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

OBJECTIVETo analyze the difference in protein profiles between human dental pulp cells (DPC) and odontogenic differentiated DPC by using proteomic approach.

METHODSHuman DPC were induced to odontoblast differentiation and total proteins in the cell lysates before and after induction were prepared. Proteins spots were isolated by two-dimensional gel electrophoresis. DeCyder V6.0 software was applied to gel image analysis. Differential protein spots were identified by peptide mass fingerprinting technique.

RESULTSForty-six protein spots were determined to be differentially expressed with twenty identified protein spots. Expression changes of the identified proteins revealed the involvement of various regulation mechanisms in odontoblast differentiation, such as cell cycle, cellular energy regulation and signal transduction.

CONCLUSIONSThe proteomic approach is a high throughput method to screen the candidate proteins involved in odontoblast differentiation of DPC.

Cell Differentiation ; Cells, Cultured ; Dental Pulp ; cytology ; metabolism ; Humans ; Odontoblasts ; cytology ; metabolism ; Proteome ; metabolism ; Proteomics ; methods

OBJECTIVETo test the capacity of the stem cells derived from human exfoliated deciduous teeth in in vitro differentiation into osteoblasts.

METHODSStem cells were isolated from the exfoliated deciduous teeth of healthy children and sorted into CD34(+)/CD117(+) cells and the remaining mixed cells using flow cytometry. After in vitro cell culture, the differentiation capacity into osteoblasts of the two groups of cells was evaluated by detecting the markers of osteoblasts using immunocytochemical techniques and fluorescent quantitative PCR. Mineralization assay was performed to identify the cell differentiation.

RESULTSThe cells isolated by typsin digestion grew in the manner of fibroblasts. After a 30-day culture of the two groups of cells, immunocytochemistry detected the expressions of osteoblast markers RUNX-2, OC, and BSP. After 40 days of cell culture, the mRNA expressions of RUNX-2, OC and BSP genes were significantly different between the two groups. At day 50 of cell culture, the CD34(+)/CD117(+) cells exhibited positivity for von Kossa's staining and alizarin red staining, but the mixed cells showed negative staining results.

CONCLUSIONThe purified CD34(+)/CD117(+) stem cells derived from exfoliated deciduous teeth of healthy children possess the capacity to differentiate into osteoblasts and form calcium deposits and mineralized nodules in vitro.

Cell Differentiation ; physiology ; Cells, Cultured ; Child ; Dental Pulp ; cytology ; Humans ; Osteoblasts ; cytology ; Osteogenesis ; physiology ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

OBJECTIVETo compare the multilineage differentiation potential of human dental pulp cells (DPC) and periodontal ligament cells (PDLC) in vitro, and to identify the stem cell characteristics.

METHODSHuman DPC and PDLC were isolated by enzymatic digestion. STRO-1 expression was investigated by flow cytometry. Cells were induced to odontogenic/osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. The multilineage differentiation capacities of DPC and PDLC were evaluated by von Kossa stain, anti-osteocalcin (OCN) and anti-dentin sialoprotein (DSP) immunocytochemistry, oil red O stain, Alcian blue stain, anti-collagen type II immunocytochemistry, and real time RT-PCR.

RESULTSColony formation was observed in DPC and PDLC, with STRO-1 positive rate of (16.5% +/- 4.2%) and (11.6% +/- 1.1%) respectively. Multilineage differentiation was demonstrated in 100% of DPC samples in contrast to 83.3% of PDLC samples. OCN, dentin sialophosphoprotein (DSPP), peroxisome-proliferator-activated receptor gamma 2 (PPARgamma2), lipoprotein lipase (LPL) and collagen type II mRNA levels in DPC and PDLC were significantly upregulated after induction (P < 0.001). There were significant differences in the ratio of upregulation of OCN and PPARgamma2 mRNAs between DPC and PDLC (P < 0.001).

CONCLUSIONSDPC and PDLC contain similar proportion of mesenchymal stem cells and possess comparable multilineage differentiation capacities.

Cell Differentiation ; Cells, Cultured ; Dental Pulp ; cytology ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Periodontal Ligament ; cytology

OBJECTIVETo isolate stem cells from human exfoliated deciduous teeth (SHEDs) and identify their phenotypes and multi-lineage differentiation potential.

METHODSHuman pulp tissue from exfoliated deciduous teeth were dissected and digested to obtain the single cell suspension. The SHEDs selected by magnetic activated cell sorting system (MACS) were identified by examination of the cell morphology and growth in vitro and detection of the expressions of the cell markers. Osteogenic and adipogenic induction was performed to test the multi-lineage differentiation potential of the cells.

RESULTSSHEDs were successfully isolated from human exfoliated deciduous teeth. SHEDs showed a lower growth rate than dental pulp cells and displayed high expressions of CD29 and CD105 but low expressions of CD34 and CD45 as shown by flow cytometry. Experiments of in vitro induction demonstrated a strong potential of the STRO-1+ SHEDs for osteogenic and adipogenic differentiation.

CONCLUSIONImmunomagnetic bead selection can be used to isolate and purify SHEDs, and the STRO-1+ SHEDs show the characteristics of stem cells with multipotent differentiation potentials.

Cell Separation ; Cells, Cultured ; Dental Pulp ; cytology ; Humans ; Immunomagnetic Separation ; methods ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

OBJECTIVETo investigate the effects of bioactive glasses (BG) including 45S5 and nano-58S on proliferation, angiogenic markers vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion and gene expression of human dental pulp cells (HDPC).

METHODSHDPC of 4th passage were cultured in Dulbecco's modified Eagle's medium (DMEM) which contained 0.1 g/L 45S5 or nano-58S ionic dissolution products. Meanwhile HDPC were cultured in DMEM without BG as control group. Proliferation of the cells was evaluated with methyl thiazolyl tetrazolium (MTT) assay on day 1, 2, 3. Quantitative real-time PCR and quantitative sandwich enzyme immunoassays were used to test VEGF and bFGF gene expression and protein secretion of HDPC on day 1, 2, 3.

RESULTSThe relative growth rate (RGR) of 45S5 and nano-58S groups were (134.5 ± 5.0)% and (146.3 ± 19.8)%, which was significantly different from that of control group (P < 0.05). The quantity of VEGF secretion of two experimental groups were (189.29 ± 4.64) and (216.18 ± 14.67) ng/L, respectively, significantly higher than that of the control group [(159.03 ± 11.69) ng/L] (P < 0.05). Furthermore, the nano-58S group secreted much more VEGF than 45S5 group (P < 0.05).bFGF secretion of HDPC was also enhanced by both 45S5 and nano-58S bioactive glasses. The VEGF gene expression of 45S5 and nano-58S on day 1 were (1.70 ± 0.19) and (1.63 ± 0.42), while the bFGF gene expressin on day 3 were (1.49 ± 0.02) and (2.30 ± 0.04), all significantly higher than that of control group (P < 0.05).

CONCLUSIONSBioactive glasses can enhance the proliferation, VEGF and bFGF secretion and gene expression of human dental pulp cells. Compared with 45S5, nano-58S showed a higher activation.

Cells, Cultured ; Dental Pulp ; cytology ; Fibroblast Growth Factor 2 ; Humans ; Tetrazolium Salts ; Vascular Endothelial Growth Factor A

Tooth loss compromises human oral health. Although several prosthetic methods, such as artificial denture and dental implants, are clinical therapies to tooth loss problems, they are thought to have safety and usage time issues. Recently, tooth tissue engineering has attracted more and more attention. Stem cell based tissue engineering is thought to be a promising way to replace the missing tooth. Mesenchymal stem cells (MSCs) are multipotent stem cells which can differentiate into a variety of cell types. The potential MSCs for tooth regeneration mainly include stem cells from human exfoliated deciduous teeth (SHEDs), adult dental pulp stem cells (DPSCs), stem cells from the apical part of the papilla (SCAPs), stem cells from the dental follicle (DFSCs), periodontal ligament stem cells (PDLSCs) and bone marrow derived mesenchymal stem cells (BMSCs). This review outlines the recent progress in the mesenchymal stem cells used in tooth regeneration.
Adult Stem Cells ; physiology ; Bone Marrow Cells ; cytology ; Dental Papilla ; cytology ; Dental Pulp ; cytology ; Dental Sac ; cytology ; Humans ; Mesenchymal Stromal Cells ; physiology ; Multipotent Stem Cells ; physiology ; Periodontal Ligament ; cytology ; Regeneration ; physiology ; Tissue Engineering ; Tooth ; physiology ; Tooth, Deciduous ; cytology

OBJECTIVETo isolate and cultivate human dental pulp stem cells (DPSCs).

METHODSPulp tissue was removed from healthy young human teeth extracted for orthodontic purposes. The pulp was digested by Type I collagenase and dispase. Then single-cell suspensions were obtained by filter and cultivated. The clones were identified by expression of STRO-1. Under the conditions of inducement, clones were identified by activity of alkaline phosphatase (ALP), formation of mineralized nodule and expression of dentin sialoprotein (DSP), and by Oil Red-O dyeing and expressing of PPARr2.

RESULTSThe clones had positive expression of STRO-1. When stimulated to differentiation, these cells took on dramatically high activity of ALP, had the ability of mineralization and expressed DSP. These cells also had ability to trans-differentiate into adipocytes.

CONCLUSIONThere are stem cells in human dental pulp tissues, which can be isolated and cultivated.

Adult ; Cell Differentiation ; Cell Separation ; methods ; Cells, Cultured ; Dental Pulp ; cytology ; Humans ; Stem Cells ; cytology ; Young Adult

Animals ; Bone Regeneration ; physiology ; Cell Differentiation ; Dental Enamel Proteins ; pharmacology ; Dental Pulp ; cytology ; Fibroblasts ; cytology ; Gingiva ; cytology ; Guided Tissue Regeneration, Periodontal ; methods ; Humans ; Induced Pluripotent Stem Cells ; cytology ; physiology ; Mouth Mucosa ; cytology ; Periodontal Ligament ; cytology ; Tissue Engineering ; methods