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vß3 Integrin Expression in Human Odontoblasts and Co-localization with Osteoadherin

Laboratory of Development of Dental Tissues, EA 1892, IFR 62, Faculty of Odontology, Lyon 1 University, G. Paradin Str., 69372 Lyon Cedex 08, France;

View larger version (101K): [in a new window]   Figure 1. Expression of v and ß3 integrin subunit genes in human dental pulp. (a) Analysis of the whole pulp by RT-PCR revealed the presence of mRNAs for both integrin subunits. PCR products migrate to a position in good agreement with their predicted sizes (v, 305 bp; ß3, 517 bp). Ethidium-bromide-stained 2% agarose gel. Digestion with restriction enzymes confirmed amplicon identity (not shown). M: molecular-weight markers (pUC Mix Marker 8, Fermentas Inc., Hanover, MD, USA). (b-e) Detection of v and ß3 integrin transcripts by in situ hybridization. v and ß3 integrin subunit mRNAs were present in mature odontoblasts (Od) (b,c) and in cells surrounding blood vessels (BV) (d,e), but not in pulp core cells (P). (f) Hybridization with the ß3 integrin subunit sense probe. No signal was detected. Bars: 40 µm.

View larger version (62K): [in a new window]   Figure 2. Immunolocalization of vß3 integrin and osteoadherin in human dental pulp. (a) An intense membrane staining was observed with the anti-vß3 integrin antibody in mature odontoblasts (Od) of the pulp horn, including processes in dentin tubules (arrows). (b) Some blood vessels (BV) were also heavily stained, whereas others were not. Isolated cells in the blood vessel wall were also positive (arrow). (c) In the same tooth section, vß3 integrin staining decreased progressively in the apical direction and became moderate in cervical odontoblasts. (d) In newly differentiated odontoblasts of the forming root, the staining was weak and less intense than in blood vessels. (e) A third molar with a mild caries lesion (C) was used for vß3 staining. The black box indicates the localization of Figs. 2f and 2g. (f) Odontoblasts aligned at the pulp periphery or included in the reactionary dentin matrix (RD) were stained with the anti-vß3 integrin antibody. (g) Blood vessels and small rounded cells scattered in the tissue (arrow) were also stained. (h) Osteoadherin was immunodetected in pulp horn odontoblasts, including processes in dentin tubules (arrows). No other cell type was stained. (i) Newly differentiated odontoblasts in the forming root were heavily stained. Osteoadherin was also detected in predentin (Pd). D: dentin. P: pulp. Bars: 50 µm.

View larger version (77K): [in a new window]   Figure 3. Immunolocalization of vß3 integrin and osteoadherin in odontoblasts differentiated in vitro. (a) Confluent cells in the middle of the culture showed strong membrane staining with the anti-vß3 integrin antibody and moderate staining of the cytoplasm. (b) Cell body and process were clearly delineated in some cells with typical odontoblast morphology. (c) At the culture periphery, where newly polarized cells aligned to constitute a typical odontoblast layer, vß3 staining was detected on the membrane of odontoblast cell bodies at the level of contact zones and intercellular junctions (arrows). (d) In extracts of odontoblast cell cultures, osteoadherin was biochemically identified as a 110-kDa molecule with the purified anti-peptide antiserum (reducing conditions). No immunoreactive band was observed when the anti-osteoadherin antibody was omitted. (e) Osteoadherin staining was detected intracellularly close to the nucleus in all odontoblasts in vitro, but not in the extracellular compartment. Bars: 100 µm (a), 50 µm (b,c), 75 µm (e).