ABSTRACT The use of tooth-derived material as a scaffold has gained attention recently due to its ease of availability and bioactive properties. Hence, the objective of this study was to determine in vitro interaction of human gingival mesenchymal stem cells (hGMSCs) with human demineralised teeth matrix (hDTM) on osteogenic potential with or without osteogenic inducers. The hGMSCs were established and characterised on their morphology, proliferation, population doubling time (PDT), viability, colony-forming ability, expression of cell surface markers and adipogenic differentiation. Further, the effect of hDTM on the biocompatibility and osteogenic differentiation ability of hGMSCs was evaluated. The hGMSCs displayed a fibroblast-like appearance and exhibited a greater proliferative activity. The cells showed > 91% viability, and PDT varied between 39.34 hours and 62.59 hours. Further, hGMSCs indicated their propensity to form clusters/ colonies, and expressed the markers, such as CD29, CD44, CD73 and CD90, but were negative for CD34 and CD45. When treated with adipogenic induction medium, hGMSCs were able to exhibit the formation of neutral lipid vacuoles. The hGMSCs cultured with hDTM did not show any cytotoxic changes including morphology and viability. Mineralisation of calcium nodules was observed in hGMSCs when cultured in osteogenic induction (OI) medium as an indication of osteogenesis. hGMSCs when cultured with hDTM confirmed the presence of a mineralised matrix. Further, when the cells were cultured with hDTM along with OI, they showed slightly enhanced differentiation into osteocytes. In conclusion, hGMSCs were shown to be biocompatible with hDTM, and demonstrated their enhanced osteogenic potential in the presence of hDTM and osteogenic supplements.
Mesenchymal Stem Cells ; Dental Pulp--cytology ; Dentin

ABSTRACT The use of tooth-derived material as a scaffold has gained attention recently due to its ease of availability and bioactive properties. Hence, the objective of this study was to determine in vitro interaction of human gingival mesenchymal stem cells (hGMSCs) with human demineralised teeth matrix (hDTM) on osteogenic potential with or without osteogenic inducers. The hGMSCs were established and characterised on their morphology, proliferation, population doubling time (PDT), viability, colony-forming ability, expression of cell surface markers and adipogenic differentiation. Further, the effect of hDTM on the biocompatibility and osteogenic differentiation ability of hGMSCs was evaluated. The hGMSCs displayed a fibroblast-like appearance and exhibited a greater proliferative activity. The cells showed > 91% viability, and PDT varied between 39.34 hours and 62.59 hours. Further, hGMSCs indicated their propensity to form clusters/ colonies, and expressed the markers, such as CD29, CD44, CD73 and CD90, but were negative for CD34 and CD45. When treated with adipogenic induction medium, hGMSCs were able to exhibit the formation of neutral lipid vacuoles. The hGMSCs cultured with hDTM did not show any cytotoxic changes including morphology and viability. Mineralisation of calcium nodules was observed in hGMSCs when cultured in osteogenic induction (OI) medium as an indication of osteogenesis. hGMSCs when cultured with hDTM confirmed the presence of a mineralised matrix. Further, when the cells were cultured with hDTM along with OI, they showed slightly enhanced differentiation into osteocytes. In conclusion, hGMSCs were shown to be biocompatible with hDTM, and demonstrated their enhanced osteogenic potential in the presence of hDTM and osteogenic supplements.
Mesenchymal Stem Cells ; Dental Pulp--cytology ; Dentin

OBJECTIVE: To compare the mRNA level of cell proliferation-related genes Twist1, SIRT1, FGF2 and TGF-β3 in placenta mesenchymal stem cells (PA-MSCs), umbilical cord mensenchymals (UC-MSCs) and dental pulp mesenchymal stem cells (DP-MSCs). METHODS: The morphology of various passages of PA-MSCs, UC-MSCs and DP-MSCs were observed by microscopy. Proliferation and promoting ability of the three cell lines were detected with the MTT method. Real-time PCR (RT-PCR) was used to determine the mRNA levels of Twist1, SIRT1, FGF2, TGF-β3. RESULTS: The morphology of UC-MSCs and DP-MSCs was different from that of PA-MSCs. Proliferation ability and promoting ability of the PA-MSCs was superior to that of UC-MSCs and DP-MSCs. In PA-MSCs, expression level of Twist1 and TGF-β3 was the highest and FGF2 was the lowest. SIRT1 was highly expressed in UC-MSCs. With the cell subcultured, different expression levels of Twist1, SIRT1, FGF2, TGF-β3 was observed in PA-MSCs, UC-MSCs and DP-MSCs. CONCLUSION Up-regulated expression of the Twist1, SIRT1 and TGF-β3 genes can promote proliferation of PA-MSCs, UC-MSCs and DP-MSCs, whilst TGF-β3 may inhibit these. The regulatory effect of Twist1, SIRT1, FGF2 and TGF-β3 genes on PA-MSCs, UC-MSCs and DP-MSCs are different.
Cell Differentiation ; Cell Proliferation/genetics* ; Cells, Cultured ; Dental Pulp/cytology* ; Female ; Fibroblast Growth Factor 2/genetics* ; Humans ; Mesenchymal Stem Cells/cytology* ; Nuclear Proteins/genetics* ; Placenta/cytology* ; Pregnancy ; Sirtuin 1/genetics* ; Transforming Growth Factor beta3/genetics* ; Twist-Related Protein 1/genetics* ; Umbilical Cord/cytology*

OBJECTIVETo evaluate the biocompatibility and viability of nonionic triblock copolymer Pluronic F-127 as a cell scaffold for osteogenic differentiation of dental pulp stem cells(DPSC).

METHODSDPSC were obtained via enzymatic digestion method and purified bylimited dilution method. The freeze dried hydrogel of 20% Pluronic F-127 was prepared and itsstructurewas observed usingscanning electron microscopy(SEM). After the encapsulation of cells of passage 3 in Pluronic F-127, the effects of hydrogel on the proliferations of DPSC were assessed with methyl thiazolyl terazolium(MTT) after one day and 3, 5, 7 days of incubations, respectively. On day 14, osteogenic abilities of DPSC encapsulated in the hydrogel were estimated by means of alizarin red S, immunocytochemical staining and real-time quantitative PCR(RT-qPCR).

RESULTSDPSC were isolated and cultured successfully in the present study. SEM observations showed that porous structures which might be suitable for cell culture. A570 values of MTT were then normalized. A570 values of the cells in 2D cultures were 0.30±0.06, 0.30±0.17, 0.35±0.04 and 0.25±0.06 and A570 values of DPSC in 3D cultures were 0.36±0.06, 0.54±0.18, 0.70±0.10 and 0.32±0.10 on day 1, 3, 5 and 7, respectively. A570 value peaks were found on day 5 in both groups. The proliferation of 3D cultured DPSC was higher than that of 2D cultured cells(P<0.05). After 14 days of osteogenic induction, there were no calcium nodules observed in the control group and the numbers of calcium nodulesin the 2D and 3D groups had no significant difference(P>0.05). Inmmunocytochemical staining demonstrated strong expression of osteoblast marker Runt-related transcription factor 2(RUNX2), type Ⅰ collagen(Col-Ⅰ) and relatively low expression of osteocalcin(OCN). Moreover, RT-qPCR showed no differences between the relative expression of ALP, RUNX-2, OCN in the 2D and 3D groups (P>0.05), but a higher relative expression of Col-Ⅰ was observed in the 3D group(P=0.023).

CONCLUSIONSPluronic F-127 is a promising cell scaffold or cell carrier for the osteobalst differentiation of dental pulp stem cells.

Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dental Pulp ; cytology ; Humans ; Hydrogel, Polyethylene Glycol Dimethacrylate ; Osteoblasts ; metabolism ; Osteocalcin ; metabolism ; Osteogenesis ; Poloxamer ; Stem Cells ; cytology ; Tissue Scaffolds

OBJECTIVETo compare the osteogenic differentiation potential and osteoclast capacity between stem cells from human exfoliated deciduous teeth (SHED) in the physiological root resorption period and dental pulp stem cells (DPSCs).

METHODSSHED and DPSCs were isolated, purified and cultured in vitro. The two stem cells were examined with ALP staining at 14 days and with alizarin red staining at 21 days of osteogenic induction, and the expressions of the genes associated with osteogenesis and osteoclastogenesis were detected using real-time PCR.

RESULTSThe isolated SHED and DPSCs both showed an elongate spindle-shaped morphology. After osteogenic induction of the cells, Alizarin red staining visualized a greater number of mineralized nodules in SHED than in DPSCs (P<0.05), and SHED also exhibited a stronger ALP activity than DPSCs (P<0.05). RT-PCR test results showed that the two stem cells expressed RANKL,OCN, ALP, OPG and Runx2 mRNA after osteogenic induction, but the expression levels of Runx2, OCN and ALP were lower in DPSCs than in SHED (P<0.05), and the ratio of RANKL/OPG was significantly higher in SHED (P<0.05).

CONCLUSIONSCompared with DPSCs, SHED has not only the ability of osteogenic differentiation but also an osteoclast capacity, which sheds light on the regulatory role of SHED in physiological root resorption bone remodeling.

Alkaline Phosphatase ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dental Pulp ; cytology ; Humans ; Osteoclasts ; cytology ; Osteogenesis ; Osteopontin ; metabolism ; RANK Ligand ; metabolism ; Real-Time Polymerase Chain Reaction ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51(+)/CD140α(+), 10.6% were CD271(+), and 0.3% were STRO-1(+)/CD146(+). Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271(+) DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271(+) DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.
Adult ; Adult Stem Cells ; cytology ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; Biomarkers ; analysis ; CD146 Antigen ; analysis ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Dental Pulp ; cytology ; Flow Cytometry ; methods ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Nerve Tissue Proteins ; analysis ; Odontogenesis ; physiology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.
Cells, Cultured ; DNA Transposable Elements ; Dental Pulp ; cytology ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Nerve Tissue Proteins ; genetics ; Tooth, Deciduous ; cytology ; Transfection

Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/β-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/β-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/β-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of β-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced β-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of β-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation.
Bone Morphogenetic Protein 2 ; physiology ; Cell Differentiation ; physiology ; Dental Pulp ; cytology ; Humans ; MAP Kinase Signaling System ; Wnt Proteins ; metabolism ; beta Catenin ; metabolism