OBJECTIVETo observe osteoclasts on the resorbing surface of human deciduous teeth.

METHODSAfter fixing the collected deciduous teeth, we prepared the tooth slices without decalcification, treated them with HE and TRAP dyestuff, and observed the osteoclasts under light and scanning electron microscope.

RESULTSThere were large quantity of various forms of overlapping and huge osteoclasts with many nuclei and silk-like protuberances on the resorbing surface of deciduous teeth. The multinucleated osteoclasts align on the surface of coarse dentin.

CONCLUSIONOn the resorbing surface of human deciduous teeth there are large amount of osteoclasts which can be used as a source of studying human osteoclast.

Humans ; In Vitro Techniques ; Osteoclasts ; cytology ; Tooth Resorption ; Tooth Root ; cytology ; Tooth, Deciduous ; cytology

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 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 isolate and identify stem cells from human exfoliated deciduous teeth (SHED).

METHODSHuman pulp tissue were dissected and digested to obtain the single cell suspension. The cell morphology was observed and the clonality of the obtained cells was assessed. The phenotype of the cells was detected by immunohistochemistry and flow cytometry (FCM), and the cell cycle was analyzed. The in vitro differentiation of the cells into adipose tissue and formation of mineralization nodules were evaluated.

RESULTSClonogenic assay showed the formation of 16-18 clones in every 10(3) plated cells derived from human exfoliated deciduous teeth. These cells were found to express the markers of mesenchymal stem cells with a multipotent differentiation potential.

CONCLUSIONThe cells isolated from human dental pulp are clonogenic and have multipotent differentiation potential, suggesting their identity of SHED.

Cell Differentiation ; physiology ; Cell Separation ; Cells, Cultured ; Child ; Female ; Humans ; Male ; Multipotent Stem Cells ; cytology ; Tooth, Deciduous ; cytology

OBJECTIVETo investigate the expression of calcitonin receptor mRNA in the osteoclasts of the resorbing deciduous teeth.

METHODSAfter fixing the collected deciduous teeth, toluidine blue was performed and tartrate-resistant acid phosphatase (TRAP) staining was used to identify the osteoclasts on the resorbing surface of human deciduous teeth and in situ hybridization of calcitonin receptor mRNA to show its existence.

RESULTSThere were a number of TRAP positive osteoclasts on the root surface which showed the expression of calcitonin receptor mRNA.

CONCLUSIONOn the resorbing surface of human deciduous teeth there are osteoclasts that express calcitonin receptor mRNA, so it is feasible to use this kind of osteoclast to test the effect of external factors on the expression of CTR mRNA.

Humans ; In Situ Hybridization ; In Vitro Techniques ; Osteoclasts ; metabolism ; RNA, Messenger ; biosynthesis ; Receptors, Calcitonin ; biosynthesis ; genetics ; Tooth, Deciduous ; cytology ; metabolism

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

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

OBJECTIVETo isolate, culture and identify odontoclasts in vitro and to establish a method of culturing human odontoclasts.

METHODSHealthy and retentive deciduous teeth were extracted, and then placed in α-minimum essential medium containing 0.1% collagenase and 0.2% dispase for 1 h.Odontoclasts were obtained and incubated from the absorbing root surfaces of deciduous teeth.Isolated cells were viewed by inverted phase contrast microscope firstly. Then, the isolated odontoclasts were morphologically observed by hematoxylin and eosin staining (HE) and tartrate-resistant acid phosphatase (TRAP) staining. The prepared teeth slices were cocultured with the isolated odontoclasts and scanning electronic microscope(SEM) was used to demonstrate the presence of resorption lacunae.

RESULTSThe isolated odontoclasts appeared as multinucleated giant cell with many vacuolus in cytoplasm. TRAP staining demonstrated that the cytoplasm of the odontoclasts was full of claret-red positive particles.Resorption lacunae on teeth slices which cocultured with odontoclasts were seen under SEM.

CONCLUSIONSEnzyme digestion is an effective method to isolate odontoclasts from absorbing root surface of deciduous teeth.

Acid Phosphatase ; metabolism ; Cells, Cultured ; Child ; Child, Preschool ; Giant Cells ; cytology ; metabolism ; ultrastructure ; Humans ; Isoenzymes ; metabolism ; Microscopy, Phase-Contrast ; Osteoclasts ; cytology ; metabolism ; ultrastructure ; Root Resorption ; Staining and Labeling ; methods ; Tartrate-Resistant Acid Phosphatase ; Tooth Root ; cytology ; Tooth, Deciduous ; cytology

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

OBJECTIVETo investigate the effect of stem cell factor on the proliferation and osteogenic differentiation of human deciduous dental pulp stem cells.

METHODHuman dental pulp tissues were harvested from extracted deciduous teeth and digested by collagenase and dispase. The stem cells from human exfoliated deciduous teeth (SHED) obtained were cultured in the presence of 3 or 10 µmol/L stem cell factor, and the proliferation of the cells was assessed by MTT assay. The influence of stem cell factor on alkaline phosphatase (ALP) activity was evaluated using ALP kit. Bone sialoprotein and osteocalcin mRNA expression in the treated cells were examined by real-time PCR.

RESULTMTT assay indicated that both 3 and 10 µmol/L stem cell factor promoted the proliferation of SHED. Stem cell factor enhanced ALP activity in the SHED, and the effect was more obvious at 10 µmol/L. Treatment of the cells with stem cell factor up-regulated the mRNA expressions of bone sialoprotein and osteocalcin.

CONCLUSIONStem cell factor can promote the proliferation and osteogenic differentiation SHED, suggesting the effect of stem cell factor in promoting tooth regeneration.

Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dental Pulp ; cytology ; Humans ; Integrin-Binding Sialoprotein ; metabolism ; Osteocalcin ; metabolism ; Osteogenesis ; Stem Cell Factor ; pharmacology ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology