Grimm Wd

Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons

The plasticity of dental pulp stem cells (DPSCs) has been demonstrated by several studies showing that they appear to self-maintain through several passages, giving rise to a variety of cells. The aim of the present study was to differentiate DPSCs to mature neuronal cells showing functional evidence of voltage gated ion channel activities in vitro. First, DPSC cultures were seeded on poly-l-lysine coated surfaces and pretreated for 48h with a medium containing basic fibroblast growth factor and the demethylating agent 5-azacytidine. Then neural induction was performed by the simultaneous activation of protein kinase C and the cyclic adenosine monophosphate pathway. Finally, maturation of the induced cells was achieved by continuous treatment with neurotrophin-3, dibutyryl cyclic AMP, and other supplementary components. Non-induced DPSCs already expressed vimentin, nestin, N-tubulin, neurogenin-2 and neurofilament-M. The inductive treatment resulted in decreased vimentin, nestin, N-tubulin and increased neurogenin-2, neuron-specific enolase, neurofilament-M and glial fibrillary acidic protein expression. By the end of the maturation period, all investigated genes were expressed at higher levels than in undifferentiated controls except vimentin and nestin. Patch clamp analysis revealed the functional activity of both voltage-dependent sodium and potassium channels in the differentiated cells. Our results demonstrate that although most surviving cells show neuronal morphology and express neuronal markers, there is a functional heterogeneity among the differentiated cells obtained by the in vitro differentiation protocol described herein. Nevertheless, this study clearly indicates that the dental pulp contains a cell population that is capable of neural commitment by our three step neuroinductive protocol.

Translational research: Palatal-derived ecto-mesenchymal stem cells from human palate: A new hope for alveolar bone and cranio-facial bone reconstruction

The management of facial defects has rapidly changed in the last decade. Functional and esthetic requirements have steadily increased along with the refinements of surgery. In the case of advanced atrophy or jaw defects, extensive horizontal and vertical bone augmentation is often unavoidable to enable patients to be fitted with implants. Loss of vertical alveolar bone height is the most common cause for a non primary stability of dental implants in adults. At present, there is no ideal therapeutic approach to cure loss of vertical alveolar bone height and achieve optimal pre-implantological bone regeneration before dental implant placement. Recently, it has been found that specific populations of stem cells and/or progenitor cells could be isolated from different dental resources, namely the dental follicle, the dental pulp and the periodontal ligament. Our research group has cultured palatal-derived stem cells (paldSCs) as dentospheres and further differentiated into various cells of the neuronal and osteogenic lineage, thereby demonstrating their stem cell state. In this publication will be shown whether paldSCs could be differentiated into the osteogenic lineage and, if so, whether these cells are able to regenerate alveolar bone tissue in vivo in an athymic rat model. Furthermore, using these data we have started a proof of principle clinical- and histological controlled study using stem cell-rich palatal tissues for improving the vertical alveolar bone augmentation in critical size defects. The initial results of the study demonstrate the feasibility of using stem cell-mediated tissue engineering to treat alveolar bone defects in humans.

Morphological characterization of periodontium-derived human stem cells.

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

Characterization and analysis of migration patterns of dentospheres derived from periodontal tissue and the palate.

BACKGROUND AND OBJECTIVE Stem cells derived from periodontal and palatal tissues may be useful for regenerative therapies of periodontal tissues. In addition to the use of single periodontium-derived stem cells (pdSCs) and palatal-derived stem cells (paldSCs), the application of pdSC and paldSC dentospheres, providing a pool of vital stem cells, may be a useful approach. As cell migration is a prerequisite for stem cells to regenerate a three-dimensional tissue environment, we characterized pdSCs and paldSCs and investigated the migratory activity of dentospheres within a three-dimensional environment. We also investigated the capacity of the dentospheres to grow on zirconium dioxide surfaces. MATERIAL AND METHODS The capacity of pdSCs and paldSCs to differentiate into the neuronal and osteogenic lineages was proved by RT-PCR and immunohistochemistry through the detection of specific lineage markers, such as alkaline phosphatase, glutamate decarboxylase 1 (also known as GAD67, the 67-kDa isoform of glutamate decarboxylase), neurofilament-M and β-III-tubulin. The expression profile of surface molecules on pdSCs and paldSCs was analyzed by flow cytometry. Adhesion and growth of pdSC/paldSC dentospheres on zirconium dioxide surfaces were determined using confocal laser-scanning microscopy. The migratory behavior of the cells was analyzed using a three-dimensional collagen matrix migration assay. RESULTS Both pdSCs and paldSCs were positive for epidermal growth factor receptor, CC chemokine receptor 2 and CXC chemokine receptor 4 expression and were able to grow on zirconium dioxide surfaces. Cell-migration experiments revealed that both stem-cell populations responded similarly to epidermal growth factor (EGF), monocyte chemotactic protein 1 (MCP-1) and stromal cell-derived factor 1alpha (SDF-1α). Stimulation with EGF resulted in an increased migratory activity of both stem-cell types, whereas the locomotory behavior of the cells was impaired by both MCP-1 and SDF-1α. CONCLUSION Dentospheres represent a pool of vital pdSCs/paldSCs. As a result of the migratory activity demonstrated, along with the capacity to grow on zirconium dioxide surfaces, dentospheres may be useful for regenerative purposes in periodontal tissues.

Influence of enamel matrix derivative on primary CD4+ T-helper lymphocyte migration, CD25 activation, and apoptosis.

BACKGROUND Enamel matrix derivative (EMD) has a low immunogenic potential. To the best of our knowledge, there are no studies on the influence of EMD on lymphocyte migration as a sensitive cellular reaction parameter. This study investigated the influence of EMD on primary T-lymphocyte migration, CD25 activation, and activation-induced cell death. METHODS After immunomagnetic-positive CD4+ lymphocyte separation from peripheral blood taken from three healthy volunteers per trial, the influence of EMD on cell locomotion was assessed in a three-dimensional collagen matrix migration model (CMMM). Direct CD4+ cell contact with EMD at concentrations of 25 and 100 microg/ml was mediated in a one-phase CMMM. We investigated the indirect influence of EMD in a two-phase CMMM: one collagen phase contained 25 and 100 microg EMD/ml, using the same concentrations, and a second adjacent phase contained T lymphocytes. After time-lapse videomicroscopy, the mean locomoting percentage of 30 randomly selected cells was analyzed. Using flow cytometry, CD25 receptor activation was assessed, and annexin V was used for apoptosis detection in lymphocytes challenged with 0, 1, 25, 50, and 100 microg EMD/ml. RESULTS The one-phase CMMM revealed a reduction and the two-phase CMMM showed a dose-dependent increase in the mean locomoting cell percentage (P <0.001). Increasing EMD concentrations resulted in dose-dependent enhanced T-cell CD25 receptor expression and in increasing apoptosis (P <0.001). CONCLUSIONS Our study showed immediate effects of EMD on primary CD4+ lymphocyte migration, CD25 activation, and apoptosis. CD4+ lymphocyte apoptosis may be a further possible background for uneventful early wound healing as seen clinically as the result of EMD application.

Does the Chronically Inflamed Periodontium Harbour Cancer Stem Cells

Trials aiming at isolating cultures and subcultures of cells from the periodontal ligament to uncover special features of such tissue-residing population are related to the past decade [1]. Moreover, the idea of cell-delivery systems, and especially with stem cells, to initiate periodontal regeneration is not new. However, describing suitable animal models for such new techniques is less documented in the literature and sometimes underestimated. The wide range of animal species allows appropriate selection of bio-models for different investigations. Each species has unique similarities and dissimilarities to humans. While many studies could ensure the initiation of periodontal regeneration using stem cells extracted from the periodontium, seeding of human periodontium-derived stem cells (pdSCs) on collagen carriers could induce major features of periodontal regeneration when implanted in experimental periodontal defects in the athymic immunodeficient rat as could be shown in our studies. However, remarkable notifications regarding the results obtained in our studies were the induction of malignant tumors (squamous cell carcinoma) in the majority of investigated animals. Considering the data presented in the literature, our studies seems to be the first that demonstrates the initiation of malignant tumors when using human pdSCs. The patients from whom the pdSCs had been extracted, the animal model used and a possible oncogenic alteration of the pdSCs themselves might all be factors behind the tumors’ initiation. In our present studies, the animal model used and the related experimental periodontal defect, as a heterologous model, could successfully present two important biological features after implantation of pdSCs; namely periodontal repair and tumorigenicity. Although not fully histo-morphometrically assessed in our studies, these features are very important for further investigations and for future more-controlled studies. However, further studies using advanced histological, immunological and genetic techniques are required to assure different supposals presented in the current studies regarding stem cell-based periodontal regeneration and stem cell-tumorigenesis.

Isolation and Characterization of Neural Crest-Derived Stem Cells From Adult Ovine Palatal Tissue

Adult mammalian craniofacial tissues contain limited numbers of post-migratory neural crest-derived stem cells. Similar to their embryonic counterparts, these adult multipotent stem cells can undergo multi-lineage differentiation and are capable of contributing to regeneration of mesodermal and ectodermal cells and tissues in vivo. In the present study, we describe for the first time the presence of Nestin-positive neural crest-derived stem cells (NCSCs) within the ovine hard palate. We show that these cells can be isolated from the palatal tissue and are able to form neurospheres. Ovine NCSCs express the typical neural crest markers Slug and Twist, exhibit high proliferative and migratory activity and are able to differentiate into α smooth muscle cells and β-III-tubulin expressing ectodermal cells. Finally, we demonstrate that oNCSCs are capable of differentiating into osteogenic, adipogenic and chondrogenic cells. Taken together, our results suggest that oNCSCs could be used as model cells to assess the efficacy and safety of autologous NCSC transplantation in a large animal model.

Translational research and therapeutic applications of neural crest-derived stem cells in regenerative periodontology

Regeneration of periodontal tissues aims to utilize tissue engineering techniques to restore lost periodontal tissues including the cementum, periodontal ligament and alveolar bone. Regenerative dentistry and its special field regenerative periodontology represent relatively new and emerging branches of translational stem cell biology and regenerative medicine focusing on replacing and regenerating dental tissues to restore or re-establish their normal function lost during degenerative diseases or acute lesions. The regeneration itself can be achieved through transplantation of autologous or allogenic stem cells, or by improving the tissue self-repair mechanisms (e.g. by application of growth factors). In addition, a combination of stem cells or stem cell-containing tissue with bone implants can be used to improve tissue integration and the clinical outcome. As the oral cavity represents a complex system consisting of teeth, bone, soft tissues and sensory nerves, regenerative periodontology relies on the use of stem cells with relatively high developmental potential. Notably, the potential use of pluripotent stem cell types such as human embryonic stem cells or induced pluripotent stem cells is still aggravated by ethical and practical problems. Thus, other cellular sources such as those readily available in the postnatal craniofacial area and particularly in oral structures offer a much better and realistic alternative as cellular regenerative sources. In this review, we summarize current knowledge on the oral neural crest-derived stem cell populations (oNCSCs) and discuss their potential in regenerative periodontology.

Treatment Strategy for Correction of Periodontal Defects Associated with Tongue Piercing: A Case Report

Intraoral piercing has increased in popularity in the last few years. This type of jewelry has been associated with periodontal and muco-gingival defects such as attachment loss and gingival recession. This case report described a 25-year-old female presented to the clinic of Periodontics at Witten/Herdecke University in Witten, Germany, with a tongue stud placed through the mid-dorsum of her tongue. The lower left central (LL1) and lower right central (LR1) incisors exhibited 6 mm and 5 mm lingual probing depth, 4mm and 3.5mm lingual recession respectively and localized radiographic bone loss in the mid-half of the roots. The treatment strategy consisted of removal of the tongue piercing, professional prophylaxis, oral hygiene instructions and an open-flap surgery of the lingual anterior region of the mandible with the application of enamel matrix derivative (EMD) and bone graft material. Results: At the 1-, 3- and 7-month recall visits, the patient’s oral hygiene has been improved, and the attachment loss appears to have stabilized. It is clear that the tongue piercing might be a risk factor for local periodontal complications.

Formal Means for the Semantic Representation of Medical Data and Clinical Knowledge

In medicine, the ever increasing need to develop subject-oriented interfaces in a natural language mainly arises from the fact that there is a great deal of findings in which the flexibility of a specialized natural language cannot be dispensed with; communication between the physician and the medical staff and the computer cannot be attained by means of artificial languages (e. g. advanced programming languages) but requires the use of a natural language.