Oriana Trubiani

Morphological and cytofluorimetric analysis of adult mesenchymal stem cells expanded ex vivo from periodontal ligament.

Many adult tissues contain a population of stem cells that have the ability of regeneration after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining physiological structure tissues and their studies have been considered very important and intriguing after having shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone, tooth-associated tissue, cardiomyocytes, but also to differentiate into cells derived from other embryonic layers, including neurons. Currently, different efforts have been focused on the identification of odontogenic progenitors from oral tissues. In this study we isolated and characterized a population of homogeneous human mesenchymal stem cells proliferating in culture with an attached well-spread morphology derived from periodontal ligament, tissue of ectomesenchymal origin, with the ability to form a specialized joint between alveolar bone and tooth. The adherent cells were harvested and expanded ex vivo under specific conditions and analysed by FACScan flow cytometer and morphological analysis was carried out by light, scanning and transmission electron microscopy. Our results displayed highly evident cells with a fibroblast like morphology and a secretory apparatus, probably indicating, that the enhanced function of the secretory apparatus of the mesenchymal stem cells may be associated with the secretion of molecules that are required to survive and proliferate. Moreover, the presence in periodontal ligament of CD90, CD29, CD44, CD166, CD 105, CD13 positive cells, antigens that are also identified as stromal precursors of the bone marrow, indicate that the periodontal ligament may turn out to be a new efficient source of the cells with intrinsic capacity to self-renewal, high ability to proliferate and differentiate, that can be utilized for a new approach to regenerative medicine and tissue engineering.

Expression profile of the embryonic markers nanog, OCT-4, SSEA-1, SSEA-4, and frizzled-9 receptor in human periodontal ligament mesenchymal stem cells

Mesenchymal stem cells (MSCs) are self‐renewing cells with the ability to differentiate into various mesodermal‐derived tissues. Recently, we have identified in adult human periodontal ligament (PDL) a population of stem cells (PDL‐MSCs) with the ability to differentiate into osteoblasts and adipocytes. The aim of the present work was to further characterize this population and the expression profile of its cells. To achieve our objective we have used flow cytometry, magnetic cell sorting, cytokine antibody array, and light and electron microscope immunostaining. Our results show that the PDL‐MSCs contain a subpopulation of frizzled‐9 (CD349) positive cells expressing a panel of key mesenchymal and embryonic markers including CD10, CD26, CD29, CD44, CD73, CD90, CD105, CD166, SSEA‐1, and SSEA‐4. They are additionally positive for nanog and Oct‐4; two critical transcription factors directing self‐renewal and pluripotency of embryonic stem cells, and they also express the cytokines EGF and IP‐10. The presence of nanog, Oct‐4, SSEA‐1, and SSEA‐4 suggests that PDL‐MSCs are less differentiated than bone marrow‐derived MSCs. Taken together, these data indicate the presence of immature MSCs in PDL and suggest that the frizzled‐9/Wnt pathway plays an important role in regulating proliferation and differentiation of these cells. J. Cell. Physiol. 225: 123–131, 2010.

Melatonin provokes cell death in human B‐lymphoma cells by mitochondrial‐dependent apoptotic pathway activation

Abstract:  Apoptosis is an important cell suicide programme involved in physiological and pathological processes. Apoptosis can be induced in different ways depending on cell type and acquired signal. Melatonin, the major secretory product of the pineal gland, participates in many important physiological functions and displays a remarkable functional versatility exhibiting antioxidant, oncostatic, anti‐aging, and immunomodulatory properties. Recently, it has been shown that, in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that may be involved in the regulation of the immune system. In this work, we examine the effect of melatonin on RAMOS‐1 human leukaemic cells. Cell growth and viability, DNA fragmentation and JC‐1, and annexin V expression have been determined. To elucidate the mechanism of action of melatonin, Western blot analyses for Bcl‐2 and caspase‐3 expression, and cytochrome c release were carried out. The results suggest that the apoptotic effect of melatonin is associated with cell‐cycle arrest, downregulation of Bcl‐2, mitochondrial membrane depolarization, cytochrome c release and activation of caspase‐3. The intrinsic (mitochondrial dependent) pathway of caspase activation is the ‘point of no return’ commitment to cell death. Taken together, our study indicates that melatonin may play a role as potential therapeutic drug in specific lymphoproliferative diseases.

Regenerative potential of human periodontal ligament derived stem cells on three-dimensional biomaterials: A morphological report

Recent studies have shown that mesenchymal stem cells obtained from periodontal ligament (PDL-MSCs) are multipotent cells that have similar features of the bone marrow and dental pulp MSCs and are capable of proliferating and producing different types of tissue such as bone and tooth associated-tissues. Human PDL-MSCs expanded ex vivo were induced to osteogenesis, seeded in three-dimensional biocompatible scaffolds (fibrin sponge, bovine-derived substitutes) and examined using light, scanning and transmission electron microscopy. Morphological observations showed extensive growth of cellular biomass partially covering the scaffolds after 4 weeks of incubation in mineralization medium. These findings indicate that periodontal ligament can be an easily and efficient autologous source of stem cells with a high expansion capacity and ability to differentiate in osteogenic cells that can colonize and grow connected to bio-compatible scaffold. It can be suggested that the use of PDL-MSCs for generating graft biomaterials is advantageous for bone tissue engineering in regenerative dentistry.

Bacterial response to the exposure of 50 Hz electromagnetic fields

To investigate the ability of prokaryotic microorganisms to activate strategies in adapting themselves to the environmental stress induced by exposure to extremely low frequency electromagnetic fields (ELF-EMF), cultures of Escherichia coli ATCC 700926 exposed at 50 Hz EMF (0.1, 0.5, 1.0 mT), and the respective sham-exposed controls were studied for: the total and culturable counts, the viability status, the antimicrobial susceptibility pattern, the morphological analysis, the genotypical and transcriptional profile. Exposed samples and controls displayed similar total and culturable counts, whereas an increased cell viability was observed in exposed samples re-incubated for 24 h outside of the solenoid compared to the corresponding controls. An exposure to 50 Hz EMF of 20-120 min produced a significant change of E. coli morphotype with a presence of coccoid cells also aggregated in clusters after re-incubation of 24 h outside of the solenoid. Atypical lengthened bacterial forms were also observed suggesting a probable alteration during cell division. No changes among DNA fingerprintings and some differences in RNA-AFLP analysis were observed for each 50 Hz EMF intensities evaluated. Our results indicate that an exposure to 50 Hz EMF acts as a stressing factor on bacteria which can represent a suitable model to investigate acute and chronic effects related to ELF-EMF exposure.

Adult mesenchymal stem cells in dental research: a new approach for tissue engineering.

Many adult tissues contain a population of stem cells that have the ability to regenerate after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues. The studies on stem cells are thought to be very important and, in fact, it has been shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone and tooth-associated tissues, but also other types of tissues. Several studies have focused on the identification of odontogenic progenitors from oral tissues, and it has been shown that the mesenchymal stem cells obtained from periodontal ligament and dental pulp could have similar morphological and phenotypical features of the bone marrow mesenchymal cells. In fact a population of homogeneous human mesenchymal stem cells derived from periodontal ligament and dental pulp, and proliferating in culture with a well-spread morphology, can be recovered and characterized. Since these cells are considered as candidates for regenerative medicine, the knowledge of the cell differentiation mechanisms is imperative for the development of predictable techniques in implant dentistry, oral surgery and maxillo-facial reconstruction. Thus, future research efforts might be focused on the potential use of this cell population in tissue engineering. Further studies will be carried out to elucidate the molecular mechanisms involved in their maintenance and differentiation in vitro and in vivo.

Proteome of human stem cells from periodontal ligament and dental pulp.

Background Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro. Methodology/Principal Findings The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4–7 and 6–9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin. Conclusion/Significance This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.

Functional interleukin-7/interleukin-7Rα, and SDF-1α/CXCR4 are expressed by human periodontal ligament derived mesenchymal stem cells

Hematopoiesis in the bone marrow (BM) is maintained by specific interactions between both hematopoietic and non‐hematopoietic stromal cells, which are mesenchymal stem cells (MSCs) capable of giving rise to several cell types. The human periodontal ligament (PDL), a tissue of ectomesenchymal origin, has been shown to also be a source of MSCs. We have investigated whether MSCs expanded from the PDL of healthy volunteers express characteristics similar to BM‐derived stem cells using structural, immunocytochemical and molecular approaches. Their ability to support the growth of hematopoietic progenitors was also analyzed. The PDL‐MSCs exhibited a fibroblast‐like morphology and their chromatin was dispersed, indicating active gene transcription. The mesenchymal‐related antigens CD90, CD29, CD166, CD105, and CD44 were homogeneously detected by cytofluorimetric analysis, whereas membrane CXCR4 was expressed only by a minority of cells. The PDL‐MSCs differentiated in vitro into osteogenic and adipogenic cells. Immunolocalization of IL‐7, IL‐7Rα, SDF‐1α, and CXCR4 resulted in a diffuse but specific labeling. RT‐PCR analysis confirmed the expression of the above‐mentioned transcripts. The cells spontaneously produced high levels of IL‐7 and SDF‐1α and were able to support the development and long‐term maintenance of BM precursor cells more efficiently than murine stromal cells and similarly to normal BM human stromal cells. We examined IL‐7 and SDF‐1α secretion pathway during adipogenic and osteogenic differentiation. IL‐7 increased during osteogenic and adipogenic differentiation, while the SDF‐1α secretion was downregulated during osteogenic differentiation but increased during adipogenic induction. Our study provides evidence that in human PDL there is an accessible niche of MSCs showing the features of BM‐derived MSCs. J. Cell. Physiol. 214: 706–713, 2008.

Characterization of an Helicobacter pylori environmental strain

Aims:  To investigate the main genotypic virulence markers and the phenotypic features of an environmental Helicobacter pylori strain, named MDC1.

Nitric oxide production during the osteogenic differentiation of human periodontal ligament mesenchymal stem cells

The critical tissues that require regeneration in the periodontium are of mesenchymal origin; therefore, the ability to identify, characterize and manipulate mesenchymal stem cells within the periodontium is of considerable clinical significance. In particular, recent findings suggest that periodontal ligament cells may possess many osteoblast-like properties. In the present study, periodontal ligament mesenchymal stem cells obtained from healthy volunteers were maintained in culture until confluence and then induced to osteogenic differentiation. Intracellular calcium ([Ca2+](i)) concentration and nitric oxide, important signalling molecules in the bone, were measured along with cell differentiation. Alkaline phosphatase activity was assayed and bone nodule-like structures were evaluated by means of morphological and histochemical analysis. Our results showed that the periodontal ligament mesenchymal stem cells underwent an in vitro osteogenic differentiation, resulting in the appearance of active osteoblast-like cells together with the formation of calcified deposits. Differentiating cells were also characterized by an increase of [Ca2+](i) and nitric oxide production. In conclusion, our data show a link between nitric oxide and the osteogenic differentiation of human periodontal ligament mesenchymal stem cells, thus suggesting that local reimplantation of expanded cells in conjugation with a nitric oxide donor could represent a promising method for treatment of periodontal defects.