Tooth Crown Morphogenesis and Cytodifferentiations: Candid Questions and Critical Comments

Teeth are probably meristic units and crown morphogenesis leads to tooth specific distribution of functional cells. Since heterodonty is derived from homodonty, one way to understand tooth morphogenesis would be to unravel the involved phenomena in homodont species and then to characterize the “put up job” of evolution leading to species specific dentitions with particular functional abilities. Interaction of paleontologists and developmental biologists should be initiated. My naive “developmental” point of view will illustrate only one facet of mouse tooth morphogenesis and cytodifferentiation. The main concern will be to try to discriminate between known facts and speculations, between hypotheses and anticipated or deduced certitudes, to call attention to conflicting data, to suggest some further investigations and to advocate the point of view that molecular interpretations should be founded on indisputable morphological data.     

Oxytocin Facilitates Dentinogenesis of Rat Dental Pulp Cells

IntroductionOxytocin (OT) is a neurohypophysial hormone that plays a role in lactation and parturition and exerts diverse biological actions via the OT receptor. Recently, several studies have reported that OT stimulates bone formation by osteoblasts in osteoporosis. We focused on OT and hypothesized that OT can stimulate the differentiation of odontoblasts as well as osteoblasts. The aim of this study was to verify whether OT is an essential factor in dentinogenesis; we examined the effects of OT on dentinogenesis using a long-term culture system of rat dental pulp cells.MethodsUsing a culture system of rat dental pulp cells with Otr knocked out by CRISPR-Cas9 genome editing, we examined the effects of OT on odontoblastlike cell differentiation as reflected by dentin formation.ResultsWe confirmed that OT stimulated mineralized nodule formation and the expression of both dentin sialoprotein and bone Gla protein messenger RNAs (mRNAs) in the culture system. Interestingly, the cultured cells treated with OT also exhibited an increase of both Wnt10a and Lef-1 mRNA. The Otr knockout cells showed inhibition of nodule formation and mRNA expression, and these phenomena remained despite OT treatment. These results indicate the following: OT regulates odontoblastlike cell differentiation via the OT receptor, it stimulates dentin formation, and the Wnt canonical pathway is closely related to these effects.ConclusionsThe present results suggest that OT can promote odontoblastlike cell differentiation, resulting in increased dentin formation, and that OT could be an important factor for dentinogenesis.     

Claudin rather than occludin is essential for differentiation in rat incisor odontoblasts

Many morphological and developmental studies have demonstrated the characteristics of tight junctions (TJs) between odontoblasts. However, detailed localization of TJ-associated proteins in odontoblasts and their functions has not yet been clarified. To elucidate the relationship between the establishment of TJ structures and the differentiation of odontoblasts during early dentinogenesis, we studied the expression and localization of constituent proteins of TJs (claudin-1, occludin, ZO-1 and ZO-2) between odontoblasts in rat lower incisors using Western blotting, immunofluorescence and immunoelectron microscopy. When the expression of claudin-1 increases at the distal portion of mature odontoblasts, the TJs form complex networks of strands, and odontoblasts differentiated by developing distal membrane domains and by secreting specific molecules for mineralization. We conclude that the TJs of odontoblasts may play an important role in the differentiation of odontoblasts in rat lower incisors during early dentinogenesis.     

DMP1 C‐terminal mutant mice recapture the human ARHR tooth phenotype

DMP1 mutations in autosomal recessive hypophosphatemic rickets (ARHR) patients and mice lacking Dmp1 display an overlapping pathophysiology, such as hypophosphatemia. However, subtle differences exist between the mouse model and human ARHR patients. These differences could be due to a species specificity of human versus mouse, or it may be that the mutant DMP1 in humans maintains partial function of DMP1. In this study we report a deformed tooth phenotype in a human DMP1 deletion mutation case. Unexpectedly, the deletion of nucleotides 1484 to 1490 (c.1484_1490delCTATCAC, delMut, resulting in replacement of the last 18 residues with 33 random amino acids) showed a severe dentin and enamel defect similar to a dentinogenesis imperfecta (DI) III–like phenotype. To address the molecular mechanism behind this phenotype, we generated delMut transgenic mice with the endogenous Dmp1 gene removed. These mutant mice did not recapture the abnormal phenotype observed in the human patient but displayed a mild rachitic tooth phenotype in comparison with that in the Dmp1‐null mice, suggesting that the DI III–like phenotype may be due to an as‐yet‐undetermined acquired gene modifier. The mechanism studies showed that the mutant fragment maintains partial function of DMP1 such as stimulating MAP kinase signaling in vitro. Last, the in vitro and in vivo data support a role of odontoblasts in the control of fibroblast growth factor 23 (FGF‐23) regulation during early postnatal development, although this regulation on P_i homeostasis is likely limited. © 2010 American Society for Bone and Mineral Research.     

Ultrastructural and immunocytochemical characterization of immortalized odontoblast MO6-G3

AIM: To investigate an immortalized murine odontoblast cell line as a potential alternative for experimental studies on dentinogenesis. METHODOLOGY: The MO6-G3 cell line was investigated morphologically over 3, 7, 11 and 42 days of culture, using histochemical localization of dentine sialoprotein (DSP), alkaline phosphatase (AP), type I collagen and actin filaments, histoenzymatic staining and biochemical investigation of AP and finally, transmission and scanning electron microscopy. RESULTS: Scanning electron micrographs showed elongated cells. Accordingly, a polarized organization of odontoblasts was observed by transmission electron microscopy, identifying distinct subcellular compartments as described in vivo. The secretion apparatus, which includes cisternae of rough endoplasmic reticulum, Golgi apparatus saccules and secretion vesicles and granules, was longitudinally organized in the supranuclear compartment ending distally in the secretory pole. A cellular process was observed. The investigation of the cytoskeleton network revealed that actin microfilaments were organized in parallel stress fibre oriented depending on the longitudinal axis of the cytoplasm. Immunofluorescent labelling showed a continuous expression of type I collagen, DSP and AP. A unipolar distribution characterized intracellular DSP immunoreactivity. Histoenzymology revealed AP active sites increasing from 3 to 11 days albeit with a moderate level of activity comparatively to the in vivo situation in dental cells. CONCLUSION: This cell line MO6-G3 not only showed the criteria of odontoblast phenotype as previously reported but also the characteristic morphodifferentiation pattern of polarized odontoblasts at the cellular level but with an apparent random distribution.     

人牙髓细胞向成牙本质细胞诱导分化中细胞周期变化

目的 建立成牙本质细胞样细胞体外培养体系,观察体外培养的牙髓细胞向成牙本质细胞分化过程中细胞周期的变化.方法 利用组织块酶解法培养人牙髓细胞并进行鉴定,使用矿化诱导液诱导其向成牙本质细胞样细胞分化.对细胞增殖情况使用流式细胞仪进行细胞周期测定.结果 ①获得的牙髓细胞均为波形丝蛋白阳性,角蛋白阴性,证明为中胚层来源的细胞.②诱导分化的牙髓细胞在2周左右进入复层生长期;3周左右开始有细胞结节形成,周围细胞呈放射状排列,部分细胞出现细长突起并呈极性排列;4周左右细胞团中央Von Kossa染色为阳性.③在诱导开始后1周,细胞处于活跃的增殖期,以后细胞增殖变慢,3周后多数细胞处于G0G1期.结论 实验成功建立了成牙本质细胞样细胞体外培养体系,所获得的成牙本质细胞样细胞具有成牙本质细胞的部分形态和生物学功能,诱导分化后细胞增殖变慢,是研究成牙本质细胞的较好模型.     

HSP27在大鼠磨牙钻磨后牙髓的表达

目的 研究热休克蛋白27(heat shock protein 27,HSP27)在成年大鼠磨牙受到钻磨刺激后不同时间牙髓组织中的表达特点及定位情况. 方法 建立大鼠牙髓炎模型,用免疫组织化学方法检测热休克蛋白27在大鼠上颌磨牙钻磨后3 d,6d,9 d的表达. 结果 热休克蛋白27在牙髓炎晚期成牙本质细胞、牙髓成纤维细胞、血管壁及内皮细胞中表达逐渐减弱.结论 热休克蛋白27的表达与牙髓炎症发生发展的程度有关,提示热休克蛋白27在牙髓损伤修复期可能是参与牙髓损伤修复的细胞因子之一.     

牙本质基质蛋白1基因反义核酸对MDPC-23细胞内、外钙离子浓度的影响

目的观察牙本质基质蛋白1(DMP1)基因反义寡核苷酸作用下成牙本质细胞系MDPC-23细胞内、外钙离子浓度的动态变化,揭示牙本质矿化机制。方法以稳定表达DMP1的MDPC-23细胞为靶细胞,10μmol/L反义DMP1(AS-ODN)为阻断剂,用Western blot法检测不同时间细胞DMP1的表达情况,并观察不同作用时间内MDPC-23细胞内游离钙离子[(Ca2+)if]、总钙离子[(Ca2+)it]和细胞外钙离子[(Ca2+)e]的动态变化。结果Western blot法检测DMP1蛋白在MDPC-23细胞的表达在AS-ODN加入后12 h时减弱,24 h后完全阻断。与正常组和正义核酸组(S-ODN)相比较(平均荧光值为87.46±39.60),AS-ODN组(Ca2+)if先升高(平均荧光值12 h处为104.10±27.06;24 h处为98.46±19.92),AS-ODN作用24 h后,(Ca2+)if又降低(平均荧光值36 h处为77.54±14.95;48 h处为68.43±22.11);(Ca2+)it明显降低,于24 h处至最低值(0.142±0.233)mmol/L(P<0.01);(Ca2+)e呈上升趋势,且与对照组差异无显著性(P>0.05)。结论反义DMP1能够影响MDPC-23细胞内(Ca2+)if和(Ca2+)it浓度,提示DMP1参与调节成牙本质细胞的钙离子代谢和转运过程,可能在牙本质矿化过程中发挥作用。     

半固态培养基法培养鼠磨牙牙胚的实验观察

目的 :观察体外培养的鼠磨牙牙胚发育过程的组织学变化。方法 :用 16d胎龄的昆明小鼠下颌第一磨牙牙胚置于RPMI- 16 40半固态培养基上培养 ,常规组织学观察。结果 :体外培养的牙胚可由帽状期进入钟状期 ,出现成牙本质细胞和成釉细胞的分化及前期牙本质的形成。结论 :半固态培养基法培养牙胚可保持其发育过程。     

细胞极性在牙发育中的研究进展

细胞极性是指由于极性蛋白复合物之间的相互协作或排斥，使细胞骨架、细胞器以及生物大分子等呈极性分布的特性。细胞各个部分实现不同生理功能需要细胞极性蛋白及分子的精密调控。细胞极性在牙发育中发挥着重要作用，特别对于成釉细胞和成牙本质细胞的分化和功能意义重大。文章就细胞极性及相关分子和通路在牙发育中的研究进展进行综述。