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PUBLISHED RESEARCH 2012


Adult Mesenchymal Stem Cells Explored in the Dental Field
Fawzy El-Sayed KM, Dörfer C, Fändrich F, Gieseler F, Moustafa MH, Ungefroren H.
2012 September 1
[Link: PubMed]

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Abstract:
During the last decade it was realized that stem cell-based therapies hold an enormous therapeutic potential, improving the life of patients with conditions ranging from neurodegenerative and traumatic diseases to regenerative medicine requiring replacement of complex structures such as bones and teeth.

Based on their ability to regenerate and/or repair damaged tissue and eventually restore organ function, multiple types of stem/progenitor cells have been discovered. In the field of periodontal regeneration and tooth engineering, several types of adult multipotent mesenchymal stem cells from various sources are currently being investigated.

These include the bone marrow stromal stem cells (BMSSCs), adipose-derived stromal cells (ADSCs), dental pulp stem cells (DPSCs), dental follicle stem cells (DFSCs), stem cells from human exfoliated deciduous teeth (SHEDs), stem cells from the apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), alveolar bone proper-derived stem cells, and gingival stem cells.

The potential of these different MSCs as precursors for regenerative purposes in the dental field is discussed in this chapter.


The Future of Jaw Replacement
Michigan School of Dentistry, the Michigan Center for Oral Health Research, and Aastrom Biosciences Inc.
2012 August 22
[Link]

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Abstract:
A recent study by the University of Michigan School of Dentistry, the Michigan Center for Oral Health Research, and Aastrom Biosciences Inc., located in Ann Arbor, may have developed a new method of re-constructing the jaw when teeth have been lost. According to the research, dentists may be able to re-grow bone using stem cells instead of replacing it with synthetic materials as we do now.

"This is potentially great news," says Dr. James A. Wells of South Charlotte Dentistry. "Patients who have experienced bone loss or are missing teeth can greatly benefit from any new advances."

"In patients with jawbone deficiencies who also have missing teeth, it is very difficult to replace the missing teeth so that they look and function naturally," said Darnell Kaigler, principal investigator and assistant professor at the U-M School of Dentistry. "This technology and approach could potentially be used to restore areas of bone loss so that missing teeth can be replaced with dental implants."

Kaigler went on to say that the treatment would be perfect for patients who have experienced bone loss due to trauma, disease, or birth defects. Jaw loss is especially difficult to treat due to the fact that the treatment must involve repairing bone, gum tissue, and in some cases even skin. The benefit to the new treatment is that instead of using synthetic materials to fix the problem, we use the patient’s own cells. The body is less likely to reject something that comes from itself.

Researchers used an experimental tissue repair treatment using stem cells to re-grow jawbone after tooth removal. Another control group was given traditional therapy so that the results could be compared.

The results of the study have been promising. Overall the stem cell group healed at a higher rate than the control group. Patients in each group were given dental implants to replace their lost teeth and then given an opportunity to heal. The stem cell group re-grew jawbone faster and with greater density than the control group. The stem cell group also needed less introduction of new bone before implants could be attached.
The research did not introduce stem cells from outside the patient’s body. Researchers collected stem cells from patients' hipbones to use in the treatment. These hip cells were then processed by special machinery at Aastrom. In this case the machinery was directed to grow the hip cells into stem cells. The researchers then simply placed the cells in the mouth and jaw of the patient and allowed the cells to do the rest.
Researchers on the project do not think that stem cell therapies for the mouth and jaw are in our near future, though. Dr. Kaigler noted that these therapies are probably five or even ten years away right now. But with the development of further research and more trials involving greater numbers of patients and larger jaw defects, this stem cell therapy will likely be available in the foreseeable future.


Osteogenic Differentiation of Dental Follicle Stem Cells
Giorgio Mori, Andrea Ballini, Claudia Carbone, Angela Oranger, Giacomina Brunetti, Adriana Di Benedetto, Biagio Rapone, Stefania Cantore, Mariasevera Di Comite, Silvia Colucci, Maria Grano, Felice R. Grassi
2012 August 13
[Link: International Journal of mediacl Sciences]

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Background:
Stem cells are defined as clonogenic cells capable of self-renewal and mul- ti-lineage differentiation. A population of these cells has been identified in human Dental Follicle (DF).
Dental Follicle Stem Cells (DFSCs) were found in pediatric unerupted wisdom teeth and have been shown to differentiate, under particular conditions, into various cell types of the mes- enchymal tissues.

Aim:
The aim of this study was to investigate if cells isolated from DF show stem features, differentiate toward osteoblastic phenotype and express osteoblastic markers.

Methods:
We studied the immunophenotype of DFSCs by flow cytometric analysis, the osteoblastic markers of differentiated DFSCs were assayed by histochemical methods and real-time PCR.

Results:
We demonstrated that DFSCs expressed a heterogeneous assortment of makers associated with stemness. Moreover DFSCs differentiated into osteoblast-like cells, pro- ducing mineralized matrix nodules and expressed the typical osteoblastic markers, Alkaline Phosphatase (ALP) and Collagen I (Coll I).

Conclusion:
This study suggests that DFSCs may provide a cell source for tissue engineering of bone.


Dental Pulp Stem Cells Differentiation Reveals New Insights in Oct4A Dynamics
Ferro F, Spelat R, D'Aurizio F, Puppato E, Pandolfi M, et al.
2012 July 23
[Link: PLoS ONE]

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Although the role played by the core transcription factor network, which includes c-Myc, Klf4, Nanog, and Oct4, in the maintenance of embryonic stem cell (ES) pluripotency and in the reprogramming of adult cells is well established, its persistence and function in adult stem cells are still debated. To verify its persistence and clarify the role played by these molecules in adult stem cell function, we investigated the expression pattern of embryonic and adult stem cell markers in undifferentiated and fully differentiated dental pulp stem cells (DPSC). A particular attention was devoted to the expression pattern and intracellular localization of the stemness-associated isoform A of Oct4 (Oct4A). Our data demonstrate that: Oct4, Nanog, Klf4 and c-Myc are expressed in adult stem cells and, with the exception of c-Myc, they are significantly down-regulated following differentiation. Cell differentiation was also associated with a significant reduction in the fraction of DPSC expressing the stem cell markers CD10, CD29 and CD117. Moreover, a nuclear to cytoplasm shuttling of Oct4A was identified in differentiated cells, which was associated with Oct4A phosphorylation. The present study would highlight the importance of the post-translational modifications in DPSC stemness maintenance, by which stem cells balance self-renewal versus differentiation. Understanding and controlling these mechanisms may be of great importance for stemness maintenance and stem cells clinical use, as well as for cancer research.


Clinical utility of stem cells for periodontal regeneration
Kim Hynes, Danijela Menicanin, Stan Gronthos, P. Mark Bartold
2012 April 17
[Link: Wiley]

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The aim of this review is to discuss the clinical utility of stem cells in periodontal regeneration by reviewing relevant literature that assesses the periodontal-regenerative potential of stem cells. We considered and described the main stem cell populations that have been utilized with regard to periodontal regeneration, including bone marrow-derived mesenchymal stem cells and the main dental-derived mesenchymal stem cell populations: periodontal ligament stem cells, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, stem cells from apical papilla and dental follicle precursor cells. Research into the use of stem cells for tissue regeneration has the potential to significantly influence periodontal treatment strategies in the future.


The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars.
Chen B, Sun HH, Wang HG, Kong H, Chen FM, Yu Q.
2012 April 17
[Link: PubMed]

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Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) in the large-scale expansion of dental pulp stem cells (DPSCs).
However, the biological effects and the optimal concentrations of PL for the proliferation and differentiation of human DPSCs remain unexplored.
We isolated and expanded stem cells from the dental pulp of extracted third molars and evaluated the effects of PL on the cells' proliferative capacity and differentiation potential in vitro and in vivo. Before testing, immunocytochemical staining and flow cytometry-based cell sorting showed that the cells derived from human dental pulp contained mesenchymal stem cell populations. Cells were grown on tissue culture plastic or on hydroxyapatite-tricalcium phosphate (HA/TCP) biomaterials and were incubated with either normal or odontogenic/osteogenic media in the presence or absence of various concentrations of human PL for further investigation. The proliferation of DPSCs was significantly increased when the cells were cultured in 5% PL under all testing conditions (P < 0.05). However, this enhancement was inconsistent when the cells were cultured in 1% PL or in 10% PL; 10% PL significantly inhibited cell proliferation and was therefore excluded from further differentiation testing.
Culture medium containing 5% PL also significantly promoted the mineralized differentiation of DPSCs, as indicated by the measurement of alkaline phosphatase activity and calcium deposition under mineral-conditioned media (P < 0.05).
Scanning electron microscopy and modified Ponceau trichrome staining showed that the cells treated with 5% PL and mineralizing media were highly capable of integrating with the HA/TCP biomaterials and had fully covered the surface of the scaffold with an extensive sheet-like structure 14 d after seeding.
In addition, 5% PL showed significantly positive effects on tissue regeneration in two in vivo transplantation models.
We conclude that the appropriate concentration of PL enhances the proliferation and mineralized differentiation of human DPSCs both in vitro and in vivo, which supports the use of PL as an alternative to FBS or a nonzoonotic adjuvant for cell culture in future clinical trials.
However, the elucidation of the molecular complexity of PL products and the identification of both the essential growth factors that determine the fate of a specific stem cell and the criteria to establish dosing require further investigation.


Comparison of stem cells properties of cells isolated from normal and inflamed dental pulps
L. O. Pereira, M. R. Rubini, J. R. Silva, D. M. Oliveira, I. C. R. Silva, M. J. Poças-Fonseca, R. B. Azevedo
2012 March 6
[Link: Wiley]

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Aim:
To compare cells from normal and inflamed human dental pulps regarding the presence of stem cells, their proliferation and differentiation potential.

Methodology:
Human dental pulp stem cells (hDPSCs) were isolated from normal (DPSC-N) and inflamed dental pulps (DPSC-I). They were compared in respect to proliferation (MTT assay), morphology and STRO-1 expression. STRO-1-positive cells were subject to proliferation (MTT and CFU counting) and morphological analyses and then submitted to odonto-osteogenic, adipogenic and condrogenic differentiation. Differentiated cells were evaluated concerning morphology and the expression, by qRT-PCR, of BSP, LPL and SOX-9 genes. The amount of mineralized matrix produced after odonto-osteogenic differentiation was compared by quantitative Alizarin Red staining.

Results:
No difference was observed in the morphology and in the proliferation rate of DPSC-N and DPSC-I either before or after separation of STRO-1-positive cells. These cells represented, 0.46% (± 0.14) and 0.43% (± 0.19) of the cell population from normal and inflamed dental pulps, respectively. Both DPSC-N and DPSC-I were capable of differentiating under the three assayed conditions and presented similar patterns for BSP, LPL and SOX-9 expression. Mineralized matrix production was also compatible. In all the quantitative experiments, differences were found between cells from each patient, either from normal or inflamed pulps. Nonetheless, there was no statistical difference between these two groups.

Conclusion:
The morphology, proliferation rate and differentiation potential of DPSC-I were similar to the observed in DPSC-N, thus demonstrating that the inflammatory process did not affect the stem cell properties that were assessed.


Osteoblastic/Cementoblastic and Neural Differentiation of Dental Stem Cells and Their Applications to Tissue Engineering and Regenerative Medicine.
Kim BC, Bae H, Kwon IK, Lee EJ, Park JH, Khademhosseini A, Hwang YS.
2012 March 6
[Link: PubMed]

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Recently, dental stem and progenitor cells have been harvested from periodontal tissues such as dental pulp, periodontal ligament, follicle, and papilla. These cells have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and multilineage differentiation capacity. These dental stem and progenitor cells are known to be derived from ectomesenchymal origin formed during tooth development. A great deal of research has been accomplished for directing osteoblastic/cementoblastic differentiation and neural differentiation from dental stem cells. To differentiate dental stem cells for use in tissue engineering and regenerative medicine, there needs to be efficient in vitro differentiation toward the osteoblastic/cementoblastic and neural lineage with well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source. This review focuses on the multilineage differentiation capacity, especially into osteoblastic/cementoblastic lineage and neural lineages, of dental stem cells such as dental pulp stem cells (DPSC), dental follicle stem cells (DFSC), periodontal ligament stem cells (PDLSC), and dental papilla stem cells (DPPSC). It also covers various experimental strategies that could be used to direct lineage-specific differentiation, and their potential applications in tissue engineering and regenerative medicine.


Human adult dental pulp stem cells enhance poststroke functional recovery through non-neural replacement mechanisms.
Leong WK, Henshall TL, Arthur A, Kremer KL, Lewis MD, Helps SC, Field J, Hamilton-Bruce MA, Warming S, Manavis J, Vink R, Gronthos S, Koblar SA.
2012 March 1
[Link: PubMed]

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Abstract:
Human adult dental pulp stem cells (DPSCs), derived from third molar teeth, are multipotent and have the capacity to differentiate into neurons under inductive conditions both in vitro and following transplantation into the avian embryo.

In this study, we demonstrate that the intracerebral transplantation of human DPSCs 24 hours following focal cerebral ischemia in a rodent model resulted in significant improvement in forelimb sensorimotor function at 4 weeks post-treatment.

At this time, 2.3 ± 0.7% of engrafted cells had survived in the poststroke brain and demonstrated targeted migration toward the stroke lesion. In the peri-infarct striatum, transplanted DPSCs differentiated into astrocytes in preference to neurons. Our data suggest that the dominant mechanism of action underlying DPSC treatment that resulted in enhanced functional recovery is unlikely to be due to neural replacement.

Functional improvement is more likely to be mediated through DPSC-dependent paracrine effects. This study provides preclinical evidence for the future use of human DPSCs in cell therapy to improve outcome in stroke patients.


Hydrogen sulfide increases hepatic differentiation in tooth-pulp stem cells.
Ishkitiev N, Calenic B, Aoyama I, Ii H, Yaegaki K, Imai T.
2012 February 27
[Link: PubMed]

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The toxicity of hydrogen sulfide (H(2)S), an oral malodorous compound, is well reported. We have recently established an experimental model of hepatic differentiation from human tooth-pulp stem cells (HTPC) using serum-free medium. The objective of the present study is to determine the effect of H(2)S on hepatic differentiation. The CD117 positive cell fraction was obtained from deciduous HTPC using magnetic cell sorting. After 3-4 passages, cells were grown in Dulbecco's modified Eagle's medium supplemented with insulin-transferrin-selenium-x (ITS-x), embryotrophic factor (ETF) and hepatocyte growth factor (HGF) for hepatic commitment (five days). For hepatic differentiation the cells were cultured in Iscove's modified Dulbecco's medium supplemented with ITS-x, ETF, oncostatin, HGF and dexamethasone for 15 days in air containing 5% CO(2), with or without H(2)S at 0.05 ng ml(-1). Cells were assayed for the expression of hepatic markers α-fetoprotein, albumin or carbamoyl phosphate synthetase, and urea concentrations and glycogen synthesis were also determined. The panel of hepatic markers was expressed more in the test groups exposed to H(2)S than in the control groups. Urea and glycogen production were also increased, especially glycogen which was approximately five times greater compared to the control (p < 0.01). We concluded that H(2)S at physiological concentrations increased the ability of HTPC to undergo hepatogenic differentiation.


Cytotoxicity of two dental materials on fibroblasts derived from human embryonic stem cells
Zhan Y, Wang XY, Li SL, Fu X, Yu GY, Cao T, Liu H.
2012 February 18
[Link: PubMed]

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OBJECTIVE:
To investigate the cytotoxicity of calcium hydroxide (CH) and composite resin on fibroblasts derived from human embryo body fibroblasts-H9 (EBf-H9), human dental pulp cells (hDPCs) and immortalized fibroblasts L929; and to evaluate the use of EBf-H9 as a cellular model for cytotoxicity screening of dental materials.
METHODS:
The EBf-H9 cells were derived from human embryonic stem cells (H9) via outgrowth of embryonic body (EB); hDPCs were isolated from healthy dental pulp, and identified by immunochemical staining. Cell Counting Kit-8 (CCK-8) assay was applied to analyze the cytotoxicity of CH and composite resin with serial concentrations on the 3 kinds of cells.
RESULTS:
Following 24 h and 48 h (or 72 h) post-treatment of CH and composite resin, the viability of L929 cells was significantly lower than that of EBf-H9 and hDPCs (P<0.05), and there was no significant difference between the last two groups (P>0.05).
CONCLUSION:
Immortalized fibroblasts L929 cells exhibited different response to CH and composite resin compared with EBf-H9 and hDPCs, and the last two cell types were similar to each other. This study indicated that fibroblasts derived from human embryonic stem cells were a potential cellular model instead of traditional immortalized murine cell line for cytotoxicity screening assay.


Cytogenetic instability of dental pulp stem cell lines.
Duailibi MT, Kulikowski LD, Duailibi SE, Lipay MV, Melaragno MI, Ferreira LM, Vacanti JP, Yelick PC.
2012 February
[Link: PubMed]

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Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.


Pro-inflammatory cytokines induce odontogenic differentiation of dental pulp-derived stem cells.
Yang X, Zhang S, Pang X, Fan M.
2012 February
[Link: PubMed]

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Postnatal dental pulp stem cells (DPSCs) represent a unique precursor population in the dental pulp, which have multipotential and harbor great potential for tissue engineering purposes. However, for therapy applications, transplanted cells are often exposed to unfavorable conditions such as cytokines released from necrotic or inflammatory cells in injured tissues. It is not clear how stem cells exposed to these conditions changes in their characteristics. In this study, the effects of pro-inflammatory cytokines, such as IL-1 and TNF, on DPSCs were investigated. Cells were treated with IL-1, TNF, or both for 3, 7, and 12 days. The cultures were evaluated for cell proliferation, ALP activity, and real-time PCR. We found that a short treatment (3 days) of pro-inflammatory cytokines induced the odontogenic differentiation of DPSCs. Furthermore, post 3 days treatment with pro-inflammatory cytokines, the cell-scaffold complexes were implanted subcutaneously in mice for 8 weeks. Histological analysis demonstrated that the cultures gave obviously mineralized tissue formation, especially for both IL-1 and TNF applied. These data suggest that IL-1 and TNF produced in the early inflammatory reaction may induce the mineralization of DPSCs.


Recommendations for using regenerative endodontic procedures in permanent immature traumatized teeth.
Garcia-Godoy F, Murray PE.
2012 February
[Link: PubMed]

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The regeneration of immature permanent teeth following trauma could be beneficial to reduce the risk of fracture and loss of millions of teeth each year. Regenerative endodontic procedures include revascularization, partial pulpotomy, and apexogenesis. Several case reports give these procedures a good prognosis as an alternative to apexification. Care is needed to deliver regenerative endodontic procedures that maintain or restore the vitality of teeth, but which also disinfect and remove necrotic tissues. Regeneration can be accomplished through the activity of the cells from the pulp, periodontium, vascular, and immune system. Most therapies use the host's own pulp or vascular cells for regeneration, but other types of dental stem cell therapies are under development. There are no standardized treatment protocols for endodontic regeneration. The purpose of this article is to review the recent literature and suggest guidelines for using regenerative endodontic procedures for the treatment of permanent immature traumatized teeth. Recommendations for the selection of regenerative and conventional procedures based on the type of tooth injury, fracture type, presence of necrosis or infection, periodontal status, presence of periapical lesions, stage of tooth development, vitality status, patient age, and patient health status will be reviewed. Because of the lack of long-term evidence to support the use of regenerative endodontic procedures in traumatized teeth with open apices, revascularization regeneration procedures should only be attempted if the tooth is not suitable for root canal obturation, and after apexogenesis, apexification, or partial pulpotomy treatments have already been attempted and have a poor prognosis.


Dental follicle cells and treated dentin matrix scaffold for tissue engineering the tooth root.
Guo W, Gong K, Shi H, Zhu G, He Y, Ding B, Wen L, Jin Y.
2012 February
[Link: PubMed]

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Tissue engineering strategies to reconstruct tooth roots are an effective therapy for the treatment of tooth loss. However, strategies to successfully regenerate tooth roots have not been developed and optimized. In the present study, rat dental follicle stem cells (DFCs) were characterized, followed by a thorough investigation of tooth roots regeneration for a combination of DFCs seeding cells, treated dentin matrix (TDM) scaffolds, and an inductive alveolar fossa microenvironment. Eighteen clones derived from single DFCs were harvested; however, only three clones were amplified successfully more than five passages and 90-95 days in culture. Following 270 days or 30 passages, the heterogeneous DFCs showed suitable characteristics for seeding cells to regenerate tooth roots. However, various features, such as variable proliferation rates, differentiation characteristics, apoptosis rates, and total lifespan were observed in DFCs and the three clones. Importantly, upon transplantation of DFCs combined with TDM for four weeks, root-like tissues stained positive for markers of dental pulp and periodontal tissues were regenerated in the alveolar fossa, but not in the skull and omental pockets. These results indicate that tooth roots were successfully regenerated and suggest that the combination of DFCs with TDM in the alveolar fossa is a feasible strategy for tooth roots regeneration. This strategy could be a promising approach for the treatment of clinical tooth loss and provides a perspective with potential applications to regeneration of other tissues and organs.


Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells.
Hosoya A, Hiraga T, Ninomiya T, Yukita A, Yoshiba K, Yoshiba N, Takahashi M, Ito S, Nakamura H.
2012 February 11
[Link: PubMed]

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The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.


Modulation of the differentiation of dental pulp stem cells by different concentrations of β-glycerophosphate.
Liu M, Sun Y, Liu Y, Yuan M, Zhang Z, Hu W.
2012 January 31
[Link: PubMed]

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Dentinogenesis is a necessary prerequisite for dental tissue engineering. One of the steps for dentinogenesis is to obtain large quantities of highly purified odontoblasts. Therefore, we have undertaken an experiment applying different concentrations of β-glycerophosphate (β-GP) to induce the differentiation of dental pulp stem cells (DPSCs) in a long-term 28-day culture. In the meanwhile, we have studied the time- and maturation-dependent expression of matrix extracellular phosphoglycoprotein (MEPE) and that of the odontoblast-like marker-dentin sialoprotein (DSP), in order to investigate an optimized mineralized condition. Western blot results revealed that the expression of DSP became lower when accompanied by the increase of the β-GP concentration, and there was also an influence on MEPE expression when different concentrations of β-GP were applied. Meanwhile, the mineralized groups had an inhibitory function on the expression of MEPE as compared with the control group. Above all, all experimental groups successfully generated mineralized nodules by Alizarin Red S and the 5 mM β-GP group formed more mineralized nodules quantitated using the CPC extraction method. In conclusion, there is a significant modulation of the β-GP during the differentiation of the DPSCs. The degree of odontoblast differentiation is β-glycerophosphate concentration dependent. A low concentration of β-GP (5 mM) has been shown to be the optimal concentration for stimulating the maturation of the DPSCs. Moreover, MEPE accompanied with DSP clearly demonstrates the degree of the differentiation.


Fibroin Scaffold Repairs Critical-Size Bone Defects In Vivo Supported by Human Amniotic Fluid and Dental Pulp Stem Cells.
Riccio M, Maraldi T, Pisciotta A, La Sala GB, Ferrari A, Bruzzesi G, Motta A, Migliaresi C, De Pol A.
2012 January 31
[Link: PubMed]

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The main aim of this study was the comparative evaluation of fibroin scaffolds combined with human stem cells, such as dental pulp stem cells (hDPSCs) and amniotic fluid stem cells (hAFSCs), used to repair critical-size cranial bone defects in immunocompromised rats. Two symmetric full-thickness cranial defects on each parietal region of rats have been replenished with silk fibroin scaffolds with or without preseeded stem cells addressed toward osteogenic lineage in vitro. Animals were euthanized after 4 weeks postoperatively and cranial tissue samples were taken for histological analysis. The presence of human cells in the new-formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Fibroin scaffolds induced mature bone formation and defect correction, with higher bone amount produced by hAFSC-seeded scaffolds. Our findings demonstrated the strong potential of stem cells/fibroin bioengineered constructs for correcting large cranial defects in animal model and is likely a promising approach for the reconstruction of human large skeletal defects in craniofacial surgery.


The enhancement of osteogenesis through the use of dental pulp pluripotent stem cells in 3D.
Atari M, Caballé-Serrano J, Gil-Recio C, Giner-Delgado C, Martínez-Sarrà E, García-Fernández DA, Barajas M, Hernández-Alfaro F, Ferrés-Padró E, Giner-Tarrida L.
2012 January 16
[Link: PubMed]

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The potential for osteogenic differentiation of dental pulp mesenchymal stem cells (DPMSCs) in vitro and in vivo has been well documented in a variety of studies. Previously, we obtained a population of cells from human dental pulp called dental pulp pluripotent stem cells (DPPSCs) that could differentiate into mesodermal, ectodermal and endodermal progenies. We compared the osteogenic capacity of DPPSCs and DPMSCs that had been isolated from the same donors (N=5) and cultivated in the same osteogenic medium in 3D (three dimensions) Cell Carrier glass scaffolds. We also compared the architecture of bone-like tissue obtained from DPPSCs and human maxillary bone tissue. Differentiation was evaluated by scanning electron microscopy, whereas the expression of bone markers such as ALP, Osteocalcin, COLL1 and Osteonectin was investigated by quantitative real time polymerase chain reaction (qRT-PCR). We also used calcium quantification, Alizarin red staining and alkaline phosphatase (ALP) activity to compare the two cell types. New bone tissue formed by DPPSCs was in perfect continuity with the trabecular host bone structure, and the restored bone network demonstrated high interconnectivity. Significant differences between DPPSCs and DPMSCs were observed for the expression of bone markers, calcium deposition and ALP activity during osteogenic differentiation; these criteria were higher for DPPSCs than DPMSCs. Both DPPSCs and differentiated tissue showed normal chromosomal dosage after being cultured in vitro and analysed using short-chromosome genomic hybridisation (short-CGH). This study demonstrates the stability and potential for the use of DPPSCs in bone tissue engineering applications.


Stage-specific embryonic antigen-4 identifies human dental pulp stem cells.
Kawanabe N, Murata S, Fukushima H, Ishihara Y, Yanagita T, Yanagita E, Ono M, Kurosaka H, Kamioka H, Itoh T, Kuboki T, Yamashiro T.
2012 January 11
[Link: PubMed]

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Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.


Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms.
Sakai K, Yamamoto A, Matsubara K, Nakamura S, Naruse M, Yamagata M, Sakamoto K, Tauchi R, Wakao N, Imagama S, Hibi H, Kadomatsu K, Ishiguro N, Ueda M.
2012 January 3
[Link: PubMed]

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Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.


Dental pulp cells derived from permanent teeth express higher levels of R-cadherin than do deciduous teeth: implications of the correlation between R-cadherin expression and restriction of multipotency in mesenchymal stem cells.
Takahashi N, Chosa N, Hasegawa T, Nishihira S, Okubo N, Takahashi M, Sugiyama Y, Tanaka M, Ishisaki A.
2012 January
[Link: PubMed]

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OBJECTIVE:
The aim of this study was to characterize the expression status of cadherins in dental pulp-derived mesenchymal progenitor/stem cells from deciduous and permanent teeth, and to determine how cadherins affect the multipotency of the progenitor/stem cells.
MATERIALS AND METHODS:
We evaluated and compared the expression status of cadherins in dental pulp-derived cells from deciduous teeth and in cells from permanent teeth by using an array of primers for amplification of RNA encoding human cell adhesion molecules and a real time PCR system. In order to elucidate how cadherins (which are differentially expressed in deciduous and permanent teeth) affect the multipotency of the dental pulp-derived progenitor/stem cells, the ability of the dental pulp cells to differentiate into adipocytes and osteoblasts was evaluated.
RESULTS:
R-cadherin was found to be vigorously expressed in the dental pulp cells derived from permanent teeth but not in the dental pulp cells derived from deciduous teeth. N-cadherin was found to be expressed essentially equally in both types of cells. The ability of the dental pulp cells of deciduous teeth to differentiate into adipocytes and osteoblasts was found to be much higher than that of cells obtained from permanent teeth.
CONCLUSION:
R-cadherin may be a key molecule for providing control over the multipotency of the dental pulp-derived mesenchymal stem cells.


Transcription factors TP53 and SP1 and the osteogenic differentiation of dental stem cells.
Oliver F, Viale-Bouroncle S, Driemel O, Reichert TE, Schmalz G, Morsczeck C.
2012 January
[Link: PubMed]

Read Abstract...

Dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle cells (DFCs) have a promising potential for tissue engineering applications including periodontal and bone regeneration. However, little is known about the molecular mechanisms underlying osteogenic differentiation. In a previous study we detected that more than 35% of genes that are regulated during osteogenic differentiation of DFCs have promoter binding sites for the transcription factors TP53 and SP1. However, the role of these transcription factors in dental stem cells is still unknown. We hypothesize that both factors influence the processes of cell proliferation and differentiation in dental stem cells. Therefore, we transiently transfected DFCs and dental pulp stem cells (SHED; Stem cells from human exfoliated decidiuous teeth) with expression vectors for these transcription factors. After overexpression of SP1 and TP53, SP1 influenced cell proliferation and TP53 osteogenic differentiation in both dental cell types. The effects on cell proliferation and differentiation were less pronounced after siRNA mediated silencing of TP53 and SP1. This indicates that the effects we observed after TP53 and SP1 overexpression are indirect and subject of complex regulation. Interestingly, upregulated biological processes in DFCs after TP53-overexpression resemble the downregulated biological processes in SHED after SP1-overexpression. Here, regulated processes are involved in cell motility, wound healing and programmed cell death. In conclusion, our study demonstrates that SP1 and TP53 influence cell proliferation and differentiation and similar biological processes in both SHED and DFCs.



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