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1.  Isolation and Characterization of Neural Crest-Derived Stem Cells from Dental Pulp of Neonatal Mice 
PLoS ONE  2011;6(11):e27526.
Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.
doi:10.1371/journal.pone.0027526
PMCID: PMC3210810  PMID: 22087335
2.  Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging 
BMC Cell Biology  2010;11:32.
Background
Dental pulp stem cells (DPSCs) can be driven into odontoblast, osteoblast, and chondrocyte lineages in different inductive media. However, the differentiation potential of naive DPSCs after serial passaging in the routine culture system has not been fully elucidated.
Results
DPSCs were isolated from human/rat dental pulps by the magnetic activated cell sorting based on STRO-1 expression, cultured and passaged in the conventional culture media. The biological features of STRO-1+ DPSCs at the 1st and 9th passages were investigated. During the long-term passage, the proliferation ability of human STRO-1+ DPSCs was downregulated as indicated by the growth kinetics. When compared with STRO-1+ DPSCs at the 1st passage (DPSC-P1), the expression of mature osteoblast-specific genes/proteins (alkaline phosphatase, bone sialoprotein, osterix, and osteopontin), odontoblast-specific gene/protein (dentin sialophosphoprotein and dentin sialoprotein), and chondrocyte-specific gene/protein (type II collagen) was significantly upregulated in human STRO-1+ DPSCs at the 9th passage (DPSC-P9). Furthermore, human DPSC-P9 cells in the mineralization-inducing media presented higher levels of alkaline phosphatase at day 3 and day 7 respectively, and produced more mineralized matrix than DPSC-P9 cells at day 14. In vivo transplantation results showed that rat DPSC-P1 cell pellets developed into dentin, bone and cartilage structures respectively, while DPSC-P9 cells can only generate bone tissues.
Conclusions
These findings suggest that STRO-1+ DPSCs consist of several interrelated subpopulations which can spontaneously differentiate into odontoblasts, osteoblasts, and chondrocytes. The differentiation capacity of these DPSCs changes during cell passaging, and DPSCs at the 9th passage restrict their differentiation potential to the osteoblast lineage in vivo.
doi:10.1186/1471-2121-11-32
PMCID: PMC2877667  PMID: 20459680
3.  Human dental pulp stem cells cultured in serum-free supplemented medium 
Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells.
Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR.
Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs.
Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs.
doi:10.3389/fphys.2013.00357
PMCID: PMC3858652  PMID: 24376422
odontoblastic differentiation; neurogenic serum-free medium; human dental pulp stem cells; neuronal progenitor; oligodendrocyte progenitor
4.  P16/p53 expression and telomerase activity in immortalized human dental pulp cells 
Cell Cycle  2011;10(22):3912-3919.
Introduction
Residing within human dental pulp are cells of an ectomesenchymal origin that have the potential to differentiate into odontoblast-like cells. These cells have a limited growth potential owing to the effects of cell senescence. This study examines the effects of immortalizing odontoblast-like cells on cell proliferation and mineralization by comparing transformed dental pulp stem cells (tDPSCs) and non-transformed dental pulp stem cells (nDPSCs).
Results
With the exogenous expression of hTERT, tDPSCs maintained a continued expression of odontogenic markers for cell proliferation and mineralization (ALP, COL-1, DMP-1, DSPP, OCN and OPN), as did nDPSCs. Oncoprotein expression was seen in both groups except for a noted absence of p16 in the tDPSCs. nDPSCs also showed lower levels of total ALP and DNA activity in comparison to tDPSCs when assayed, as well as low telomerase activity readings.
Methods
Using a retroviral vector, exogenous human telomerase reverse transcriptase (hTERT) was expressed in tDPSCs. Both cell groups were cultured, and their telomerase activities were determined using a telomerase quantification assay. Also examined, were the expression of genes involved in proliferation and mineralization, such as human alkaline phosphatase (ALP), β-actin, collagen I (col-1), core binding factor (cbfa)-1, dentin matrix protein (DMP-1), dentin sialophosphoprotein (DSPP), GAPDH, hTERT, osteocalcin (OCN), osteopontin (OPN) as well as oncoproteins involved in senescence (p16, p21 and p53) using RT-PCR. DNA and alkaline phosphate activity was also assayed in both cell groups.
Conclusion
These results indicate maintenance of odontoblast-like differentiation characteristics after retroviral transformation with hTERT and suggest a possible link with a reduced p16 expression.
doi:10.4161/cc.10.22.18093
PMCID: PMC3266118  PMID: 22067611
dental pulp stem cells; odontoblasts; immortalization; senescence; oncoproteins; cell proliferation; cell mineralization
5.  Bobby Sox homology regulates odontoblast differentiation of human dental pulp stem cells/progenitors 
Background
Transcription factors have been implicated in regulating the differentiation of odontoblasts from dental pulp stem cells/progenitors (DPSCs/progenitors), but their regulatory network is not completely understood.
Result
New transcription factors that control the odontoblast differentiation of human DPSCs/progenitors were analyzed using a microarray. The result revealed bobby sox homolog (BBX) to be expressed most strongly during odontoblast differentiation. Validation using RT-PCR also revealed the strong expression of BBX during the odontoblast differentiation of DPSCs/progenitors. BBX expression was also detected in adult molar odontoblasts and other tissues, including the heart, kidney, testis, and bone marrow. To understand the role of BBX in odontoblast differentiation, BBX variant 1 and 2 cDNA were cloned and overexpressed in DPSCs/progenitors. The results showed that the overexpression of BBX cDNA in DPSCs/progenitors induced substantial mineralization and expression of the odontoblast marker genes, such as ALP, OPN, BSP, DMP1, and DSPP. The knockdown of BBX using shRNA, however, did not affect mineralization, but the expression of ALP and DSPP was decreased substantially. Meanwhile overexpression or knockdown of BBX did not modulate proliferation of DPSCs/progenitors.
Conclusion
Our results suggest that BBX plays an important role during the odontoblast differentiation of human DPSCs/progenitors.
doi:10.1186/1478-811X-12-35
PMCID: PMC4062286  PMID: 24885382
BBX; Transcription factor; DPSC
6.  The Role of Lysyl Oxidase-like 2 in the Odontogenic Differentiation of Human Dental Pulp Stem Cells 
Molecules and Cells  2013;35(6):543-549.
Adult human dental pulp stem cells (hDPSCs) are a unique population of precursor cells those are isolated from postnatal dental pulp and have the ability to differentiate into a variety of cell types utilized for the formation of a reparative dentin-like complex. Using LC-MS/MS proteomics approaches, we identified the proteins secreted from the differentiating hDPSCs in mineralization media. Lysyl oxidase-like 2 (LOXL2) was identified as a protein that was down-regulated in the hDPSCs that differentiate into odontoblast-like cells. The role of LOXL2 has not been studied in dental pulp stem cells. LOXL2 mRNA levels were reduced in differentiating hDPSCs, whereas the levels of other LOX family members including LOX, LOXL1, LOXL3, and LOXL4, are increased. The protein expression and secretion levels of LOXL2 were also decreased during odontogenic differentiation. Recombinant LOXL2 protein treatment to hDPSCs resulted in a dose-dependent decrease in the early differentiation and the mineralization accompanying with the lower levels of odontogenic markers such as DSPP, DMP-1 and ALP. These results suggest that LOXL2 has a negative effect on the differentiation of hDPSCs and blocking LOXL2 can promote the hDPSC differentiation to odontoblasts.
doi:10.1007/s10059-013-0080-3
PMCID: PMC3887878  PMID: 23677379
human dental pulp stem cells; lysyl oxidase-like 2; odontogenic differentiation
7.  Osteoblasts can induce dental pulp stem cells to undergo osteogenic differentiation 
Cytotechnology  2012;65(2):223-231.
Recent studies have shown that, in numerous species, systemically administered bone marrow-derived mesenchymal stem cells undergo site-specific differentiation. This suggests that osteoblasts, by means of cytokine secretion, may promote dental pulp stem cells (DPSCs) to undergo osteogenesis. The objective of this study was to assess the potential synergistic interaction effect of osteoblasts on DPSCs for promotion of osteogenesis. Stem cells, derived from dental pulp of healthy human donors, were co-cultured with calvaria osteoblasts using a culture insert system. The proliferation rate, calcium deposition, osteogenic-related gene expression of induced DPSCs, including Runx-2, bone sialoprotein, osteocalcin and collagen-1, were assayed using MTT, Alizarin Red S staining and reverse transcriptase polymerase chain reaction, respectively. Co-cultured DPSCs had the highest rate of proliferation compared with those cultured in absence of osteoblasts. The morphology and ultrastructure of DPSCs in the co-cultures showed improvement, with co-cultured DPSCs becoming more osteoblast-like as compared with DPSCs cultured alone, and the mineralization potential of co-cultured DPSCs was enhanced compared with DPSCs cultured alone. Furthermore, osteogenic-related genes were significantly over-expressed in co-cultured DPSCs after osteogenic induction. The results demonstrate that DPSCs successfully differentiate towards osteoblasts and that the paracrine interaction of osteoblasts is likely to contribute to DPSC differentiation. It is believed that this study demonstrates certain useful applications for DPSCs in bone tissue engineering.
doi:10.1007/s10616-012-9479-5
PMCID: PMC3560885  PMID: 22806484
Dental pulp stem cells (DPSCs); Osteogenesis; Mineralized tissue; Co-culture
8.  Blockade of TLR2 Inhibits Porphyromonas gingivalis Suppression of Mineralized Matrix Formation by Human Dental Pulp Stem Cells 
Journal of Endodontics  2011;37(6):812-818.
Introduction
Human dental pulp stem/progenitor cells (hDPSC) can differentiate into odontoblast-like cells and express dentin sialophosphoprotein (DSPP) and osteocalcin (OCN); thus, they may be used to regenerate dentin. However, residual bacterial components in the root canal may suppress this activity.
Purpose
This study investigated the effect of a Porphyromonas gingivalis component on the expression of DSPP and OCN by stimulated hDPSCs and the influence of blockade of TLR2-mediated P. gingivalis host recognition.
Methods
Stimulated hDPSCs were exposed to varying concentrations of P. gingivalis lipopolysaccharide (LPS), and the expression of DSPP and OCN was measured. Similar groups of stimulated hDPSCs were exposed to TLR2 blocking agents before exposure to LPS.
Results
hDPSCs exposed to 5, 10, and 20 µg/mL LPS exhibited a dose-dependent reduction in the expression of DSPP (3.19 ± 0.18, 2.60 ± 0.49, and 1.15 ± 0.29, respectively) and OCN (3.51 ± 1.18, 2.60 ± 0.67 and 1.66 ± 0.89, respectively). The expression of DSPP and OCN after exposure to 20 µg/mL of LPS was significantly lower than measured for unexposed stimulated cells (analysis of variance and post hoc Tukey test, P < .05). The blockade of TLR2 using an extra- and intracellular agent affected DSPP (4.67 ± 0.97 and 5.29 ± 1.66, respectively) and OCN (5.25 ± 1.69 and 5.82 ± 2.38, respectively) expression at levels comparable to stimulated cells unexposed to 20 µg/mL LPS (6.32 ± 2.47 and 4.70 ± 1.60 for DSPP and OCN, respectively).
Conclusions
The suppressing effect of P. gingivalis on mineralized matrix formation by hDPSCs is confirmed, and this suppression can be moderated by TLR2 blockade.
doi:10.1016/j.joen.2011.03.013
PMCID: PMC3282592  PMID: 21787495
Dental pulp stem cells; dentin sialophosphoprotein; osteocalcin; Porphyromonas gingivalis; TLR2
9.  Wnt/β-catenin Inhibits Dental Pulp Stem Cell Differentiation 
Journal of dental research  2008;87(2):126-130.
Dental pulp stem cells (DPSCs) are a unique precursor population isolated from post-natal human dental pulp and have the ability to regenerate a reparative dentin-like complex. Canonical Wnt signaling plays a critical role in tooth development and stem cell self-renewal through β-catenin. In this study, the regulation of odontoblast-like differentiation of DPSCs by canonical Wnt signaling was examined. DPSCs were stably transduced with canonical Wnt-1 or the active form of β-catenin, with retrovirus-mediated infection. Northern blot analysis found that Wnt-1 strongly induced the expression of matricellular protein osteopontin, and modestly enhanced the expression of type I collagen in DPSCs. Unexpectedly, Wnt-1 inhibited alkaline phosphatase (ALP) activity and the formation of mineralized nodules in DPSCs. Moreover, over-expression of β-catenin was also sufficient to suppress the differentiation and mineralization of DPSCs. In conclusion, our results suggest that canonical Wnt signaling negatively regulates the odontoblast-like differentiation of DPSCs.
PMCID: PMC2906770  PMID: 18218837
Wnt; mineralization; stem cell; dental pulp; osteopontin
10.  Mechanical Stretch Increases the Proliferation While Inhibiting the Osteogenic Differentiation in Dental Pulp Stem Cells 
Tissue Engineering. Part A  2012;19(5-6):625-633.
Dental pulp stem cells (DPSCs), which can differentiate into several types of cells, are subjected to mechanical stress by jaw movement and occlusal forces. In this study, we evaluated how the uniaxial mechanical stretch influences proliferation and differentiation of DPSCs. DPSCs were isolated and cultured from male Sprague-Dawley rats. Cultured DPSCs were identified by surface markers and the differentiation capabilities as adipocytes or osteoblasts. To examine the response to mechanical stress, uniaxial stretch was exposed to cultured DPSCs. We evaluated the impact of stretch on the intracellular signaling, proliferation, osteogenic differentiation, and gene expressions of DPSCs. Stretch increased the phosphorylation of Akt, ERK1/2, and p38 MAP kinase as well as the proliferation of DPSCs. The stretch-induced proliferation of DPSCs was abolished by the inhibition of the ERK pathway. On the other hand, stretch significantly decreased the osteogenic differentiation of DPSCs, but did not affect the adipogenic differentiation. We also confirmed mRNA expressions of osteocalcin and osteopontin were significantly suppressed by stretch. In conclusion, uniaxial stretch increased the proliferation of DPSCs, while suppressing osteogenic differentiation. These results suggest a crucial role of mechanical stretch in the preservation of DPSCs in dentin. Furthermore, mechanical stretch may be a useful tool for increasing the quantity of DPSCs in vitro for regenerative medicine.
doi:10.1089/ten.tea.2012.0099
PMCID: PMC3566654  PMID: 23153222
11.  The odontogenic differentiation of human dental pulp stem cells on nanofibrous poly(L-lactic acid) scaffolds in vitro and in vivo 
Acta biomaterialia  2010;6(10):10.1016/j.actbio.2010.04.009.
The aim of this study was to investigate the odontogenic differentiation of human dental pulp stem cells (DPSCs) on nanofibrous (NF) poly(L-lactic acid) (PLLA) scaffolds in vitro and in vivo. Highly porous NF-PLLA scaffolds which mimic the architecture of collagen type I (Col I) fibers were fabricated by the combination of a phase separation technique and a porogen leaching method. The human DPSCs were then seeded onto the scaffolds and cultured in different media for odontogenic differentiation: “Control” medium without supplements; “DXM” medium containing 10−8 M dexamethasone (DXM), 50 μg/mL ascorbic acid and 5 mM β-glycerophosphate; “BMP-7 + DXM” medium containing 10−8 M DXM, 50 μg/mL ascorbic acid, 5 mM β-glycerophosphate plus 50 ng/mL bone morphogenetic protein 7 (BMP-7). For odontogenic differentiation study in vitro, alkaline phosphatase (ALP) activity quantification, reverse transcription polymerase chain reaction (RT-PCR), scanning electron microscopy (SEM), von Kossa staining and calcium content quantification were carried out. While both “DXM” medium and “BMP-7+DXM” medium induced the DPSCs to odontoblast-like cells, the “BMP-7 + DXM” medium had greater inducing capacity than the “DXM” medium. Consistent with the in vitro studies, “BMP-7 + DXM” group presented more extracellular matrix (ECM) and hard tissue formation than “DXM” group after 8 weeks of ectopic implantation in nude mice. Differentiation of DPSCs to odontoblast-like cells was identified by the positive immunohistochemical staining for dentin sialoprotein (DSP). In conclusion, odontogenic differentiation of DPSCs can be achieved on NF-PLLA scaffolds both in vitro and in vivo; the combination of BMP-7 and DXM induced the odontogenic differentiation more effectively than DXM alone. The NF-PLLA scaffold and the combined odontogenic inductive factors provide excellent environment for DPSCs to regenerate dental pulp and dentin.
doi:10.1016/j.actbio.2010.04.009
PMCID: PMC3885689  PMID: 20406702
Dental pulp stem cells (DPSCs); Nanofibrous poly(L-lactic acid) (PLLA) scaffold; Odontogenic differentiation; Dentin regeneration; Bone morphogenetic protein 7 (BMP-7)
12.  A Hydrogel Scaffold That Maintains Viability and Supports Differentiation of Dental Pulp Stem Cells 
Objectives
The clinical translation of stem cell-based Regenerative Endodontics demands further development of suitable injectable scaffolds. Puramatrix™ is a defined, self-assembling peptide hydrogel which instantaneously polymerizes under normal physiological conditions. Here, we assessed the compatibility of Puramatrix™ with dental pulp stem cell (DPSC) growth and differentiation.
Methods
DPSC cells were grown in 0.05 to 0.25% Puramatrix™. Cell viability was measured colorimetrically using the WST-1 assay. Cell morphology was observed in 3-D modeling using confocal microscopy. In addition, we used the human tooth slice model with Puramatrix™ to verify DPSC differentiation into odontoblast-like cells, as measured by expression of DSPP and DMP-1.
Results
DPSC survived and proliferated in Puramatrix™ for at least three weeks in culture. Confocal microscopy revealed that cells seeded in Puramatrix™ presented morphological features of healthy cells, and some cells exhibited cytoplasmic elongations. Notably, after 21 days in tooth slices containing Puramatrix™, DPSC cells expressed DMP-1 and DSPP, putative markers of odontoblastic differentiation.
Significance
Collectively, these data suggest that self-assembling peptide hydrogels might be useful injectable scaffolds for stem cell-based Regenerative Endodontics.
doi:10.1016/j.dental.2012.08.002
PMCID: PMC3515741  PMID: 22901827
Tissue engineering; Hydrogel; Dental pulp; Regenerative Endodontics; Odontoblast; Stem cells
13.  Runx2, Osx, and Dspp in Tooth Development 
Journal of Dental Research  2009;88(10):904-909.
The transcription factors Runx2 and Osx are necessary for osteoblast and odontoblast differentiation, while Dspp is important for odontoblast differentiation. The relationship among Runx2, Osx, and Dspp during tooth and craniofacial bone development remains unknown. In this study, we hypothesized that the roles of Runx2 and Osx in the regulation of osteoblast and odontoblast lineages may be independent of one another. The results showed that Runx2 expression overlapped with Osx in dental and osteogenic mesenchyme from E12 to E16. At the later stages, from E18 to PN14, Runx2 and Osx expressions remained intense in alveolar bone osteoblasts. However, Runx2 expression was down-regulated, whereas Osx expression was clearly seen in odontoblasts. At later stages, Dspp transcription was weakly present in osteoblasts, but strong in odontoblasts where Osx was highly expressed. In mouse odontoblast-like cells, Osx overexpression increased Dspp transcription. Analysis of these data suggests differential biological functions of Runx2, Osx, and Dspp during odontogenesis and osteogenesis. Abbreviations: E, embryonic day; PN, post-natal day; Dspp, dentin sialophosphoprotein; Osx, Osterix.
doi:10.1177/0022034509342873
PMCID: PMC3045537  PMID: 19783797
Runx2; Osx; Dspp; odontoblast; osteoblast; tooth development
14.  β-Catenin Enhances Odontoblastic Differentiation of Dental Pulp Cells through Activation of Runx2 
PLoS ONE  2014;9(2):e88890.
An intense stimulus can cause death of odontoblasts and initiate odontoblastic differentiation of stem/progenitor cell populations of dental pulp cells (DPCs), which is followed by reparative dentin formation. However, the mechanism of odontoblastic differentiation during reparative dentin formation remains unclear. This study was to determine the role of β-catenin, a key player in tooth development, in reparative dentin formation, especially in odontoblastic differentiation. We found that β-catenin was expressed in odontoblast-like cells and DPCs beneath the perforation site during reparative dentin formation after direct pulp capping. The expression of β-catenin was also significantly upregulated during odontoblastic differentiation of in vitro cultured DPCs. The expression pattern of runt-related transcription factor 2 (Runx2) was similar to that of β-catenin. Immunofluorescence staining indicated that Runx2 was also expressed in β-catenin–positive odontoblast-like cells and DPCs during reparative dentin formation. Knockdown of β-catenin disrupted odontoblastic differentiation, which was accompanied by a reduction in β-catenin binding to the Runx2 promoter and diminished expression of Runx2. In contrast, lithium chloride (LiCl) induced accumulation of β-catenin produced the opposite effect to that caused by β-catenin knockdown. In conclusion, it was reported in this study for the first time that β-catenin can enhance the odontoblastic differentiation of DPCs through activation of Runx2, which might be the mechanism involved in odontoblastic differentiation during reparative dentin formation.
doi:10.1371/journal.pone.0088890
PMCID: PMC3919828  PMID: 24520423
15.  Odontogenic Differentiation of Human Dental Pulp Stem Cells Stimulated by the Calcium Phosphate Porous Granules 
Journal of Tissue Engineering  2011;2011:812547.
Effects of three-dimensional (3D) calcium phosphate (CaP) porous granules on the growth and odontogenic differentiation of human dental pulp stem cells (hDPSCs) were examined for dental tissue engineering. hDPSCs isolated from adult human dental pulps were cultured for 3-4 passages, and populated on porous granules. Cell growth on the culture dish showed an ongoing increase for up to 21 days, whereas the growth on the 3D granules decreased after 14 days. This reduction in proliferative potential on the 3D granules was more conspicuous under the osteogenic medium conditions, indicating that the 3D granules may induce the odontogenic differentiation of hDPSCs. Differentiation behavior on the 3D granules was confirmed by the increased alkaline phosphatase activity, up-regulation of odontoblast-specific genes, including dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) by quantitative polymerase chain reaction, and greater level of dentin sialoprotein synthesis by western blot. Moreover, the cellular mineralization, as assessed by Alizarin red S and calcium quantification, was significantly higher in the 3D CaP granules than in the culture dish. Taken all, the 3D CaP porous granules should be useful for dental tissue engineering in combination with hDPSCs by providing favorable 3D substrate conditions for cell growth and odontogenic development.
doi:10.4061/2011/812547
PMCID: PMC3136101  PMID: 21772958
16.  Nano-Structured Gelatin/Bioactive Glass Hybrid Scaffolds for the Enhancement of Odontogenic Differentiation of Human Dental Pulp Stem Cells 
Tooth decay is one of the most common chronic disorders throughout the world. Regenerating decayed dentin/pulp structure requires the design of novel scaffolding materials that mimic the architecture of natural dental extracellular matrix (ECM) and provide suitable environments for the attachment, proliferation, differentiation, and biomineralization of dental pulp stem cells (DPSCs). In this work, we developed an approach to prepare three-dimensional (3D) nano-fibrous gelatin/silica bioactive glass (NF-gelatin/SBG) hybrid scaffolds that mimic the nano-structured architecture and chemical composition of natural dental ECM. This approach involved the combination of a thermally induced phase separation, sol-gel, and porogen leaching process, and synthesized hybrid scaffolds possessing natural ECM-like architecture, high porosity, well-defined pore size and interconnectivity, and improved mechanical strength. An in vitro cell culture study showed that human DPSCs had a significantly higher proliferation rate on NF-gelatin/SBG scaffolds compared to NF-gelatin scaffolds under the same conditions. Furthermore, the integration of SBG into the hybrid scaffold significantly promoted the differentiation and biomineralization of the human DPSCs. The alkaline phosphatase (ALP) activity and expressions of marker genes for odontogenic differentiation (Col I, ALP, OCN, DSPP and DMP-1) were all significantly higher in the NF-gelatin/SBG than in the NF-gelatin group. Those results were further confirmed by hematoxylin and eosin (H&E) and von Kossa staining, as evidenced by greater ECM secretion and mineral deposition in the hybrid scaffold. In summary, the biomimetic NF-gelatin/SBG hybrid scaffolds provide an excellent environment for the growth and differentiation of human DPSCs and are promising candidates for dentin/pulp tissue regeneration.
doi:10.1039/C3TB21002B
PMCID: PMC3789537  PMID: 24098854
17.  Proteomic Analysis of Mesenchymal Stem Cells from Normal and Deep Carious Dental Pulp 
PLoS ONE  2014;9(5):e97026.
Dental pulp stem cells (DPSCs), precursor cells of odontoblasts, are ideal seed cells for tooth tissue engineering and regeneration. Our previous study has demonstrated that stem cells exist in dental pulp with deep caries and are called carious dental pulp stem cells (CDPSCs). The results indicated that CDPSCs had a higher proliferative and stronger osteogenic differentiation potential than DPSCs. However, the molecular mechanisms responsible for the biological differences between DPSCs and CDPSCs are poorly understood. The aim of this study was to define the molecular features of DPSCs and CDPSCs by comparing the proteomic profiles using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our results revealed that there were 18 protein spots differentially expressed between DPSCs and CDPSCs in a narrow pH range of 4 to 7. These differently expressed proteins are mostly involved in the regulation of cell proliferation, differentiation, cell cytoskeleton and motility. In addition, our results suggested that CDPSCs had a higher expression of antioxidative proteins that might protect CDPSCs from oxidative stress. This study explores some potential proteins responsible for the biological differences between DPSCs and CDPSCs and expands our understanding on the molecular mechanisms of mineralization of DPSCs in the formation of the dentin-pulp complex.
doi:10.1371/journal.pone.0097026
PMCID: PMC4014579  PMID: 24809979
18.  Mouse-Induced Pluripotent Stem Cells Differentiate into Odontoblast-Like Cells with Induction of Altered Adhesive and Migratory Phenotype of Integrin 
PLoS ONE  2013;8(11):e80026.
Methods for differentiating induced pluripotent stem (iPS) cells into odontoblasts generally require epithelial–mesenchymal interactions. Here, we sought to characterize the cells produced by a ‘hanging drop’ technique for differentiating mouse iPS cells into odontoblast-like cells that requires no such interaction. Cells were cultured by the hanging drop method on a collagen type-I (Col-I) scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4) without an epithelial–mesenchymal interaction. We evaluated the expression of odontoblast-related mRNA and protein, and the proliferation rate of these cells using reverse-transcription polymerase chain reaction, immunofluorescence staining, and BrdU cell proliferation enzyme-linked immunosorbent assay, respectively. The differentiated cells strongly expressed the mRNA for dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (Dmp-1), which are markers of mature odontoblasts. Osteopontin and osteocalcin were not expressed in the differentiated cells, demonstrating that the differentiated iPS cells bore little resemblance to osteoblasts. Instead, they acquired odontoblast-specific properties, including the adoption of an odontoblastic phenotype, typified by high alkaline phosphatase (ALP) activity and calcification capacity. The cell-surface expression of proteins such as integrins α2, α6, αV and αVβ3 was rapidly up-regulated. Interestingly, antibodies and siRNAs against integrin α2 suppressed the expression of DSPP and Dmp-1, reduced the activity of ALP and blocked calcification, suggesting that integrin α2 in iPS cells mediates their differentiation into odontoblast-like cells. The adhesion of these cells to fibronectin and Col-I, and their migration on these substrata, was significantly increased following differentiation into odontoblast-like cells. Thus, we have demonstrated that integrin α2 is involved in the differentiation of mouse iPS cells into odontoblast-like cells using the hanging drop culture method, and that these cells have the appropriate physiological and functional characteristics to act as odontoblasts in tissue engineering and regenerative therapies for the treatment of dentin and/or dental pulp damage.
doi:10.1371/journal.pone.0080026
PMCID: PMC3823835  PMID: 24244598
19.  Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures 
The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, Matrigel™ and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in Matrigel™ DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.
doi:10.4081/ejh.2010.e46
PMCID: PMC3167326  PMID: 21263745
dental pulp stem cell; mesenchymal stem cells; osteogenic differentiation; 3D scaffolds.
20.  Runx2, Osx, and Dspp in Tooth development 
Journal of dental research  2009;88(10):904-909.
The transcription factors Runx2 and Osx are necessary for osteoblast and odontoblast differentiation, while Dspp is important for odontoblast differentiation. The relationship among Runx2, Osx, and Dspp during tooth and craniofacial bone development remains unknown. In this study, we hypothesized that the roles of Runx2 and Osx in the regulation of osteoblast and odontoblast lineages may be independent of one another. The results showed that Runx2 expression overlapped with Osx in dental and osteogenic mesenchyme from E12 to E16. At the later stages, from E18 to PN14, Runx2 and Osx expressions remained intense in alveolar bone osteoblasts. However, Runx2 expression was down-regulated, whereas Osx expression was clearly seen in odontoblasts. At later stages, Dspp transcription was weakly present in osteo-blasts, but strong in odontoblasts where Osx was highly expressed. In mouse odontoblast-like cells, Osx overexpression increased Dspp transcription. Analysis of these data suggests differential biological functions of Runx2, Osx, and Dspp during odontogenesis and osteogenesis.
doi:10.1177/0022034509342873
PMCID: PMC3045537  PMID: 19783797
Runx2; Osx; Dspp; odontoblast; osteoblast; tooth development
21.  Mutant DLX 3 disrupts odontoblast polarization and dentin formation 
Developmental biology  2010;344(2):682-692.
Tricho-dento-osseous (TDO) syndrome is an autosomal dominant disorder characterized by abnormalities in the thickness and density of bones and teeth. A 4-bp deletion mutation in the Distal-Less 3 (DLX3) gene is etiologic for most cases of TDO. To investigate the in vivo role of mutant DLX3 (MT-DLX3) on dentin development, we generated transgenic (TG) mice expressing MT-DLX3 driven by a mouse 2.3 Col1A1 promoter. Dentin defects were radiographically evident in all teeth and the size of the nonmineralized pulp was enlarged in TG mice, consistent with clinical characteristics in patients with TDO. High-resolution radiography, microcomputed tomography, and SEM revealed a reduced zone of mineralized dentin with anomalies in the number and organization of dentinal tubules in MT-DLX3 TG mice. Histological and immunohistochemical studies demonstrated that the decreased dentin was accompanied by altered odontoblast cytology that included disruption of odontoblast polarization and reduced numbers of odontoblasts. TUNEL assays indicated enhanced odontoblast apoptosis. Expression levels of the apoptotic marker caspase-3 were increased in odontoblasts in TG mice as well as in odontoblastic-like MDPC-23 cells transfected with MT-DLX3 cDNA. Expression of Runx2, Wnt 10A, and TBC1D19 colocalized with DLX3 expression in odontoblasts, and MT-DLX3 significantly reduced expression of all three genes. TBC1D19 functions in cell polarity and decreased TBC1D19 expression may contribute to the observed disruption of odontoblast polarity and apoptosis. These data indicate that MT-DLX3 acts to disrupt odontoblast cytodifferentiation leading to odontoblast apoptosis, and aberrations of dentin tubule formation and dentin matrix production, resulting in decreased dentin and taurodontism.
In summary, this TG model demonstrates that MT-DLX3 has differential effects on matrix production and mineralization in dentin and bone and provides a novel tool for the investigation of odontoblast biology.
doi:10.1016/j.ydbio.2010.05.499
PMCID: PMC2945701  PMID: 20510228
DLX3; Transgenic mouse; Dentin mineralization; Taurodontism, TDO, odontoblast polarization; apoptosis, TBC1D19
22.  Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs 
BMC Cell Biology  2011;12:39.
Background
Dental pulp stem/stromal cells (DPSCs) are categorized as adult stem cells (ASCs) that retain multipotent differentiation capabilities. DPSCs can be isolated from individuals at any age and are considered to be true personal stem cells, making DPSCs one of the potential options for stem cell therapy. However, the properties of DPSCs from individuals with an inherited genetic disorder, such as Huntington's disease (HD), have not been fully investigated.
Results
To examine if mutant huntingtin (htt) protein impacts DPSC properties, we have established DPSCs from tooth germ of transgenic monkeys that expressed both mutant htt and green fluorescent protein (GFP) genes (rHD/G-DPSCs), and from a monkey that expressed only the GFP gene (rG-DPSCs), which served as a control. Although mutant htt and oligomeric htt aggregates were overtly present in rHD/G-DPSCs, all rHD/G-DPSCs and rG-DPSCs shared similar characteristics, including self-renewal, multipotent differentiation capabilities, expression of stemness and differentiation markers, and cell surface antigen profile.
Conclusions
Our results suggest that DPSCs from Huntington monkeys retain ASC properties. Thus DPSCs derived from individuals with genetic disorders such as HD could be a potential source of personal stem cells for therapeutic purposes.
doi:10.1186/1471-2121-12-39
PMCID: PMC3189880  PMID: 21910887
Adult stem cells; animal model; DPSCs; Huntington's disease; transgenic HD monkeys; cell therapy
23.  In Vitro Differentiation and Mineralization of Dental Pulp Stem Cells on Enamel-Like Fluorapatite Surfaces 
Tissue Engineering. Part C, Methods  2012;18(11):821-830.
Our previous studies have shown good biocompatibility of fluorapatite (FA) crystal surfaces in providing a favorable environment for functional cell–matrix interactions of human dental pulp stem cells (DPSCs) and also in supporting their long-term growth. The aim of the current study was to further investigate whether this enamel-like surface can support the differentiation and mineralization of DPSCs, and, therefore, act as a potential model for studying the enamel/dentin interface and, perhaps, dentine/pulp regeneration in tooth tissue engineering. The human pathway-focused osteogenesis polymerase chain reaction (PCR) array demonstrated that the expression of osteogenesis-related genes of human DPSCs was increased on FA surfaces compared with that on etched stainless steel (SSE). Consistent with the PCR array, FA promoted mineralization compared with the SSE surface with or without the addition of a mineralization promoting supplement (MS). This was confirmed by alkaline phosphatase (ALP) staining, Alizarin red staining, and tetracycline staining for mineral formation. In conclusion, FA crystal surfaces, especially ordered (OR) FA surfaces, which mimicked the physical architecture of enamel, provided a favorable extracellular matrix microenvironment for the cells. This resulted in the differentiation of human DPSCs and mineralized tissue formation, and, thus, demonstrated that it may be a promising biomimetic model for dentin-pulp tissue engineering.
doi:10.1089/ten.tec.2011.0624
PMCID: PMC3483051  PMID: 22563788
24.  Rescue of Odontogenesis in Dmp1-deficient Mice by Targeted Re-expression of DMP1 Reveals Roles for DMP1 in Early Odontogenesis and Dentin Apposition in vivo 
Developmental biology  2006;303(1):191-201.
Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a three-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting that the effects of DMP1 on mineralization are locally mediated. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.
doi:10.1016/j.ydbio.2006.11.001
PMCID: PMC2059935  PMID: 17196192
DMP1 transgenic mice; odontogenesis; dentin apposition rate; tooth development; mineralization
25.  Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential 
Regenerative medicine  2010;5(4):617-631.
Background
Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs).
Aims
To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo.
Materials & methods
DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model.
Results
The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice.
Conclusion
DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo.
doi:10.2217/rme.10.30
PMCID: PMC3035701  PMID: 20465527
cytokine; dental pulp stem cell; IL-β; immunocompromised mice; inflamed pulp; pulp/dentin complex; tissue regeneration; TNF-α

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