Interference of tooth differentiation with interposed filters

doi:10.1016/0012-1606(77)90085-9

Developmental Biology

Volume 58, Issue 1, 1 July 1977, Pages 197-203

https://doi.org/10.1016/0012-1606(77)90085-9 Get rights and content

Abstract

The effect of interposed Nuclepore filters on the epithelio-mesenchymal interaction in embryonic mouse tooth was studied. Filters with pore sizes of 0.6 and 0.2 μm allowed differentiation of odontoblasts and ameloblasts in the bell-stage tooth germ. This differentiation progressed more rapidly when the 0.6-μm pore size filter was used. Nuclepore filters with 0.1-μm pores prevented differentiation. Electron microscopic examination revealed penetration of cell processes into the filter pores. Cytoplasmic material could be seen in the 0.6-μm pore-size filter within 3 days of cultivation, whereas, in the 0.2-μm filter pores, penetration was slight. After 6 days of cultivation, cytoplasmic material was found at all levels of the 0.2-μm pore-size filter, but not in the channels of the 0.1-μm pore-size filters, preventing differentiation. It is concluded that the 0.1-μm pore-size filter blocks tooth development at the level of mesenchymal cell differentiation into odontoblasts. It is suggested that this differentiation requires a close association between the interacting mesenchymal and epithelial cells.

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Citation Excerpt :
Some three decades ago, leading investigators in the field of odontogenesis focused their attention on reciprocal epithelial–mesenchymal signal exchanges during tooth formation. These investigators postulated the exchange of biochemical signals, RNA, and other effector molecules believed to be responsible for instructing epithelia‐fate or mesenchyme‐fate during tooth development (Kollar and Baird, 1970a,b; Kollar and Lumsden, 1979; Slavkin and Bringas, 1976; Thesleff et al., 1977). Moreover, they postulated that signaling occurring in the crown of the tooth might also occur during root formation (Thomas and Kollar, 1989).
Extracellular matrix proteins control the formation of the inorganic component of hard tissues including bone, dentin, and enamel. The structural proteins expressed primarily in the enamel matrix are amelogenin, ameloblastin, enamelin, and amelotin. Other proteins, like biglycan, are also present in the enamel matrix as well as in other mineralizing and nonmineralizing tissues of mammals. In addition, the presence of sulfated enamel proteins, and “tuft” proteins has been examined and discussed in relation to enamel formation. The structural proteins of the enamel matrix must have specific protein–protein interactions to produce a matrix capable of directing the highly ordered structure of the enamel crystallites. Protein–protein interactions are also likely to occur between the secreted enamel proteins and the plasma membrane of the enamel producing cells, the ameloblasts. Such protein–protein interactions are hypothesized to influence the secretion of enamel proteins, establish short‐term order of the forming matrix, and to mediate feedback signals to the transcriptional machinery of these cells. Membrane‐bound proteins identified in ameloblasts, and which interact with the structural enamel proteins, include Cd63 (cluster of differentiation 63 antigen), annexin A2 (Anxa2), and lysosomal‐associated glycoprotein 1 (Lamp1). These and related data help explain the molecular and cellular mechanisms responsible for the removal of the organic enamel matrix during the events of enamel mineralization, and how the enamel matrix influences its own fate through signaling initiated at the cell surface. The knowledge gained from enamel developmental studies may lead to better dental and nondental materials, or materials inspired by Nature. These data will be critical to scientists, engineers, and dentists in their pursuits to regenerate an entire tooth. For tooth regeneration to become a reality, the protein–protein interactions involving the key dental proteins must be identified and understood. The scope of this review is to discuss the current understanding of protein–protein interactions of the developing enamel matrix, and relate this knowledge to enamel biomineralization.
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Organ culture systems have proven extremely useful techniques in studies that investigate the process of normal and abnormal development. The explant of tissues into an organ culture system is one of the few techniques that maintains three-dimensional cellular interactions under conditions that simultaneously permit controlled experimental manipulation in vitro. In this article we outline a procedure for growing “faces” in culture. In this system, the facial primordia prepared from embryonic mice or chicks can be maintained in culture for up to 7–8 days. During this time, the facial primordia continue to grow, differentiate, fuse and develop into organized structures that closely resemble those observed in situ. The procedure is relatively simple, requiring only a stable substratum, culture medium, sufficient oxygenation and incubation of the organ system at 37°C. The advantages and disadvantages of the procedure are presented, along with detailed methods to help troubleshoot some of the common pitfalls of organ culture systems.
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Epithelial-mesenchymal interaction is a prerequisite for tooth morphogenesis. To study this interaction, inner enamel epithelium and dental papilla mesenchyme of molar tooth germs from a 16.5-day mouse embryo were dissociated enzymatically and cultured alone or after recombination. Characteristic matrix protein synthesized and secreted by recombined tooth germ was determined quantitatively by enzyme-linked immunosorbent assay. The protein was detected in the culture of recombined tooth germ but not of dissociated enamel epithelium alone. The amount of enamel protein increased until 8 days in culture. Morphological differentiation of the recombined epithelial rudiment into ameloblasts and enamel protein production were confirmed.
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1994, Developmental Biology
The expression of tissue-specific enamel matrix genes is believed to require both instructive and permissive interactions of enamel organ epithelium with dental papilla mesenchyme and/or extracellular matrix during a restricted period of development. Biosynthesis of amelogenin gene products has been found to be associated with the terminal differentiation of inner enamel organ epithelium. The developing mouse first mandibular molar was used for a detailed examination of the temporal initiation and developmental pattern of amelogenin transcription. These studies define temporally instructive versus permissive influences on amelogenin transcription. During in vivo development, amelogenin transcripts were detected in late cap (15 days in utero; E15) through bell stage (E16 through E19) mouse molar tooth formation utilizing reverse transcription coupled to polymerase chain reaction amplification. Alternatively spliced amelogenin transcripts were detected in late bell stage (E18) molars. Amelogenin transcripts were also detected in isolated late cap stage (E15) enamel organ epethelium dissected free of dental papilla mesenchyme and cultured within a substitute basement membrane gel, but not in identical cap stage enamel organ epithelium cultured on plastic or a laminin-coated filter. Amelogenin transcripts were also found in early cap stage (E14) isolated enamel organ epithelium cultured within a basement membrane gel, but were not detected in enamel organ epithelium isolated from earlier stages of odontogenesis and cultured within a basement membrane gel. The results of these experiments indicate that a basement membrane gel is a useful extracellular substrate which provides permissive interactions required for the expression of amelogenin transcripts by enamel organ epithelium and that instructive interactions which determine enamel organ epithelium to become committed to amelogenin transcription occur prior to the early cap stage (E14) of odontogenesis. The results also suggest that continued interactions of enamel organ epithelium with dental papilla mesenchyme serve to regulate amelogenin transcription and post-transcriptional amelogenin RNA splicing in a complex manner during odontogenesis.
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^☆: This work was supported by the National Research Council for Medical Sciences, Finland, the Sigrid Jusélius Foundation, and the Finska Läkaresällskapet.

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Brief noteInterference of tooth differentiation with interposed filters☆

Abstract

Exp. Cell Res

J. Ultrastruct. Res

J. Ultrastruct. Res

J. Ultrastruct. Res

Arch. Oral Biol

Develop. Biol

Exp. Cell Res

J. Anat

Anat. Rec

Brief note
Interference of tooth differentiation with interposed filters☆