Dental stem cells (DSCs), a distinct subset of mesenchymal stem cells (MSCs), are isolated from dental tissues, such as dental pulp, exfoliated deciduous teeth, periodontal ligament, and apical papilla. They have emerged as a promising source of stem cell therapy for tissue regeneration and autoimmune disorders. The main types of DSCs include dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), and stem cells from apical papilla (SCAP). Each type exhibits distinct advantages: easy access via minimally invasive procedures, multi-lineage differentiation potential, and excellent ethical acceptability. DSCs have demonstrated outstanding clinical efficacy in oral and maxillofacial regeneration, and their long-term safety has been verified. In oral tissue regeneration, DSCs are highly effective in oral tissue regeneration for critical applications such as the restoration of dental pulp vitality and periodontal tissue repair. A defining advantage of DSCs lies in their ability to integrate with host tissues and promote physiological regeneration, which render them a better option for oral tissue regenerative therapies. Beyond oral applications, DSCs also exhibit promising potential in the treatment of systemic diseases, including type Ⅱ diabetes and autoimmune diseases due to their immunomodulatory effects. Moreover, extracellular vesicles (EVs) derived from DSCs act as critical mediators for DSCs' paracrine functions. Possessing regulatory properties similar to their parental cells, EVs are extensively utilized in research targeting tissue repair, immunomodulation, and regenerative therapy-offering a "cell-free" strategy to mitigate the limitations associated with cell-based therapies. Despite these advancements, standardizing large-scale manufacturing, maintaining strict quality control, and clarifying the molecular mechanisms underlying the interaction of DSCs and their EVs with recipient tissues remain major obstacles to the clinical translation of these treatments into broad clinical use. Addressing these barriers will be critical to enhancing their clinical applicability and therapeutic efficacy. In conclusion, DSCs and their EVs represent a transformative approach in regenerative medicine, and increasing clinical evidence supports their application in oral and systemic diseases. Continuous innovation remains essential to unlocking the widespread clinical potential of DSCs.
Humans ; Dental Pulp/cytology* ; Translational Research, Biomedical ; Mesenchymal Stem Cells/cytology* ; Periodontal Ligament/cytology* ; Stem Cells/cytology* ; Regeneration ; Tooth, Deciduous/cytology* ; Cell Differentiation ; Tissue Engineering/methods* ; Regenerative Medicine

OBJECTIVE: Stem cells from human exfoliated teeth (SHED) were sorted by magnetically activated cell sorting (MACS) technique to obtain the CD146 positive and negative cell subpopulation. Then the biological characteristics of these subpopulations were compared to explore their specific application potential in tissue engineering. METHODS: In this study, freshly extracted deciduous teeth without any caries or dental pulp disease were obtained. SHED was isolated using enzyme digestion method and then sorted by MACS, CD146 positive cells and CD146 negative cells were obtained after cell sorting. The biological characteristics of the unsorted mixed cells, CD146 positive subpopulation and CD146 negative subpopulation were compared. The proliferation ability was detected through cell counting kit-8 (CCK-8) and colony-forming unit (CFU). After osteogenic induction, alizarin red staining was performed and the gene expression of osteogenic related markers was detected by quantitative real-time polymerase chain reaction(qPCR). After adipogenic induction, oil-red O staining was performed and the gene expression of adipogenic related markers was detected. After neurogenic differentiation induction, the expression of neural markers was detected by immunofluorescence and the gene expression of neural markers was detected by qPCR. RESULTS: SHED of the fifth passage was sorted by MACS. And the CD146 positive cell subpopulation and CD146 negative cell subpopulation were obtained. CCK8 assay showed that the proliferative tendency of the three cell groups was consistent, but the proliferation potential of CD146 positive and negative cell subpopulations was significantly lower than that of the unsorted cells. The colony forming rates of the unsorted mixed cell group, CD146 positive and negative populations were 28.6%±3%,17.1%±2.3% and 27.5%±2.5%, respectively. After 21 days of osteogenic induction, alizarin red staining and qPCR showed that the CD146 positive cell population had more mineralized nodule formation and expressed higher level of osteogenic related genes compared with the other two groups. After 21 days of adipogenic induction, oil red O staining and qPCR results showed that the CD146 negative subpopulation produced more lipid droplets and the expression of lipid related genes increased more significantly. After 14 days of neural induction, cell immunofluorescence and qPCR results showed that the unsorted mixed cell group and CD146 positive subpopulation expressed glial cell marker, and the expressions of neural precursor cells and neuronal marker increased significantly in negative subpopulation. CONCLUSION The unsorted mixed cells showed better proliferative potential than CD146 positive and negative subpopulations. The CD146 positive subpopulation was most potent in osteogenic differentiation; it was more suitable for bone tissue engineering. The CD146 negative cells had stronger adipogenic differentiation potential than the other two cell groups; different subpopulations differed in neural differentiation.
Bone and Bones ; CD146 Antigen/analysis* ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stem Cells ; Neural Stem Cells ; Neurons ; Osteogenesis ; Staining and Labeling ; Tissue Engineering ; 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

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 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

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 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

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 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 establish three-dimensional culture model of human dental mesenchymal cells and bioengineer in vivo with ceramic bovine bone (CBB) and Collagraft as scaffolds.

METHODSHuman dental mesenchymal cells induced upon stimulation of bFGF and IGF-1 or TGF-beta(1) were implanted onto CBB and Collagraft containing the same kinds of growth factors respectively. Then cell/scaffold constructs were transplanted into nude mice to establish in vivo culture model of dental mesenchymal cells. Control groups were set up at the same time. After 4 weeks or 10 weeks, the implants were taken out for histological and immunohistochemical analysis.

RESULTSWithin 10-week implant tissues, typical dentin-pulp complex-like structures were generated in scaffolds containing growth factors. Human dentin sialoprotein (DSP) was expressed in the newly formed dentin. This phenomenon wasn't observed in control groups and 4-week implants.

CONCLUSIONSDentin-pulp complex-like structures could be bioengineered successfully with human dental mesenchymal cells and CBB or Collagrafts containing growth factors in nude mice.

Animals ; Calcium Phosphates ; Cattle ; Cells, Cultured ; Collagen ; Dental Pulp ; Dentin ; Humans ; Male ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Odontogenesis ; Tissue Engineering ; methods ; Tooth, Deciduous ; cytology ; embryology