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Objective To compare proteomics and biological function of human dentin matrix molecules (hDMMs) and bovine dentin matrix molecules (bDMMs). Design Dentin powder from human or bovine teeth (n = 4) was demineralized in 10% (v/v) ethylenediaminetetraacetic acid for 7 days. The extracts were dialyzed, lyophilized and proteins were characterized using liquid chromatography-tandem mass spectrometry and shotgun proteomic analysis. To study biological function, mouse-derived undifferentiated dental pulp cells (OD21) were treated with 0.01, 0.1 or 1 μg/mL of hDMMs or bDMMs and proliferation was measured after 24 hours and 48 hours using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell migration was assessed after 24 hours using a Boyden chamber. Alizarin Red S staining was used to evaluate mineral formation. Results There were 307 proteins identified, of which 93 proteins were common to both species. Gene Ontology functional analysis demonstrated similar pattern of biological process in both species which consisted mainly of tissue development and biomineralization. hDMMs and bDMMs both enhanced cell proliferation. After 24 hours, all concentrations of bDMMs promoted cell proliferation (p ≤ 0.05), while hDMMs did not affect proliferation. After 48 hours, groups with 1μg/mL of bDMMs and 0.01μg/mL of hDMMs had increased cell proliferation compared to control (p ≤ 0.0001). All concentrations of hDMMs and bDMMs enhanced cell migration and mineralization (p ≤ 0.0001). Conclusion bDMMs has similar biological functions as hDMMs. Moreover, bDMMs stimulated cell proliferation, migration and differentiation similar to hDMMs.

Objective Dentine sialophosphoprotein (DSPP) was initially thought to be unique for dentine formation during tooth development, whilst recent reports have shown a much broader expression pattern such as in bone, periodontium and inner ear. Our goal was to explore its expression and potential impact during early nasal cartilage formation in comparison with tooth development. Study design We investigated DSPP expression in the nasal cartilage by immunohistochemistry and in situ hybridisation. We also cloned a 719 bp partial DSPP cDNA from nasal cartilage and analysed its homology to the published mouse DSPP cDNA sequence. In addition, quantitative RT-PCR was undertaken to compare the expression pattern of DSPP in nasal cartilage and tooth germs during embryonic development. Results The expression of DSPP in mouse nasal chondrocytes was detected using in situ hybridisation and immunohistochemical staining. The quantitative RT-PCR data showed that expression levels of DSPP in nasal cartilage are similar to that of tooth: low at E18, and increased during development with the peak level at P3. Furthermore, DSPP levels in nasal cartilage are lower than tooth but higher than bone. Conclusion DSPP is expressed in nasal cartilage, and a similar temporal expression pattern in cartilage and tooth indicates the potential importance of DSPP during development.

As a mineralised connective tissue, dentine is well adapted to its functional role as a major structural component of the tooth. Although similar in composition to bone, dentine matrix is not remodelled physiologically and traditionally, has been regarded as a rather inert tissue. Nevertheless, dentine–pulp demonstrates strong regenerative potential which allows it to respond to disease and traumatic injury. Such responses are strongly influenced by cell–matrix interactions and modified by disease processes, including infection and inflammation. The identification of many bioactive molecules bound within dentine matrix has allowed their potential involvement in regenerative and other tissue responses to be better understood and new opportunities to be recognised for novel clinical therapies.

Objective Vascular endothelial growth factor (VEGF) has been implicated in the regulation of dental pulp and dentine repair. Therapeutic ultrasound was shown to be effective for fracture repair. We investigated whether low frequency ultrasound influences the production of VEGF by odontoblast-like cells. Moreover, we examined the direct effects of VEGF on odontoblast-like cell proliferation. Design MDPC-23, an established odontoblast-like cell line, was exposed to increasing intensities of 30 kHz ultrasound using an ultrasonic tip probe. Results After 24 h cell culture, WST-1 analysis of cell viability and number showed a dose-dependent decrease in the number of viable cells with increasing ultrasound power. However, the relative concentration of VEGF as analysed by ELISA and normalised to cell number was significantly increased in the culture supernatants indicating an ultrasound-induced stimulation of odontoblastic VEGF secretion. Analysis of VEGF gene expression by sqRT-PCR revealed the expression of the main VEGF isoforms in the MDPC-23 cells, i.e. VEGF 120 and VEGF 164 as well as to a minor extent VEGF 188 . Low power ultrasound increased gene expression of all VEGF isoforms. Addition of recombinant VEGF to the cell cultures significantly stimulated cell proliferation. Gene expression of the VEGF receptors Flt1/VEGFR1 and KDR/VEGFR2 was detected in the MDPC-23, suggesting the possibility that VEGF may act on the odontoblast-like cells in an autocrine manner. Conclusions Our results indicate that ultrasound promoted VEGF expression and production by odontoblast-like cells and that VEGF may have autocrine effects on these cells. It is proposed that ultrasound may influence odontoblast activity and dentine repair by modulating production of endogenous growth factors in the dentine-pulp complex.

Isolation of sufficient quantities of pure populations of odontoblasts from healthy and diseased teeth will facilitate our understanding of dentinogenesis during development and repair. Here we describe a novel Piezo-power microdissection (PPMD) technique for the isolation of pure populations of odontoblasts and pulpal tissue from formalin-fixed, paraffin-embedded, mature, healthy and carious human teeth. Odontoblasts and pulpal tissue gene expression were subsequently studied in ribonucleic acid isolated from PPMD preparations using a semi-quantitative reverse transcription polymerase chain reaction approach. Data confirmed that the genes for dentine sialophosphoprotein and Nestin are preferentially expressed in odontoblasts, whilst the genes for both collagen-1alpha and collagen-3alpha were expressed preferentially in pulpal tissue, particularly in carious samples. PPMD provides a novel and powerful approach to isolate pure populations of dental tissues and cells from fixed specimens for subsequent downstream molecular analyses.

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 .

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 TGF beta s-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. (C) 2009 Elsevier Ltd. All rights reserved.

Summary Objective: The function of apoptosis and its regulation in odontoblasts remain unclear. In this study, we characterize the possible role of transforming growth factor (TGF)-β 1 in the induction of apoptosis and the molecular mechanisms that mediate TGF-β1-induced apoptosis in odontoblasts. Methods: Annexin V/propidium iodide staining, cell Death Detection ELISA and DNA ladder were used to examine the effect of TGF-β1 on apoptosis in a mouse odontoblast-like cell line, MDPC-23. Stable cell clones expressing Smad2 or Smad3 dominant negative mutants, or wild-type Smad7 were constructed to investigate the role of Smad proteins in the mediation of apoptosis by TGF-β1 in MDPC-23 cells. The TGF-β1-induced transcriptional activity in stable cell clones expressing Smad proteins was analyzed by a transient transfected TGF-β-responsive reporter gene, p3TP-Lux. Results: TGF-β1 can induce apoptotic cell death in MDPC-23 cells in a dose-dependent manner. Transfection of dominant negative mutant forms of Smad2 or Smad3 blocked TGF-β1-induced apoptosis; moreover, the Smad3 mutant was more efficient than the Smad2 mutant. Transfection of Smad7, an inhibitory Smad, also significantly inhibited TGF-β1-induced apoptosis of these cells. Over-expression of Smad3 dominant negative mutant or Smad7 significantly inhibited TGF-β1-induced transcriptional activity. Conclusion: These results suggest that Smad proteins are involved in TGF-β1-induced apoptosis of odontoblast cells.

Cranial neural crest-derived ectomesenchymal cells represent a population of pluripotent stem cells giving rise to many of the various oro-facial and dental tissues. The factors determining the terminal fate of these cells are still unclear. The potentiality of human embryonic ectomesenchymal cells from the first branchial arch have been investigated when isolated and grown in a three-dimensional (3D)-collagen gel culture system in the presence of dentin matrix-derived non-collagenous proteins (DNCP) and TGFbeta-1. Functional differentiation of cells showing some characteristics of odontoblast-like cells could be observed when the cells were cultured with DNCP+TGFbeta-1 or DNCP, however, only cytological differentiation was observed during culture with TGFbeta-1 alone. The characteristics of these cells was assessed by morphological appearance, expression of the odontoblast phenotype marker dentin sialophosphoprotein (DSPP), increased alkaline phosphatase levels and formation of mineralised nodules in vitro. The results indicate that these embryonic cells from the first branchial arch are capable of responding to the inductive stimulus of DNCP or DNCP+TGFbeta-1 when isolated and grown in the 3D collagen gel culture system. The capacity of the isolated cells to differentiate into mineralizing cells showing some characteristics of odontoblast-like cells under these growth conditions highlights the potential of such approaches for tissue engineering strategies for hard-tissue regeneration after injury.

Objective : Transforming growth factor-β (TGF-β) regulates odontoblast differentiation and stimulates dentine extracellular matrix synthesis. However, until recently, the molecular mechanisms of action of TGF-β have been unknown. Smad proteins have recently been identified as intracellular signalling mediators of TGF-β. In this study, we characterise the role of Smad proteins as mediators of TGF-β in a mouse odontoblast cell line MDPC-23. Methods : Transcription of Smads was detected by RT-PCR. The change of intracellular location of Smad proteins treated by TGF-β1 was evaluated immunocytochemically. Smad function and its role in transcription of dentin sialophosphoprotein (DSPP) were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. Results : MDPC-23 cells expressed Smad2, Smad3 and Smad4 mRNA. Endogenous Smad2, Smad3 and Smad4 rapidly translocated from the cytoplasm into the nucleus in response to TGF-β1. The activity of the TGF-β-responsive p3TP-Lux reporter construct was stimulated by 12.7-fold with TGF-β1 treatment. Over-expression of wild-type Smad3 promoted TGF-β1-induced luciferase activity, whereas dominant negative Smad3 inhibited it. TGF-β1 also inhibited the activity of DSPP promoter luciferase reporter construct containing the sequence between −791 bp and +54 bp of the mouse DSPP gene. Over-expression of wild-type Smad3 potentiate the inhibitory effect of TGF-β1 on transcriptional regulation of DSPP, while dominant negative Smad3 decreased the effect. In contrast to Smad3, wild-type Smad2 or its dominant negative mutant had little effect on TGF-β1 regulation of the promoter activity of DSPP. Conclusions : Smad2, Smad3 and Smad4 are present and activated by TGF-β1 in MDPC-23 cells. The Smad pathway is functional in these cells and Smad3 appears to be involved in down-regulation of DSPP by TGF-β1. These findings raise the possibility that Smad signalling plays a role in dentinogenesis.

Knowledge of the molecular events that occur in carious disease has so far been constrained due to difficulties in obtaining sufficient quantities of the dental tissues and cells involved. Our histological findings indicate that a pulp-odontoblast cellular complex can be obtained from carious and healthy human teeth when exposed to low-temperatures prior to pulpal extirpation and from rodent teeth processed at room-temperature. In contrast, pulpal tissue extracted from room-temperature processed human teeth and low-temperature processed rodent teeth resulted in the odontoblast layer remaining attached to the pulp chamber. Semi-quantitative RT-PCR (sq-RT-PCR) analysis confirmed that markers previously shown to be preferentially expressed in odontoblasts, namely dentin sialophosphoprotein (DSPP) and Nestin, amplified more readily from the extracted pulp-odontoblast complex, as compared to pulpal tissue alone, in both human and rodent samples. Subsequent gene expression analysis of collagen-1alpha and collagen-3alpha indicated levels were significantly higher in carious pulpal tissue. In addition, analysis characterising the expression of members of the transforming growth factor and bone morphogenic protein families and their receptors indicated in general, that these genes were expressed by healthy odontoblasts and up-regulated in both pulpal cells and odontoblasts in response to carious injury. Use of this temperature-sensitive dental tissue preparation procedure allows detection of differential gene expression in odontoblasts and other pulpal cells in healthy and carious tissue.