Dentin matrix proteins (DMPs) enhance differentiation of BMMSCs via ERK and P38 MAPK pathways.

Dentin, the predominant mineralized tissue of the tooth, comprises an extracellular matrix of collagen and a heterogeneous mixture of non-collagenous components, many of which have cellular signaling properties. These properties may be important in signaling stem cell involvement in tissue regeneration following injury and the present study investigates their morphogenic effects on differentiation of Bone Marrow Stromal Stem Cells (BMMSCs) in vitro. Non-collagenous dentin matrix proteins (DMPs) were isolated from healthy human teeth and their effects on BMMSCs behavior examined during in vitro culture. In vitro, DMPs enhanced alkaline phosphatase activity and mineralization in BMMSCs cultures as well as increasing the expression of dentinogenic and osteogenic differentiation markers (including runt-related transcription factor 2, osterix, bone sialoprotein, dentin sialophosphoprotein and osteocalcin) at both transcript and protein levels, with 10 µg/mL DMPs being the optimal stimulatory concentration. Expression of phosphor-ERK/phosphor-P38 in BMMSCs was up-regulated by DMPs and, in the presence of the ERK1/2- and p38-specific inhibitors, the differentiation of BMMSCs was inhibited. These data indicate that DMPs promote the dentinogenic/osteogenic differentiation of BMMSCs via the ERK/p38 MAPK pathways.

Differentiation of BMMSCs into odontoblast-like cells induced by natural dentine matrix.

OBJECTIVE: To assess the odontogenic potential of bone marrow mesenchymal stem cells (BMMSCs) to differentiate into odontoblast-like cells under the morphogenetic influence of dentine matrix as a possible basis for new stem cell-mediated therapeutic approaches to pulp diseases. DESIGN: BMMSCs were harvested from the whole bone marrow and cells at passages 3-5 were used for subsequent experiments. For in vitro studies, 1×10(4) cells were seeded on the surface of dentine slabs and co-cultured for 2 weeks in 24-well plates, then fixed, decalcified, embedded in paraffin and serial sections were processed for analyses. Haematoxylin-eosin (HE) staining was used for the morphological analysis of BMMSCs on the dentine slabs. The protein expression of dentine sialoprotein (DSP) in co-cultured BMMSCs was detected by immunohistochemical (IHC) staining. For in vivo studies, 5×10(6) cells were collected as cell pellets, seeded onto dentine slices and transplanted into renal capsules for 6 weeks. Histological analyses of harvested tissues were performed as described for the in vitro studies. Total RNA and protein were extracted from harvested tissues and Dspp/DSP expression was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. RESULTS: After 2 weeks of co-culture with dentine slabs, BMMSCs demonstrated good viability in terms of morphological appearance and some showed polarization and extension of their cytoplasmic processes into dentine tubules with DSP expression. In vivo study demonstrated similar morphological changes and DSP expression in cells adjacent to dentine. RT-PCR and Western blot also demonstrated that the expression of Dspp/DSP in the co-cultured BMMSCs groups was higher than in the control groups. CONCLUSION: Dentine matrix can signal morphogenic induction of differentiation of BMMSCs into odontoblast-like cells in vivo and in vitro.

Angiogenic activity of dentin matrix components.

UNLABELLED: Angiogenesis is key to both the development and regeneration of the dentin-pulp complex. OBJECTIVE: We hypothesized that proangiogenic signaling molecules sequestered in dentin matrix can be solubilised to induce angiogenic events. METHODS: Matrix components were extracted from powdered sound human dentin with EDTA and their dose-dependent (0.0001-5 mg/mL) effects examined in endothelial cells in an in vitro angiogenic tube formation assay, proliferation assay, and transcriptional regulation of the VEGF and VEGF-R2 genes. RESULTS: Lower concentrations of dentin matrix components were found to show proangiogenic activity, whereas higher concentrations suppressed angiogenic activity. CONCLUSION: This study highlights that the release of dentin matrix components after dental injury can contribute to the angiogenic events that support pulp regeneration.

Phenotype and behaviour of dental pulp cells during expansion culture.

OBJECTIVE: Primary pulp cell cultures are frequently used to study cellular responses, odontogenic potential and stem cell responses. Their isolation and expansion via a range of technical approaches are widely reported. The purpose of this study was to investigate the influence of isolation approach and extended expansion on cell phenotype and behaviour. DESIGN: To determine viable cell isolation, enzymatic dissociation was performed on rodent incisor pulps using collagenase, trypsin, hyaluronidase and ficin. Extended expansion culture of released cells was performed in DMEM and alpha-MEM media. Cultures were subsequently analysed for gene expression, cell proliferation, cell morphology and differentiation capacity up to passage 20. RESULTS: Data indicated that incubation of extirpated and mechanically minced rodent pulpal tissue with 0.25% Trypsin:EDTA and subsequent culture in alpha-MEM medium provided optimal conditions for maximal cell growth and expansion. Under these conditions, extended culture decreased cellular proliferative capacity up to passage 7, whilst higher passages demonstrated recovered growth rates. In general gene expression analysis of osteogenic and dentinogenic associated markers decreased with increasing passage number. Notably expression of TGFbetas-1, -2 and -3 increased up to passage 10 as did the stem cell and pericyte/myofibroblast markers, CD74, Neuroserpin and alpha-SMA. Analysis of molecular phenotypes indicated little difference in lineage differentiation capacity between earlier and later passages. CONCLUSIONS: The present study characterizes conditions for primary pulp cell isolation and expansion and indicates that both earlier and later passages maintain differentiation capacity. Continued passage however may result in selection for cells with a pericyte/myofibroblast phenotype.

The erosive potential of flavoured sparkling water drinks.

OBJECTIVE: The potential role of acidic drinks in the aetiology of dental erosion is well recognized. Whilst the wide-scale consumption of bottled waters is unlikely to contribute significantly to erosion, the role of flavoured sparkling water drinks is unclear. The aim of this study was to determine the pH, titratable acidity and in vitro erosive potential of a selection of these drinks drawn from the UK market to identify what dietary advice would be appropriate in relation to their consumption. METHODS: pH was measured using a pH electrode and titratable acidity recorded by titration with 0.1-m NaOH. Erosive potential was assessed using an in vitro dissolution assay with hydroxyapatite powder and electron microscopic examination of surface enamel of extracted human teeth, following exposure to the flavoured sparkling waters for 30 min. RESULTS: All of the flavoured waters tested showed appreciable titratable acidity (0.344-0.663 mmol) and low pH (2.74-3.34). In the hydroxyapatite dissolution assay, all of the waters demonstrated erosive potential (89-143%) similar to or greater than that of pure orange juice, an established erosive drink. Exposure of the extracted teeth to the flavoured waters resulted in surface changes consistent with erosive dissolution. CONCLUSIONS: Flavoured sparkling waters should be considered as potentially erosive, and preventive advice on their consumption should recognize them as potentially acidic drinks rather than water with flavouring.