Effects of ascorbate-deficiency on collagen secretion and resorption in cultured mouse incisor germs

The effects of ascorbic acid deficiency on mouse incisors, grown in vitro, has been investigated at the histological and cytological levels. In this model, continuously growing mouse incisors are characterized by the existence of different type of predentin-dentin matrix on its lingual (root-analogue) and labial (crown-analogue) surface and the absence of enamel on the lingual surface. Our observations indicated that ascorbate-deficiency affected the behavior of mouse tooth germs in vitro: odontoblast differentiation was disturbed and morphological evidence for odontoblast-mediated collagen resorption were observed. An abnormal amorphous predentin-dentin matrix existed and the basement membrane was prematurely disrupted. The dentin mineralization, as well as functional differentiation of ameloblasts were strongly hampered. Chronic deficiency led to disorganization of the dental tissues.     

免疫组化和扫描电镜研究成牙本质细胞突

目的：观察成牙本质细胞突在牙本质小管中的分布。方法：取24颗无龋或釉质龋的恒前磨牙，20颗免疫组化染色，4颗扫描电镜观察。结果：阳性染色的波形蛋白结构在牙本质小管内呈连续较直的长条状。在冠部至釉牙本质界，在根部达牙本质小管末端。其分布趋势是从牙本质内层到外层逐渐减少，阳性表达程度逐渐减弱。扫描电镜观察成牙本质细胞突达釉牙本质界。结论：成牙本质细胞突贯穿牙本质全层达牙本质小管末端。     

修复性牙本质形成的大鼠模型

目的：建立修复性牙本质形成的动物实验模型,观察修复性牙本质形成的组织开矿学特征。方法：在Ｗｉｓｔａｒ大鼠第一磨牙近中面制备窝洞,分别观察３、１５、３０ｄ,标本切片,ＨＥ染色,显微镜下观察。结果：术后３ｄ未见修复性牙本质形成。１５ｄ后可见修复性牙本工可见骨样牙本质。窝洞下原代成牙本质细胞由新分化的成牙本质细胞样细胞取代。３０ｄ后,修复性牙本质明显增加。新分化的成牙本质细胞样细胞发生极化。结论：大鼠模     

巢蛋白在人牙齿发育过程中表达的免疫组化研究

目的:探讨巢蛋白(Nestin)在人牙齿发育过程中的表达模式和作用.方法:制备人牙齿发育各阶段以及人成牙本质细胞标本,采用SP法进行巢蛋白的免疫组织化学染色.结果:巢蛋白主要表达于功能性成牙本质细胞和成釉器.结论:巢蛋白参与人牙齿发育过程,特别是成牙本质细胞分化和基质分泌,可以作为功能性成牙本质细胞的标志物.     

Identification of Regulatory Elements Necessary for the Expression of the COL1A1 Promoter in Murine Odontoblasts

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies).

Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct.

These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.     

Expression of Vesicular Nucleotide Transporter in Rat Odontoblasts

Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X₃, an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp.     

Dentin Mineralization and the Role of Odontoblasts in Calcium Transport

Dentin is formed by two simultaneous processes, in which the odontoblasts are instrumental—the formation of the collagenous matrix, and mineral crystal formation in this matrix. This pattern of formation is similar to that of bone, another mineralized connective tissue. Dentin and bone also have chemical compositions which are similar but with distinct differences. It is of fundamental importance to understand how the ions constituting the inorganic phase are transported from the circulation to the site of mineral formation and how this transport is regulated. For dentinogenesis, calcium is essentially the only ion for which data are available. Recent evidence suggests that a major portion of the Ca²⁺ ions are transported by a transcellular route, thus being under cellular control. The cells maintain a delicate Ca²⁺ ion balance by the concerted action of transmembraneous transport mechanisms, including Ca-ATPase, Na⁺/Ca²⁺ exchangers and calcium channels, and of intracellular Ca²⁺-binding proteins. The net effect of this is a maintenance of a sub-micromolar intracellular Ca²⁺ activity, and an extracellular accumulation of Ca²⁺ ions in predentin, at the mineralization front. Predentin can be regarded as a zone of formation and maturation of the scaffolding collagen web of the dentin organic matrix. In addition to collagen, it contains little but proteoglycan. Simultaneous with mineral formation, additional non-collagenous macromolecules are added to the extracellular matrix of dentin, these presumably being transported within the odontoblast process. Among these are highly phosphorylated dentin phosphoprotein (phospho-phoryn) and another pool of proteoglycan. The functionality of this may be explained by the fact that polyanionic macromolecules are capable of inducing the formation of hydroxyapatite at ionic conditions resembling those in vivo. They can also inhibit mineral growth and regulate crystal size.     

Characterization of Protein Kinases Involved in Dentinogenesis

Protein phosphorylation and dephosphorylation control many different cell functions as well as responses to internal and external signals. It has also been shown that highly phosphorylated acidic proteins have an important role in matrix mediated biomineralization, perhaps functioning as nucleators for crystal formation. Dentine phosphoprotein (DPP) is one of such proteins which is exclusively synthesized by the odontoblast cells and therefore a likely candidate to play a significant role in normal and abnormal dentine biomineralization. These studies are directed at characterizing the protein kinases involved in dentinogenesis and in particular the enzyme(s) responsible for DPP phosphorylation. In this report we present data which indicate that there are several different types of kinases in the odontoblast-enriched dental papilla mesenchyme (DPM), some of which can phosphorylate DPP, such as casein kinase I and II. However, a different DPP-kinase activity was identified. This enzyme(s) appears to be different from other reported kinases, and it is the only kinase that can phosphorylate both phosphorylated DPP and enzymatically dephosphorylated DPP.