Dentin Non-collagenous Proteins Research Articles

Combined Application of Dentin Noncollagenous Proteins and Odontogenic Biphasic Calcium Phosphate in Rabbit Maxillary Sinus Lifting.

Teeth can be used as a raw material for preparing bone substitutes due to their similar chemical composition to bone. The objective of our study was to evaluate the effect of odontogenic biphasic calcium phosphate (BCP) incorporating dentin noncollagenous proteins (DNCPs) on osteogenesis and stability in maxillary sinus augmentation. The composition, structure and morphology of the odontogenic BCP were tested by X-ray powder diffraction (XRD), Brunauer-Emmett-Teller, and scanning electron microscopy methods. The biocompatibility and osteoinduction of DNCPs and materials were examined in vitro and their bone regeneration capacity was verified in vivo. The results showed that the cells adhered and proliferated well on the DNCP-loaded BCP scaffold. The odontogenic BCP and DNCPs promoted osteogenic differentiation of cells, The new bone formation in the BCP groups and DNCP subgroups was significantly higher than the new bone formation in the control, and the new bone quality was better. The bone regeneration effect of odontogenic BCP was similar to the effect of deproteinized bovine bone mineral, but β-TCP did not maintain the height and volume of bone reconstruction. In conclusion, the combined application of DNCPs and odontogenic BCP is an effective strategy for tissue engineering osteogenesis in the maxillary sinus region. The biomimetic strategy could provide a new approach for patients requiring maxillary sinus lifting.

Phosphorylated PAMAM dendrimers: an analog of dentin non-collagenous proteins, enhancing the osteo/odontogenic differentiation of dental pulp stem cells.

Polyamidoamine (PAMAM) dendrimers have well-defined structures, with monodispersity and easily modified surface groups, and they have broad applications in biomedicine. In this study, phosphorylated PAMAM (P-PAMAM) dendrimers were synthesized based on the idea of mimicking the phosphorylated proteins of dentin non-collagenous proteins (DNCP). Then, proliferation and osteo/odontogenic differentiation effects of P-PAMAM on dental pulp stem cells (DPSCs) were investigated and were compared with DNCP. P-PAMAM was synthesized via the Mannich-type reaction. DNCP were extracted directly from human dentin with ethylenediaminetetraacetic acid (EDTA) solution. Then, the conditioned medium of P-PAMAM and DNCP were prepared respectively and applied to DPSCs. Proliferation of P-PAMAM was investigated with CCK-8, flow cytometry, and EdU test. Osteo/odontogenic differentiation of P-PAMAM was analyzed using alkaline phosphatase activity and staining, RT-PCR, western blot, alizarin red staining, and immunofluorescence staining. Fourier transform infrared spectroscopy and 1H nuclear magnetic resonance revealed that PAMAM were successfully phosphorylated. Western blot verified that the extracted DNCP contained dentin-related proteins DSPP, OPN, and BMP2. In cell proliferation, there was no apparent difference between P-PAMAM, DNCP, and Control groups (P > 0.05). P-PAMAM and DNCP upregulated related genes and proteins expression (DSPP/DSPP, COL-1/COL-1, ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN) and matrix mineralization. Still, the potential was lower than that of DNCP (P < 0.05). P-PAMAM dendrimers, as a biomimetic analog of DNCP, promote osteo/odontogenic differentiation of DPSCs without influencing their proliferation at a low concentration. This preliminary study about P-PAMAM dendrimers is expected to provide a more convenient bioactive macromolecular material for the regeneration of the pulp-dentin complex.

DSPP dosage affects tooth development and dentin mineralization.

Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.

The composite sandwich structure of dNCPs polyelectrolyte multilayers induced the osteogenic differentiation of PDLSCs in vitro.

This study reported about the fabrication of dentin non-collagenous proteins (dNCPs) polyelectrolyte multilayers and evaluated its osteogenic potential. The composite sandwich structure of dNCPs polyelectrolyte multilayers was generated on the surface of polycaprolactone electrospinning membranes by the Layer-by-Layer self-assembly technique. The dNCPs-coated membranes comprised the experimental group and the non-coated membranes acted as the control. Nanofiber morphologies of both membranes were observed under scanning electron microscope. The release of dNCPs was evaluated by ELISA kit. Periodontal ligament stem cells (PDLSCs) were seeded on both membranes. The morphology changes and proliferation of cells were tested. The expressions of osteogenic-related genes and proteins were evaluated by RT-PCR, alkaline phosphatase (ALP) activity assay, and immunofluorescence staining. dNCPs-coated membranes displayed significantly different fiber morphology than the non-coated membranes. A stable release of dentin phosphoprotein was maintained from day 4 to day 15 in the experimental group. Cells on dNCPs-coated membranes were found to have cuboidal or polygonal shapes. The proliferative rate of cells was significantly lower in the experimental group from day 4 to day 9 (p<0.05). However, cells on the dNCPs-coated membranes demonstrated a significantly higher ALP content and expression levels of osteogenic gene and proteins than the controls (p<0.05). These results indicated that dNCPs polyelectrolyte multilayers could induce the osteogenic differentiation of PDLSCs in vitro.

Protocols for Studying Formation and Mineralization of Dental Tissues In Vivo: Extraction Protocol for Isolating Dentin Matrix Proteins from Developing Teeth.

The organic material in developing dentin is 90% type I collagen and 10% non-collagenous proteins. The key to understanding dentin biomineralization is to study how these proteins collectively precipitate and organize hydroxyapatite crystals. The first step in characterizing the proteins within a mineralizing matrix is to efficiently extract and isolate the essential molecular participants and elucidate their structural and biochemical properties. In this study, we expanded previous approaches to develop an improved strategy for the extraction of extracellular matrix proteins from the dentin of developing teeth. Proteins in dentin powder were sequentially extracted in the order Tris-guanidine buffer, HCl-formic acid solution, acetic acid-NaCl solution, Tris-NaCl buffer, and a second Tris-guanidine buffer. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by gelatin or casein zymography, and by Western blot analysis using dentin sialoprotein (DSP)- or dentin glycoprotein (DGP)-specific antibodies. This approach was used to purify assorted porcine dentin non-collagenous proteins.

The functional significance of dentin sialoprotein-phosphophoryn and dentin sialoprotein

Phosphophoryn (PP) and dentin sialoprotein (DSP) are the most dominant non-collagenous proteins in dentin. PP is an extremely acidic protein that can function as a mineral nucleator for dentin mineralization. DSP was first identified in 1981, yet its functional significance is still controversial. Historically, these two proteins were considered to be independently synthesized and secreted by dental pulp cells into the developing dentin matrix. However, with the identification of the DSP coding sequence in 1994, followed 2 years later by the finding that the PP coding sequence was located immediately downstream from the DSP sequence, it became immediately clear that DSP and PP proteins were derived from a single DSP-PP (i.e., dentin sialophosphoprotein, DSPP) transcript. Since DSPP cDNA became available, tremendous progress has been made in studying DSP-PP mRNA distribution and DSP generation from the DSP-PP precursor protein at specific cleavage sites by protease tolloid-related-1 (TLR1) or bone morphogenetic protein 1 (BMP1). The functions of DSP-PP and DSP were investigated via DSP-PP knockout (KO) and DSP knockin in DSP-PP KO mice. In addition, a number of in vitro studies aimed to elucidate DSPP and DSP function in dental pulp cells.

In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75+ stem cells with dental follicle cell conditioned medium

Cranial neural crest-derived cells (CNCCs) play important role in epithelial–mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75+) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75+ CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features to cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75+ cells, suggesting their differentiation along cementoblast-like lineage. p75+ stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial–mesenchymal interactions in tooth morphogenesis.

Dentin Sialophosphoprotein–derived Proteins in the Dental Pulp

Porcine dentin sialophosphoprotein (DSPP), the most abundant noncollagenous protein in dentin, is critical for proper mineralization of tooth dentin. DSPP is processed by proteases into 3 major domains: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). There are at least 2 mRNA variants expressed from the Dspp gene: one encodes the full-length DSPP protein (DSP+DGP+DPP); the other encodes only DSP. The shorter transcript is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region (DGP and DPP are encoded by exon 5). We fractionated DSPP-derived proteins from the dental pulp of developing porcine incisors using heparin chromatography. DSP was identified, but little DPP could be detected in any fractions. BMP-1 digestion of DSPP-derived proteins extracted from dental pulp did not generate new DPP bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (indicating an absence of intact DSPP), although the results suggested another BMP-1 cleavage site within DSP. We further purified DSPP-derived protein by reversed-phase high-performance liquid chromatography. Its amino acid composition was similar to DSP. Expression of the full-length Dspp mRNA by quantitative real-time polymerase chain reaction analysis was significantly higher in odontoblasts than in pulp, while expression of the DSP-only mRNA was almost equal in odontoblasts and in the body of the pulp. Expression of the full-length Dspp mRNA was also significantly higher than the expression of DSP-only mRNA in odontoblasts. Both the full-length and the DSP-only Dspp mRNA showed only trace expression in the pulp tip. We conclude that use of the 3′ polyadenylation signal in exon 5 predominates in fully differentiated odontoblasts, while both polyadenylation signals are used throughout odontoblast differentiation.

DPP and DSP are Necessary for Maintaining TGF-β1 Activity in Dentin

Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-β activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-β receptor inhibitor. We then purified TGF-β-unbound or -bound DPP and DSP by reverse-phase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-β isoform bound to DPP and DSP was identified as being TGF-β1 by both ELISA and LC-MS/MS analysis. We incubated carrier-free human recombinant TGF-β1 (CF-hTGF-β1) with TGF-β-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-β1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-β1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-β to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-β1. We conclude that both DPP and DSP help retain TGF-β1 activity in porcine dentin.

Immunohistochemical localization of the NH2-terminal and COOH-terminal fragments of dentin sialoprotein in mouse teeth

Dentin sialoprotein (DSP) is a major non-collagenous protein in dentin. Mutation studies in human, along with gene knockout and transgenic experiments in mice, have confirmed the critical role of DSP for dentin formation. Our previous study reported that DSP is processed into fragments in mouse odontoblast-like cells. In order to gain insights into the function of DSP fragments, we further evaluated the expression pattern of DSP in the mouse odontoblast-like cells using immunohistochemistry and western blot assay with antibodies against the NH(2)-terminal and COOH-terminal regions of DSP. Then, the distribution profiles of the DSP NH(2)-terminal and COOH-terminal fragments and osteopontin (OPN) were investigated in mouse teeth at different ages by immunohistochemistry. In the odontoblast-like cells, multiple low molecular weight DSP fragments were detected, suggesting that part of the DSP protein was processed in the odontoblast-like cells. In mouse first lower molars, immunoreactions for anti-DSP-NH(2) antibody were intense in the predentin matrix but weak in mineralized dentin; in contrast, for anti-DSP-COOH antibody, strong immunoreactions were found in mineralized dentin, in particular dentinal tubules but weak in predentin. Therefore, DSP NH(2)-terminal and COOH-terminal fragments from odontoblasts were secreted to different parts of teeth, suggesting that they may play distinct roles in dentinogenesis. Meanwhile, both DSP antibodies showed weak staining in reactionary dentin (RD), whereas osteopontin (OPN) was clearly positive in RD. Therefore, DSP may be less crucial for RD formation than OPN.

Expression and purification recombinant human dentin sialoprotein in Escherichia coli and its effects on human dental pulp cells

Dentin sialoprotein (DSP) is cleaved from dentin sialophosphoprotein (DSPP) and most abundant dentinal non-collagenous proteins in dentin. DSP is believed to participate in differentiation and mineralization of cells. In this study, we first constructed recombinant human DSP (rhDSP) in Escherichia coli (E. coli) and investigated its odontoblastic differentiation effects on human dental pulp cells (hDPCs). Cell adhesion activity was measured by crystal violet assay and cell proliferation activity was measured by MTT assay. To assess mineralization activity of rhDSP, Alizarin Red S staining was performed. In addition, the mRNA levels of collagen type І (Col І), alkaline phosphatase (ALP), and osteocalcin (OCN) were measured due to their use as mineralization markers for odontoblast-/osteoblast-like differentiation of hDPCs. The obtained rhDSP in E. coli was approximately identified by SDS–PAGE and Western blot. Initially, rhDSP significantly enhanced hDPCs adhesion activity and proliferation (p<0.05). In Alizarin Red S staining, stained hDPCs increased in a time-dependent manner. This odontoblastic differentiation activity was also verified through mRNA levels of odontoblast-related markers. Here, we first demonstrated that rhDSP may be an important regulatory ECM in determining the hDPCs fate including cell adhesion, proliferation, and odontoblastic differentiation activity. These findings indicate that rhDSP can induce growth and differentiation on hDPCs, leading to improve tooth repair and regeneration.

Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro

Adipose tissue-derived stem cells (ADSCs), which are easily harvested and show excellent pluripotency potential, have generated considerable interest in regenerative medicine. In this study, the differentiation of ADSCs was assessed after treatment with dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs). ADSCs exhibited a fibroblast-like morphology and high proliferative capacity. However, after treatment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and in vitro mineralization behaviour of ADSCs were significantly enhanced. Mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype. These results demonstrate that ADSCs acquired cementoblast features in vitro with dNCPs/DFCCM treatment and could be a potential source of cementogenic cells for periodontal regeneration.

Possible role of DMP1 in dentin mineralization

Dentin Matrix Protein 1 (DMP1), the essential noncollagenous proteins in dentin and bone, is believed to play an important role in the mineralization of these tissues, although the mechanisms of its action are not fully understood. To gain insight into DMP1 functions in dentin mineralization we have performed immunomapping of DMP1 in fully mineralized rat incisors and in vitro calcium phosphate mineralization experiments in the presence of DMP1. DMP1 immunofluorescene was localized in peritubular dentin (PTD) and along the dentin-enamel boundary. In vitro phosphorylated DMP1 induced the formation of parallel arrays of crystallites with their c-axes co-aligned. Such crystalline arrangement is a hallmark of mineralized collagen fibrils of bone and dentin. Interestingly, in DMP1-rich PTD, which lacks collagen fibrils, the crystals are organized in a similar manner. Based on our findings we hypothesize, that in vivo DMP1 controls the mineral organization outside of the collagen fibrils and plays a major role in the mineralization of PTD.

Imperfect hybrid layers created by an aggressive one‐step self‐etch adhesive in primary dentin are amendable to biomimetic remineralization in vitro

Degradation of hybrid layers created in primary dentin occurs as early as 6 months in vivo. Biomimetic remineralization utilizes "bottom-up" nanotechnology principles for interfibrillar and intrafibrillar remineralization of collagen matrices. This study examined whether imperfect hybrid layers created in primary dentin can be remineralized. Coronal dentin surfaces were prepared from extracted primary molars and bonded using Adper Prompt L-Pop and a composite. One-millimeter-thick specimen slabs of the resin-dentin interface were immersed in a Portland cement-based remineralization medium that contained two biomimetic analogs to mimic the sequestration and templating functions of dentin noncollagenous proteins. Specimens were retrieved after 1-6 months. Confocal laser scanning microscopy was used for evaluating the permeability of hybrid layers to Rhodamine B. Transmission electron microscopy was used to examine the status of remineralization within hybrid layers. Remineralization at different locations of the hybrid layers corresponded with quenching of fluorescence within similar locations of those hybrid layers. Remineralization was predominantly intrafibrillar in nature as interfibrillar spaces were filled with adhesive resin. Biomimetic remineralization of imperfect hybrid layers in primary human dentin is a potential means for preserving bond integrity. The success of the current proof-of-concept, laterally diffusing remineralization protocol warrants development of a clinically applicable biomimetic remineralization delivery system.

Molecular evolution of dentin phosphoprotein among toothed and toothless animals

BackgroundDentin sialophosphoprotein (DSPP) is the largest member of the SIBLING family and is the most abundant noncollagenous protein in dentin. DSPP is also expressed in non-mineralized tissues including metabolically active ductal epithelia and some cancers. Its function, however, is poorly defined. The carboxy-terminal fragment, dentin phosphoprotein (DPP) is encoded predominantly by a large repetitive domain that requires separate cloning/sequencing reactions and is, therefore, often incomplete in genomic databases. Comparison of DPP sequences from at least one member of each major branch in the mammalian evolutionary tree (including some "toothless" mammals) as well as one reptile and bird may help delineate its possible functions in both dentin and ductal epithelia.ResultsThe BMP1-cleavage and translation-termination domains were sufficiently conserved to permit amplification/cloning/sequencing of most species' DPP. While the integrin-binding domain, RGD, was present in about half of species, only vestigial remnants of this tripeptide were identified in the others. The number of tandem repeats of the nominal SerSerAsp phosphorylation motif in toothed mammals (including baleen whale and platypus which lack teeth as adults), ranged from ~75 (elephant) to >230 (human). These repeats were not perfect, however, and patterns of intervening sequences highlight the rapidity of changes among even closely related species. Two toothless anteater species have evolved different sets of nonsense mutations shortly after their BMP1 motifs suggesting that while cleavage may be important for DSPP processing in other tissues, the DPP domain itself may be required only in dentin. The lizard DSPP had an intact BMP1 site, a remnant RGD motif, as well as a distinctly different Ser/Asp-rich domain compared to mammals.ConclusionsThe DPP domain of DSPP was found to change dramatically within mammals and was lost in two truly toothless animals. The defining aspect of DPP, the long repeating phosphorylation domain, apparently undergoes frequent slip replication and recombination events that rapidly change specific patterns but not its overall biochemical character in toothed animals. Species may have to co-evolve protein processing mechanisms, however, to handle increased lengths of DSP repeats. While the RGD domain is lost in many species, some evolutionary pressure to maintain integrin binding can be observed.

The Biological Effect of Dentin Noncollagenous Proteins (DNCPs) on the Human Periodontal Ligament Stem Cells (HPDLSCs) In Vitro and In Vivo

It was recognized that periodontal progenitor cells penetrate disintegrated Hertwig's epithelial root sheath, and contact with root dentin give rise to periodontium formation. Clinically, direct contact of the conditioned or denuded root surfaces with periodontal cells seems to be a prerequisite for periodontal regeneration. In this study, we investigated the biological effect of dentin noncollagenous proteins (DNCPs) on the human periodontal ligament stem cells (HPDLSCs) in vitro and in vivo. Chemical-conditioned root dentin (CCRD) was prepared by process of partly demineralization and deproteinization. Treated HPDLSCs with DNCPs showed increased proliferation and adhesion ability. Induced HPDLSCs presented several features of cementoblast differentiation, as indicated by morphologic changes, enhanced alkaline phosphatase (ALP) activity, increased matrix mineralization, and upregulated expression of mineralization-associated genes. Incubation of treated HPDLSC aggregate in vivo revealed that cementum-like tissues formed along the CCRD surface with fibrous tissue adjacent to or inserted into it, but untreated HPDLSCs cannot form similar structure. To our knowledge, this is the first study to apply active proteins derived from dentin with periodontal stem cells to construct periodontal structure, which may shed light on human periodontal tissue regeneration.

The effect of odontoblast conditioned media and dentin non-collagenous proteins on the differentiation and mineralization of cementoblasts in vitro

Objective Cementum is an important mineralized tissue in root formation, however, the precise mechanism of cementum formation remains undetermined. The purpose of this study was to evaluate the effect of odontoblast conditioned media (CM) and dentin non-collagenous proteins (dNCPs) on the differentiation and mineralization of cementoblastic OCCM-30 cells. Methods The CM of ameloblastic ALCs, odontoblastic MDPC-23 and OD-11, osteoblastic MG-63, and fibroblastic NIH3T3 cells were transferred to OCCM-30 cells. dNCPs were extracted directly from porcine and human dentin and applied to OCCM-30 cells. The results were evaluated through the analysis of the morphologic appearance, expression of cementum matrix genes, and the formation of mineralized nodules in vitro. Results dNCPs hardly influenced proliferation, cell cycle modification, and chemotaxis of cementoblasts. Mineralization of cementoblasts was accelerated with dNCPs and CM from odontoblastic MDPC-23 and OD-11. RT-PCR analysis revealed the earlier and stronger expression of bone sialoprotein (BSP), alkaline phosphatase (ALP), and osteocalcin (OC) mRNAs in the MDPC23- and OD11-CM-treated OCCM-30 cells than those in the control OCCM-30 cells. The level of gene expression was also significantly higher in the dNCP-treated group than the control group. Conclusion These results suggest that dentin matrix proteins, or the secreted products of odontoblasts, induced cementoblast differentiation and mineralization. These findings may contribute to the development of a periodontal treatment that includes cementum regeneration.

Effect of Dentin Phosphoprotein on Phosphate-Induced Apoptosis of Odontoblast-Like Cells

Dentin phosphoprotein, the major noncollagenous protein in dentin, has effects on differentiation of odontoblast-like cells. This study was designed to investigate the effect of the protein on apoptosis of the cells. The odontoblast-like cells were prepared from the pulp cells of rat incisors. Apoptosis was detected by measuring caspase-3 activity by using DEVD-AMC as a fluorescent substrate. The cells formed calcification nodules in the presence of 2-glycerophosphate and expressed dentin sialophosphoprotein. Apoptosis was not observed in the cells through the differentiation stages. Then, apoptosis was induced by raising inorganic phosphate concentration in the medium. Elevation of phosphate concentration to 5 mM reduced the number of viable cells and increased caspase-3 activity, indicating the induction of apoptosis. Addition of bovine dentin phosphoprotein in the medium suppressed phosphate-induced apoptosis. Phosvitin and poly(Asp) had similar antiapoptotic effects.

Dentin phosphoprotein frameshift mutations in hereditary dentin disorders and their variation patterns in normal human population

Background:Dentin phosphoprotein (DPP) is the most abundant non-collagenous protein in dentin, which is highly phosphorylated and plays key roles in dentin biomineralisation. The aetiology of isolated hereditary dentin disorders in...

Dentin non‐collagenous proteins (dNCPs) can stimulate dental follicle cells to differentiate into cementoblast lineages

Although the mechanism of cementogenesis is an area full of debate, the DFCs (dental follicle cells) are thought to be the precursors of cementoblasts. At the onset of cementogenesis, DFCs come into contact with the root dentin surface and undergo subsequent differentiation. But the exact effects of dentin or dentin matrix on DFCs remain an open question. In the present study, we hypothesized that dNCPs (dentin non-collagenous proteins) extracted from dentin could stimulate DFCs to differentiate into cementoblast lineages. DFCs were isolated from tooth germs of SD (Sprague-Dawley) rats and then co-cultured with dNCPs. Treated DFCs presented several features of cementoblast lineages in vitro, as indicated by morphological changes, decreased proliferation, enhanced ALP (alkaline phosphatase) activity and increased matrix mineralization. The expression of mineralization-associated proteins and genes were up-regulated after induction, whereas the expression of specific markers of odontoblast were not detected. Incubation of treated DFC pellets in vivo revealed that a large amount of cementum-like tissues was formed within the novel dentin carriers, which were quite distinct from the newly formed osteodentin secreted by DPSCs (dental pulp stem cells). The negative expression of DSP (dentin sialoprotein) also excluded the possibility of producing dentin matrix by treated DFCs. dNCPs can stimulate DFCs to differentiate into cementoblast lineages. The present study provides new insights into the mechanism of cementogenesis.