A Novel Organ Culture Method to Study the Function of Human Odontoblasts in vitro: Gelatinase Expression by Odontoblasts is Differentially Regulated by TGF-β1

Abstract

Odontoblasts cannot be cultured by traditional cell culture methods, thus restricting in vitro studies. Here we present an organ culture method for human odonto-blasts that utilizes the pulp chamber as a culture crucible. Crowns of human third molars were dissected, pulp was gently removed, and the odontoblasts attached to and in the walls of the pulp chambers were cultured in serum-free OPTI-MEM medium, or DMEM/Ham's F12 medium containing 10% serum. Pulp tissues were cultured separately. Cell content and morphology were analyzed by SEM, and the removed pulps were examined by light microscopy. Proteins secreted into the medium with or without TGF-beta1 supplementation were metabolically labeled with [35S]methionine, and the total protein content was assessed by TCA precipitation and SDS-PAGE/fluorography. To assess the role of gelatinolytic enzymes on dentin matrix remodeling, we used enzymography to analyze the effect of TGF-beta1 on gelatinase A and B expression. SEM revealed odontoblasts in pulp chambers after 5 days of culture, with only few or no fibroblasts, and no alterations in the odontoblast cell morphology or differences between the cells cultured in serum-free and serum-containing media. Rarely were any odontoblasts present in pulp tissue. Radiolabeling revealed protein synthesis and secretion until day 6 in both the odontoblast and pulp cultures, with no marked differences between TGF-beta1-treated and control cultures. The level of gelatinase A remained constant up to 7 days, while gelatinase B expression was always low and decreased with time in culture. However, gelatinase B levels were markedly increased upon TGF-beta1 treatment of cells and remained high to day 7. The results suggest that this method provides a novel technique for the study of human odontoblasts in vitro and that odontoblasts can be cultured even in serum-free conditions.

Evaluation of plasmatic MMP-8, MMP-9, TIMP-1 and MPO levels in obese and lean women

“…We observed reduced MMP-8 levels in obese women, which may be explained by the negative regulation of MMP-8 expression by biomolecules such as transforming growth factor-beta 1 (TGF-β1) that are elevated in obesity [28] and that strongly downregulate MMP-8 [29], while stimulating MMP-9 expression [30]. To exclude the possibility of decreased MMP-8 levels due to reduced neutrophil activation, we measured the plasma MPO activity.…”

Matrix Metalloproteinases and Other Matrix Proteinases in Relation to Cariology: The Era of ‘Dentin Degradomics'

“…Only 15 years later, mature human odontoblasts were shown to express MMP-2 and MMP-9 [Tjäderhane et al, 1998b], MMP-2, MMP-9 and MMP-8 were identified in carious dentin, and the role of MMPs in the degradation of dentin collagen was demonstrated [Tjäderhane et al, 1998a]. At the same time, the activation of latent salivary pro-MMPs by exposure to low pH followed by neutralization (so-called acid activation) was also shown [Tjäderhane et al, 1998a;Sulkala et al, 2001].…”

Pre-eruptive Intracoronal Resorption of Dentin in a Permanent Molar: Case Report

“…Only 15 years later, mature human odontoblasts were shown to express MMP-2 and MMP-9. 20,21 Resorption in the crown dentin may be due to these factors. These factors should be investigated in future studies.…”