Anne Poliard

Inflammatory and immunological aspects of dental pulp repair

The repair of dental pulp by direct capping with calcium hydroxide or by implantation of bioactive extracellular matrix (ECM) molecules implies a cascade of four steps: a moderate inflammation, the commitment of adult reserve stem cells, their proliferation and terminal differentiation. The link between the initial inflammation and cell commitment is not yet well established but appears as a potential key factor in the reparative process. Either the release of cytokines due to inflammatory events activates resident stem (progenitor) cells, or inflammatory cells or pulp fibroblasts undergo a phenotypic conversion into osteoblast/odontoblast-like progenitors implicated in reparative dentin formation. Activation of antigen-presenting dendritic cells by mild inflammatory processes may also promote osteoblast/odontoblast-like differentiation and expression of ECM molecules implicated in mineralization. Recognition of bacteria by specific odontoblast and fibroblast membrane receptors triggers an inflammatory and immune response within the pulp tissue that would also modulate the repair process.

All hepatocytes are involved in the expression of the albumin gene in the normal adult rat: A demonstration by in situ hybridization and immunoperoxidase techniques

Immunoperoxidase techniques have yielded conflicting results concerning the percentage of hepatocytes engaged in albumin production in normal adult rats. In addition, the question of whether functional differences in the synthesis of plasma proteins exist within the hepatic lobule remains to be determined. To clarify these questions, we have searched for gene albumin transcripts by in situ hybridization, and for the corresponding protein by immunoperoxidase, on adjacent liver sections. We observed that all hepatocytes contain albumin transcripts as well as the albumin protein, without any detectable zonal variation within the liver lobule. Taken altogether, these results demonstrate that every hepatocyte, whatever its location in the hepatic lobule, is actively engaged in albumin gene expression.

Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

The pluripotent mesoblastic C1 cell line was used under serum‐free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone‐dependent Sox9 upregulation is essential for chondrogenesis.

Serotonergic 5-HT2B Receptor Controls Tissue-nonspecific Alkaline Phosphatase Activity in Osteoblasts via Eicosanoids and Phosphatidylinositol-specific Phospholipase C

In previous studies, we observed that mice knocked out for the serotonin-2B receptor (5-HT2BR) show defects in bone homeostasis. The present work focuses on the downstream targets relaying the anabolic function of this receptor in osteoblasts. A functional link between the 5-HT2BR and the activity of the tissue-nonspecific alkaline phosphatase (TNAP) is established using the C1 osteoprogenitor cell line. During C1 osteogenic differentiation, both 5-HT2BR and TNAP mRNA translations are delayed with respect to extracellular matrix deposition. Once the receptor is expressed, it constitutively controls TNAP activity at a post-translational level along the overall period of mineral deposition. Indeed, pharmacological inhibition of the 5-HT2BR intrinsic activity or shRNA-mediated 5-HT2BR knockdown prevents TNAP activation, but not its mRNA translation. In contrast, agonist stimulation of the receptor further increases TNAP activity during the initial mineralization phase. Building upon our previous observations that the 5-HT2BR couples with the phospholipase A2 pathway and prostaglandin production at the beginning of mineral deposition, we show that the 5-HT2BR controls leukotriene synthesis via phospholipase A2 at the terminal stages of C1 differentiation. These two 5-HT2BR-dependent eicosanoid productions delineate distinct time windows of TNAP regulation during the osteogenic program. Finally, prostaglandins or leukotrienes are shown to relay the post-translational activation of TNAP via stimulation of the phosphatidylinositol-specific phospholipase C. In agreement with the above findings, primary calvarial osteoblasts from 5-HT2BR-null mice exhibit defects in TNAP activity.

Matricellular molecules and odontoblast progenitors as tools for dentin repair and regeneration.

This review summarizes the in vivo experiments carried out by our group after implantation of bioactive molecules (matricellular molecules) into the exposed pulp of the first maxillary molar of the rat or the mandibular incisor of rats and mice. We describe the cascade of recruitment, proliferation and terminal differentiation of cells involved in the formation of reparative dentin. Cloned immortalized odontoblast progenitors were also implanted in the incisors and in vitro studies aimed at revealing the signaling pathways leading from undifferentiated progenitors to fully differentiated polarized cells. Together, these experimental approaches pave the way for controlled dentin regenerative processes and repair.

Short-term effects of amelogenin gene splice products A+4 and A-4 implanted in the exposed rat molar pulp

In order to study the short-time effects of two bioactive low-molecular amelogenins A+4 and A-4, half-moon cavities were prepared in the mesial aspect of the first maxillary molars, and after pulp exposure, agarose beads alone (controls) or beads soaked in A+4 or A-4 (experimental) were implanted into the pulp. After 1, 3 or 7 days, the rats were killed and the teeth studied by immunohistochemistry. Cell proliferation was studied by PCNA labeling, positive at 3 days, but decreasing at day 7 for A+4, whilst constantly high between 3 and 7 days for A-4. The differentiation toward the osteo/odontoblast lineage shown by RP59 labeling was more apparent for A-4 compared with A+4. Osteopontin-positive cells were alike at days 3 and 7 for A-4. In contrast, for A+4, the weak labeling detected at day 3 became stronger at day 7. Dentin sialoprotein (DSP), an in vivo odontoblast marker, was not detectable until day 7 where a few cells became DSP positive after A-4 stimulation, but not for A+4. These results suggest that A +/- 4 promote the proliferation of some pulp cells. Some of them further differentiate into osteoblast-like progenitors, the effects being more precocious for A-4 (day 3) compared with A+4 (day 7). The present data suggest that A +/- 4 promote early recruitment of osteogenic progenitors, and evidence functional differences between A+4 and A-4.

Autoregulatory loop of Msx1 expression involving its antisense transcripts

The Msx1 homeogene plays an important role in epithelial–mesenchymal interactions leading organogenesis. Msx1 gene is submitted to bidirectional transcription generating a long non‐coding antisense (AS) RNA potentially involved in Msx1 expression regulation. RT‐Q‐PCR and RNA‐FISH studies indicated that transient overexpression of the Msx1 AS transcript in 705IC5 mouse odontoblasts decreased the abundance of endogenous Msx1 S mRNA at the post‐transcriptional level. Conversely, Msx1 overexpression increased the AS RNA level probably by activating AS transcription. In vivo mapping by RT‐PCR evidenced both Msx1 RNAs in all adult mouse tissues tested raising the issue of Msx1 function during adulthood. The expression patterns of the two RNAs were similar, confirming the tight S/AS relationship. In particular, both Msx1 mRNAs and Msx1 protein were similarly distributed in eyes, and were found in regions with a common ectodermic origin and in cells potentially involved in regeneration. In conclusion, we report that Msx1 S RNA is negatively controlled by its AS RNA at a post‐transcriptional level, and that the AS RNA is retrocontrolled positively by Msx1. The tight link between Msx1 S and AS RNAs constitutes a regulatory loop resulting in a fine‐tuned expression of Msx1 which appears to be significant for adult homeostasis. J. Cell. Physiol. 220: 303–310, 2009.

An In vivo Model for Short-Term Evaluation of the Implantation Effects of Biomolecules or Stem Cells in the Dental Pulp

The continuously growing rodent incisor is a widely used model to investigate odontogenesis and mineralized tissue formation. This study focused on evaluating the mouse mandibular incisor as an experimental biological tool for analyzing in vivo the capacity of odontoblast-like progenitors or bioactive molecules to contribute to reparative dentinogenesis. We describe here a surgical procedure allowing direct access to the forming part of the incisor dental pulp Amelogenin peptide A+4 adsorbed on agarose beads, or dental pulp progenitor cells were implanted in the pulp following this procedure. After 10 days A+4 induced the formation of an osteodentin occluding almost the totality of the pulp compartment. Implantation of progenitor cells leads to formation of islets of osteodentin-like structures located centrally in the pulp. These pilot studies validate the incisor as an experimental model to test the capacity of progenitor cells or bioactive molecules to induce the formation of reparative dentin.

Regulation of Gene Expression in Developmental and Oncogenic Processes: The Albumin Alpha-Fetoprotein Locus in Mammals

The molecular mechanisms underlying malignant cell transformation are hardly understood. An attractive hypothesis is that the acquisition of the malignant phenotype might be brought about by the activation of genes (e.g. oncogenes) whose expression is normally restricted to actively proliferating embryonic cells. Insight into the mechanisms controlling gene expression during developmental and oncogenic processes appears then essential to our understanding of neoplastic transformation.