The role of asporin in mineralization of human dental pulp stem cells

Human adult dental pulp stem cells (hDPSCs) are a unique precursor population isolated from postnatal dental pulp and have the ability to regenerate a reparative dentin-like complex. In this study, we investigated the role of Asporin in hDPSCs, which was identified as a matrix protein in our previous dentin proteomic analysis. We isolated a clonogenic, highly proliferative population of cells from adult human dental pulp. These isolated hDPSCs were confirmed by fluorescence activated cell sorting (FACS) using stem cell-specific markers and have shown multilineage differentiation potential. The localization of Asporin was identified by immunohistochemistry in the globular calcification region in the junction of predentin and dentin. The gene and protein expression levels of Asporin were enhanced at the early stage of and then reduced during the late stage of differentiation of hDPSCs in mineralization media. ASPN knock-down using a lentiviral system suppressed the mineralization of hDPSCs. These results suggest that ASPN plays positive roles in the mineralization of hDPSCs and predentin to dentin.     

Long non-coding RNA H19/SAHH axis epigenetically regulates odontogenic differentiation of human dental pulp stem cells

Long noncoding RNAs (lncRNAs) are emerging as important regulators in molecular processes and may play vital roles in odontogenic differentiation of human dental pulp stem cells (hDPSCs). However, their functions remain to be elucidated. As lncRNA H19 is one of the most classical lncRNA, which plays essential roles in cellular differentiation, thus we explored the effects and mechanisms of H19 in odontogenic differentiation of hDPSCs. Stable overexpression and knockdown of H19 in hDPSCs were constructed using recombinant lentiviruses containing H19 and short hairpin-H19 expression cassettes, respectively. Alkaline phosphatase (ALP) assay, Alizarin red staining assay, von kossa staining, quantitative polymerase chain reaction (qPCR), Western blot analysis, and immunofluorescent staining results indicated that overexpression of H19 in hDPSCs positively regulates the odontogenic differentiation of hDPSCs, while knockdown of H19 in hDPSCs inhibits odontogenic differentiation of hDPSCs. Further, we found that H19 promotes the odontogenic differentiation of hDPSCs through S-adenosylhomocysteine hydrolase (SAHH) epigenetically regulates the methylation and expression of distal-less homeobox (DLX3) gene. Herein, for the first time, we determined that H19/SAHH axis epigentically regulates odontogenic differentiaiton of hDPSCs by inhibiting the DNA methyltransferase 3B (DNMT3B)-mediated methylation of DLX3. Our findings provide a new insight into how H19/SAHH axis play its role in odontogenic differentiation of hDPSCs, and would be helpful in developing therapeutic approaches for dentin regeneration based on stem cells.     

Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.     

4-Methylumbelliferone promotes the migration and odontogenetic differentiation of human dental pulp stem cells exposed to lipopolysaccharide in vitro

Hyaluronic acid (HA), a major component of the extracellular matrix, is essential to inflammatory regulation. 4-Methylumbelliferone (4-mu), as the specific inhibitor of HA synthesis, is an anti-inflammatory in multiple systems. However, there have been no studies, to our knowledge, regarding 4-mu treatment in pulp inflammation. Therefore, the purpose of this study was to investigate the effects of 4-mu on biological behaviors in human dental pulp stem cells (hDPSCs) exposed to lipopolysaccharide (LPS) in vitro. hDPSCs were exposed to LPS to construct the inflammation model in vitro. Immunocytochemistry, quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, scratch/Transwell assay, and alizarin red staining/alkaline phosphatase staining were selected to explore the effect of 4-mu on the expression of inflammatory factors, cell proliferation, cell migration, and the odontogenic differentiation ability of hDPSCs. LPS stimulated hDPSCs to highly express the related inflammatory factors and CD44 (the major HA receptor), which were all inhibited by 0.1 mM of 4-mu. In addition, the cell proliferation ability of hDPSCs was suppressed by 4-mu, while cell migration and odontogenic differentiation abilities were significantly improved under inflammation. In conclusion, 4-mu suppressed inflammatory cytokines in inflamed hDPSCs and had a positive effect on the migration and odontogenic differentiation of hDPSCs.     

Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.     

Stathmin inhibits proliferation and differentiation of dental pulp stem cells via sonic hedgehog/Gli

The mineralization of dental pulp stem cells is an important factor in the tissue engineering of teeth, but the mechanism is not yet obvious. This study aimed to identify the effect of Stathmin on the proliferation and osteogenic/odontoblastic differentiation of human dental pulp stem cells (hDPSCs) and to explore whether the Shh signalling pathway was involved in this regulation. First, Stathmin was expressed in the cytoplasm and on the cell membranes of hDPSCs by cell immunofluorescence. Then, by constructing a lentiviral vector, the expression of Stathmin in hDPSCs was inhibited. Treatment with Stathmin shRNA (shRNA‐Stathmin group) inhibited the ability of hDPSCs to proliferate, as demonstrated by a CCK8 assay and flow cytometry analysis, and suppressed the osteogenic/odontoblastic differentiation ability, as demonstrated by alizarin red S staining and osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) activity, compared to that of hDPSCs from the control shRNA group. Molecular analyses showed that the Shh/GLI1 signalling pathway was inhibited when Stathmin was silenced, and purmorphamine, the Shh signalling pathway activator, was added to hDPSCs in the shRNA‐Stathmin group, real‐time PCR and Western blotting confirmed that expression of Shh and its downstream signalling molecules PTCH1, SMO and GLI1 increased significantly. After activating the Shh signalling pathway, the proliferation of hDPSCs increased markedly, as demonstrated by a CCK8 assay and flow cytometry analysis; osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) expression also increased significantly. Collectively, these findings firstly revealed that Stathmin‐Shh/GLI1 signalling pathway plays a positive role in hDPSC proliferation and osteogenic/odontoblastic differentiation.     

The role of preameloblast-conditioned medium in dental pulp regeneration

Pulp regeneration using human dental pulp stem cells (hDPSCs) maintains tooth vitality compared with conventional root canal therapy. Our previous study demonstrated that preameloblast-conditioned medium (PA-CM) from murine apical bud cells induces the odontogenic differentiation of hDPSCs and promoted dentin formation in mouse subcutaneous tissue. The purpose of the present study is to evaluate the effects of PA-CM with human whole pulp cells on pulp regeneration in an empty root canal space. Human pulp cells were seeded in the pulp cavities of 5 mm-thick human tooth segments with or without PA-CM treatment, and then transplanted subcutaneously into immunocompromised mice. In the pulp cell-only group, skeletal muscle with pulp-like tissue was generated in the pulp cavity. A reparative dentin-like structure with entrapped cells lined the existing dentin wall. However, in the PA-CM-treated group, only pulp-like tissue was regenerated without muscle or a reparative dentin-like structure. Moreover, human odontoblast-like cells exhibited palisade arrangement around the pulp, and typical odontoblast processes elongated into dentinal tubules. The results suggest that PA-CM can induce pulp regeneration of human pulp cells with physiological structures in an empty root canal space.     

Role of epithelial-stem cell interactions during dental cell differentiation

Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.     

A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.     

Gangliosides are involved in neural differentiation of human dental pulp-derived stem cells

Human dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the neural differentiation of hDPSCs. When hDPSCs were cultured under neural differentiation conditions, expression of neural cell marker genes such as Nestin, MAP-2, and NeuN was detected. Immunostaining and high-performance thin-layer chromatography analysis showed that an increase in ganglioside biosynthesis was associated with neural differentiation of hDPSCs. Specifically, a significant increase in GD3 and GD1a expression was observed during neural differentiation. To confirm the role of gangliosides in neural differentiation, ganglioside biosynthesis was inhibited in hDPSCs by knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg), which prevented differentiation into neural cells. These results suggest that gangliosides may play a role in the neural differentiation process of hDPSCs.     

LncRNA CALB2 sponges miR-30b-3p to promote odontoblast differentiation of human dental pulp stem cells via up-regulating RUNX2

Illuminating the mechanisms of odontoblast differentiation of human dental pulp stem cells (hDPSCs) is the key to find therapeutic clues to promote odontogenesis. LncRNAs play a regulatory role in odontoblast differentiation. Here, we identified a novel lncRNA, named lncRNA CALB2. It was up-regulated in odontoblast-differentiated hDPSCs and potentially interacted with miR-30b-3p and RUNX2. Via gain- and loss-of-function approaches, we found lncRNA CALB2 significantly promoted the odontoblast differentiation of hDPSCs. Then, dual luciferase reporter assay and RNA immunoprecipitation assay revealed that both lncRNA CALB2 and RUNX2 mRNA could directly bind to miR-30b-3p via the same binding sites. Interestingly, miR-30b-3p in hDPSCs was down-regulated and RUNX2 was up-regulated during odontoblast differentiation. Moreover, lncRNA CALB2 knockdown significantly reduced the protein level of RUNX2, DSPP and DMP-1, while miR-30b-3p inhibitor rescued the reduction. Furthermore, miR-30b-3p exerted an inhibitory effect on odontoblast differentiation, which could be reversed by lncRNA CALB2. Collectively, these findings indicate that the newly identified lncRNA CALB2 acts as a miR-30b-3p sponge to regulate RUNX2 expression, thus promoting the odontoblast differentiation of hDPSCs. LncRNA CALB2/miR-30b-3p/RUNX2 axis could be a novel therapeutic target for accelerating odontogenesis.     

TGF-beta3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development

Several members of the transforming growth factor (TGF)-beta superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-beta1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-beta3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-beta3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-beta3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-beta3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-beta3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-beta3 soaked beads in vivo. These results indicate for the first time that TGF-beta3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.     

Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells (hDPSCs) on four commercially available scaffold biomaterials.METHODS: hDPSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. hDPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureOss (Allograft), Cerabone (Xenograft), PLLA (Synthetic), and OSTEON II Collagen (Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy (SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase (ALP) activity assay, respectively.RESULTS: All scaffold biomaterials except SureOss (Allograft) supported hDPSC adhesion, proliferation and differentiation. hDPSCs seeded on PLLA (Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hDPSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA (Synthetic) and OSTEON II Collagen (Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone (Xenograft) and OSTEON II Collagen (Composite) scaffolds, the hDPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape.CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hDPSCs. Hence, it may be useful in combination with hDPSCs for cell-based reconstructive therapy.