殖相关标志物和干细胞标志物的表达水平。采用石蜡切片免疫组织化学染色观察增殖细胞核抗原(proliferating cell nuclear antigen, Pcna)、釉原蛋白(amelogenin, Amelx)、甲基转移酶ZESTE增强子同源物2(enhancer of zeste homolog 2, Ezh2)、组蛋白H3第27位赖氨酸的三甲基化修饰(histone H3 trimethylated at lysine 27, H3K27me3)阳性染色分布及水平。使用细胞增殖(cell counting kit-8, CCK-8)试剂盒对外源性添加不同浓度(0.01、0.1、1 mmol/L)尼古丁以及Ezh2抑制剂GSK126的DESC进行细胞活力检测。结果 PNE组孕鼠较对照组更容易出现不良妊娠结局, P0时子代PNE组小鼠体质量[(0.99±0.02)g]显著低于子代对照组[(1.20±0.04)g](P<0.001);P25时, 子代PNE组小鼠体质量[(9.65±1.32)g]显著低于子代对照组[(15.26±1.70)g](P<0.001), 死产率[(46.40±9.30)%]显著高于对照组(0)(P<0.001)。P14和P25时, 子代PNE组小鼠下颌切牙釉质矿化沉积点与第一磨牙近中面的距离数值[分别为(-1 349±45)、(-506±380)μm]均较子代对照组[分别为(-1 192±147)、(504±198)μm]显著减小(P<0.05, P<0.001), 提示矿化沉积点延迟。子代PNE组小鼠切牙釉柱及釉柱间质较对照组排列松散无序, 显微硬度[(245.7±18.4)MPa]较子代对照组[(371.9±28.7)MPa]显著降低(P<0.001)。HE染色显示子代PNE组小鼠前成釉细胞(pre-ameloblast, Pre-Am)区域排列较对照组紊乱, 矿化沉积点推迟, 暂时性扩增细胞(transit-amplifying cell, TA)及Pre-Am区域延长。免疫组织化学染色显示, 子代PNE组小鼠唇侧颈环(labial cervical loop, LaCL)整体Pcna表达较子代对照组显著增多(P<0.05), H3K27me3较对照组显著富集(P<0.01), 对应区域可见Ezh2表达显著增加(P<0.01), 同时成釉细胞胞质中Amelx阳性信号减弱。体外实验添加1 mmol/L 尼古丁能显著上调子代PNE组DESC的Pcna基因表达水平(P<0.01), 同时显著下调DESC标志物B淋巴瘤Mo-MLV插入蛋白1、富亮氨酸重复序列免疫球蛋白样结构域1和成釉细胞向分化的相关基因Amelx等的基因表达水平(P<0.05, P<0.05, P<0.01)。此外, 与0 h时相比, 干预24 h后1 mmol/L尼古丁可显著增强DESC的增殖活性(P<0.001), 添加10 μmol/L Ezh2抑制剂GSK126可挽救1 mmol/L尼古丁对DESC带来的增殖促进作用。结论孕期尼古丁暴露可能导致成釉向谱系定向分化过程延迟, 致子代小鼠DESC增殖及分化异常, 并且影响H3K27me3表观遗传修饰水平, 造成子代牙釉质发育障碍, 提示孕期尼古丁暴露是牙发育的危险环境因素。

DKK1与牙齿发育和口腔疾病关系的研究进展

Dickkopf家族(DKK)是进化中高度保守的古老体系,通过抑制Wnt通路的信号传导来发挥调节作用。DKK1作为DKK家族的重要成员具有广泛的生物学功能,可以通过调节细胞生长、迁移、分化来控制机体发育和疾病发展,在胚胎发育和肿瘤形成等方面研究较为成熟。随着近年来人们对口腔健康的重视和一些口腔疾病发病率逐年上涨,学者们对疾病的分子机制领域研究逐渐深入,DKK1通过调节Wnt通路这一重要机制在口腔医学领域也逐渐得到重视,并被认为是一个潜在的肿瘤治疗靶点。本文通过综述近年来国内外学者对DKK1在牙齿发育和口腔的常见疾病如口腔肿瘤、口腔黏膜下纤维化等方面的研究成果,分析其中可能的分子机制,为学者们未来进一步研究牙齿形态发育的精密调控机制和口腔疾病的防治提供理论参考与思路。