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Establishment of in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells

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Abstract

We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel’s method. We succeeded in establishing an in vitro culture system for evaluating dentin–pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1–3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp–dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.

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Acknowledgments

The authors cordially thank Dr. Helena Ritchie for providing Dspp plasmids. This work was supported in part by Grants-in-Aid for Scientific Research (B) (Nos. 22390341 and 25293371 to H.O.) from JSPS.

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  1. Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan

    Hiroko Ida-Yonemochi, Mitsushiro Nakatomi & Hayato Ohshima

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  1. Hiroko Ida-Yonemochi
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  2. Mitsushiro Nakatomi
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  3. Hayato Ohshima
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Correspondence to Hayato Ohshima.

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418_2014_1200_MOESM1_ESM.jpg

Supplementary Fig. 1 Macroscopic view of tooth slice culture. a A tooth slice is put onto a membrane filter and cultured in a Trowell system. b A whole-mount immunohistochemistry for nestin in the sliced tooth at 7 days after slice culture. (JPEG 359 kb)

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Ida-Yonemochi, H., Nakatomi, M. & Ohshima, H. Establishment of in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells. Histochem Cell Biol 142, 323–333 (2014). https://doi.org/10.1007/s00418-014-1200-7

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