Helena H. Ritchie

IN VITRO DIFFERENTIATION AND MINERALIZATION OF HUMAN DENTAL PULP CELLS INDUCED BY DENTIN EXTRACT

SummaryIn this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], β-glycerophosphate [β-GP]) and MS+DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS+DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, β-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.

The Expression and Role of Stromal Cell–derived Factor-1α–CXCR4 Axis in Human Dental Pulp

Recent reports have suggested that the stromal cell-derived factor (SDF)-1alpha-CXCR4 axis has a direct effect on stem and progenitor cell recruitment in muscle and neural tissue repair after injury. No information is available about SDF-1alpha or CXC chemokine receptor 4 (CXCR4) in dental tissues. The aim of this study was to assess the expression of SDF-1alpha and its receptor, CXCR4, in healthy or inflamed human dental pulp and to evaluate the effects of SDF-1alpha on dental pulp cells (DPCs) in both proliferation and migration in vitro. Immunohistochemical staining and reverse-transcription polymerase chain reaction detected weak expression of SDF-1alpha and CXCR4 in healthy dental pulp and strong expression of SDF-1alpha and CXCR4 in inflamed dental pulp. A methylthiazol tetrazolium assay showed that SDF-1alpha could not promote DPCs proliferation. A transmigration assay, however, indicated that SDF-1alpha enhanced DPCs migration, which could be abolished by anti-CXCR4 antibodies. Taken together, these results imply that the SDF-1alpha-CXCR4 axis may play a role in the recruitment of CXCR4-positive DPCs toward the damaged sites.

Effects of EDTA on the Hydration Mechanism of Mineral Trioxide Aggregate

Ethylenediaminetetraacetic acid (EDTA) is commonly used during the preparation of obstructed root canals that face a high risk of root perforation. Such perforations may be repaired with mineral trioxide aggregate (MTA). Due to EDTA’s ability to chelate calcium ions, we hypothesized that EDTA may disrupt the hydration of MTA. Using scanning electron microscopy and energy-dispersive x-ray spectroscopy, we found that MTA specimens stored in an EDTA solution had no crystalline structure and a Ca/Si molar ratio considerably lower than those obtained for specimens stored in distilled water and normal saline. Poor cell adhesion in EDTA-treated MTA was also noted. X-ray diffraction indicated that the peak corresponding to portlandite, which is normally present in hydrated MTA, was not shown in the EDTA group. The microhardness of EDTA-treated specimens was also significantly reduced (p < 0.0001). These findings suggest that EDTA interferes with the hydration of MTA, resulting in decreased hardness and poor biocompatibility.

Hypoxia Promotes Mineralization of Human Dental Pulp Cells

INTRODUCTION Dental pulp can be exposed to hypoxic conditions in case of trauma or inflammation. Dental pulp cells (DPCs) have mineralization potential, which plays a key role in pulp repair and reparative dentinogenesis process. Little information is available about DPC mineralization in hypoxic condition. The purpose of this study was to assess the influence of hypoxia on DPC mineralization to pave the way for a better understanding of dental pulp regeneration and reparative dentin formation. METHODS Human DPCs were obtained by using tissue explant technique in vitro and cultured in normoxia (20% O(2)) or hypoxia (5% O(2)). Cell viability was investigated by methyl-thiazol-tetrazolium assay. Cell mineralization was assessed by von Kossa staining and alizarin red S staining. Important mineral genes such as osteocalcin (OCN), dentin matrix acidic phosphoprotein-1 (DMP-1), bone sialoprotein (BSP), and dentin sialophosphoprotein (DSPP) were determined by real-time polymerase chain reaction. RESULTS Cell viability of DPCs increased more in hypoxia than in normoxia from day 3 to day 5. Von Kossa staining and alizarin red S staining showed DPCs in hypoxia had higher mineralization activity than in normoxia. Expression of mRNAs for OCN, DMP-1, BSP, and DSPP was greater in hypoxia than in normoxia. CONCLUSIONS These results imply that hypoxia promotes DPC mineralization.

A novel rat 523 amino acid phosphophoryn: nucleotide sequence and genomic organization

Phosphophoryns (PP), the major noncollagenous proteins (NCPs) in dentin, are believed to play a crucial role in mineral nucleation and hydroxyapatite growth during dentin mineralization. Previously we identified two mature rat PP transcripts, one coding for a 240 amino acid protein (designated as PP(240)) (H.H. Ritchie, L.-H. Wang, J. Biol. Chem. 271 (1996) 21695-21698), and another coding for a 171 amino acid protein (PP(171)) (H. Ritchie, L. Wang, Biochim. Biophys. Acta 1493 (2000) 27-32). We now have identified a third novel dentin sialoprotein (DSP)-PP cDNA transcript that encodes a 523 amino acid protein (PP(523)) with typical PP characteristics including DSS and DS motifs suitable as potential casein kinase I and II phosphorylation sites. Based on amino acid composition, the PP(523) protein product is identical to native rat HP2. We also show that the PP(523) sequence is identical to the corresponding genomic DNA sequence. Taken together, the existence of multiple DSP-PP transcripts, each significantly different from the other in net negative charge, suggests that dentin mineralization processes may be under fine-tune control by these PP protein isoforms.

Phosphate and calcium uptake by rat odontoblast-like MRPC-1 cells concomitant with mineralization.

It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this study was to characterize cellular Pi and Ca2+ uptake in the novel rat odontoblast‐like cell line mineralizing rat pulpal cell line (MRPC) 1 during mineralization to see if changes in the ion transport activity would occur as the cultures develop and begin forming a mineralized matrix. MRPC‐1 cells were cultured in chemically defined medium containing ascorbate and Pi, and cultures were specifically analyzed for cellular Pi and Ca2+ uptake activities and expression of type II high‐capacity Na+‐Pi cotransporters. The odontoblast‐like phenotype of the cell line was ascertained by monitoring the expression of collagen type I and dentin phosphopoprotein (DPP). Mineralized nodule formation started at day 9 after confluency and then rapidly increased. Ca2+ uptake by the cells showed a maximum during the end of the proliferative phase (days 5–7). Pi uptake declined to a basal level during proliferation and then was up‐regulated simultaneously with the onset of mineralization to a level fourfold of the basal uptake, suggesting an initiating and regulatory role for cellular Pi uptake in mineral formation. This up‐regulation coincided with a conspicuously increased glycosylation of NaPi‐2a, indicating an activation of this Na+‐Pi cotransporter. The study showed that MRPC‐1 cells express an odontoblast‐like phenotype already at the onset of culture, but that to mineralize the collagenous extracellular matrix (ECM) that formed, a further differentiation involving their ion transporters is necessary.

Development of an odontoblast in vitro model to study dentin mineralization.

The aim of the present work was to characterize the odontoblastic proliferation, differentiation, and matrix mineralization in culture of the recently established M2H4 rat cell line. Proliferation was assessed by cell counts, differentiation by RT-PCR analysis, and mineralization by alizarin red staining, atomic absorption spectrometry, and FTIR microspectroscopy. The results showed that M2H4 cell behavior closely mimics in vivo odontoblast differentiation, with, in particular, temporally regulated expression of DMP-1 and DSPP. Moreover, the mineral phase formed by M2H4 cells was similar to that in dentin from rat incisors. Finally, because in mice, transforming growth factor (TGF)-β1 over-expression in vivo leads to an hypomineralization similar to that observed in dentinogenesis imperfecta type II, effects of TGF-β1 on mineralization in M2H4 cell culture were studied. Treatment with TGF-β1 dramatically reduced mineralization, whereas positive control treatment with bone morphogenetic protein-4 enhanced it, suggesting that M2H4 cell line is a promising tool to explore the mineralization mechanisms in physiopathologic conditions.

Dentin-pulp complex responses to carious lesions.

To understand the molecular events underlying the dentin-pulp complex responses to carious progression, we systematically analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries. Dentin matrix proteins analyzed included collagen type I, phosphophoryn (PP) and dentin sialoprotein (DSP), all of which play decisive roles in the dentin mineralization process. Human non-carious and carious third molar teeth were freshly collected, demineralized, and processed for hematoxylin and eosin staining. The ABC-peroxidase method was used for immunohistochemical staining of collagen type I, PP and DSP proteins using specific antibodies. In situ hybridization was also performed. In contrast to elongated odontoblasts in non-carious teeth, odontoblasts subjacent to dentin caries were cuboidal and fewer in number. The predentin zone was also dramatically reduced in teeth with dentin caries. The staining intensity for collagen type I, PP and DSP in the dentin-pulp complex increased progressively from non-carious teeth, to teeth with enamel and dentin caries. In situ hybridization studies showed DSP-PP mRNA expression in odontoblasts and dental pulp that was consistent with our immunohistochemical results. These results suggest that carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type I, PP and DSP proteins. This response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation.

The presence of multiple rat DSP-PP transcripts

Phosphoproteins or phosphophoryns (PPs) are the most abundant (>50%) non-collagenous proteins (NCPs) in dentin. PPs bind to calcium and hydroxyapatite and are believed to play a crucial role in dentin mineralization. Dentin sialoprotein (DSP), a highly glycosylated protein, comprised 5-8% of NCPs in dentin. The coding sequences for these two major NCPs are known to be contiguously located (i.e. DSP-PP) at the cDNA and genomic DNA levels in both rat and mouse. Previous studies have demonstrated the presence of multiple DSP-PP transcripts in the total RNA of adult rat incisors. To further understand the nature of these multiple transcripts, we performed reverse transcription-PCR and obtained a PP cDNA variant which encoded a 171 amino acid peptide (PP(171)) that shares many of the same characteristics as that of the published rat PP(240) sequence [Ritchie, H.H. and Wang, L.-H., J. Biol. Chem. 271 (1996) 21695-21698]. Due to its reduced size, as compared to PP(240), this cDNA encodes a phosphorylated protein with a reduced negative charge that may differentially affect mineralization processes. We provide evidence that there are multiple DSP-PP transcripts with various sizes of PP sequences in rat.

Dynamic Processing of Recombinant Dentin Sialoprotein-Phosphophoryn Protein

Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two noncollagenous proteins classically linked to dentin but more recently found in bone, kidney, and salivary glands. These two proteins are derived from a single copy DSP-PP gene. Although this suggests that the DSP-PP gene is first transcribed into DSP-PP mRNAs, which later undergo processing to yield the DSP and PP proteins, this mechanism has not yet been demonstrated because of the inability to identify a DSP-PP precursor protein from any cell or tissue sample. To study this problem, we utilized a baculovirus expression system to produce recombinant DSP-PP precursor proteins from a DSP-PP240 cDNA, which represents one of several endogenous DSP-PP transcripts that influence various tooth mineralization phases. Our in vitro results demonstrate that DSP-PP240 precursor proteins are produced by this system and are capable of self-processing to yield both DSP and PP proteins. We further demonstrated that purified recombinant DSP-PP240, purified recombinant PP240, and the native highly phosphorylated protein (equivalent to the PP523 isoform) have proteolytic activity. These newly identified tissue proteases may play key roles in tissue modeling during organogenesis.