Jacopo Pizzicannella

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.

Does optical coherence tomography identify arterial healing after stenting? An in vivo comparison with histology, in a rabbit carotid model

Objective: To verify whether optical coherence tomography (OCT) can accurately monitor the occurrence of arterial healing after stenting. Setting: Delayed stent endothelialisation may predispose to stent thrombosis. OCT is a high-resolution intravascular imaging technique that accurately identifies stent struts and arterial tissues. Design and interventions: Eight New Zealand white rabbits underwent the implantation of single bare metal stents (diameter 2–2.5 mm, length 8–13 mm) in the right common carotid artery through the external carotid artery. After a median of 11 days (range 2–28), the stented arteries were visualised by OCT, with images acquired at a pull-back speed of 0.5 mm/sec. The rabbits were then euthanised, vessels were formalin-fixed and finally processed for histopathology. Results: We analysed 32 cross-sections from eight stented carotid arteries, for a total of 384 stent struts. OCT detected all of the stent struts in 30 of 32 cross-sections (93.7%), and correctly identified the presence/absence of tissue for every strut. Histological and OCT measurements of mean neointima thickness (0.135 (SD 0.079) mm and 0.145 (SD 0.085) mm, respectively, p = NS) were similar and closely related (r = 0.85, p<0.001). Neointima area progressively increased with longer time intervals from stent deployment to sacrifice; histological and OCT measurements were similar for each time interval. The intra-observer and interobserver reproducibility of OCT neointima measurements were excellent (R2 = 0.90 and 0.88, respectively). Conclusions: OCT is a promising means for monitoring stent strut coverage and vessel wall healing in vivo, the relevance of which will become even more significant with the increasing use of drug-eluting stents.

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.

Endothelial committed oral stem cells as modelling in the relationship between periodontal and cardiovascular disease

In the present study we have mimicked, in vitro, an inflammatory process using Lipopolysaccharide derived from Porphyromonas Gingivalis (LPS‐G) and human Periodontal Ligament Stem Cells induced to endothelial differentiation (e‐hPDLSCs). The research project has been organized into the three following steps: i) induction of hPDLSCs toward endothelial differentiation; ii) evaluation of the molecular signaling pathway involved in the response to the LPS‐G, and iii) functional response evaluation of the living construct constituted by porcine decellularized valve/e‐hPDLSCs treated with LPS‐G. Obtained results showed that 5 μg/ml LPS‐G stimulus provokes: a slowdown of cell growth starting from 24 hr and the release of IL6, IL8, and MCP1 molecules. Signaling network analyzed showed the activation of TLR4/ NFkB/ERK1/2/p‐ERK1/2 signaling mediated by MyD88 in LPS‐G stimulated e‐hPDLSCs, moreover a time course put in evidence a nuclear traslocation of ERK1/2 and p‐ERK1/2 in differentiated samples. Following, the ability of e‐hPDLSCs to expand and colonize the decellularized porcine heart valves was appraised at ultrastructural level. Considering that, the Reactive Oxygen Species (ROS) play an important role in the progression and development of cardiovascular disease (CVD), in LPS‐G living construct model e‐hPDLSCs/decellularized porcine heart valves (dPHV), ROS production was assessed. Time lapse experiments evidenced that LPS‐G provokes in e‐hPDLSCs a rapid and sustained increase in ROS generation, negligible on undifferentiated cells. From obtained data, by multiparametric analyses, a reasonable conclusion may be that the inflammation process activated by LPS‐G can affect endothelial cells and could represent in vivo a possible pathological and predictor state of CVD.

5-Aza Exposure Improves Reprogramming Process Through Embryoid Body Formation in Human Gingival Stem Cells

Embryoid bodies (EBs) are three-dimensional aggregates formed by pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells. They are used as an in vitro model to evaluate early extraembryonic tissue formation and differentiation process. In the adult organisms, cell differentiation is controlled and realized through the epigenetic regulation of gene expression, which consists of various mechanisms including DNA methylation. One demethylating agent is represented by 5-Azacytidine (5-Aza), considered able to induce epigenetic changes through gene derepression. Human gingival mesenchymal stem cells (hGMSCs), an easily accessible stem cells population, migrated from neural crest. They are particularly apt as an in vitro study model in regenerative medicine and in systemic diseases. The ability of 5-Aza treatment to induce hGMSCs toward a dedifferentiation stage and in particular versus EBs formation was investigated. For this purpose hGMSCs were treated for 48 h with 5-Aza (5 μM). After treatment, hGMSCs are organized as round 3D structures (EBs-hGMSCs). At light and transmission electron microscopy, the cells at the periphery of EBs-hGMSCs appear elongated, while ribbon-shaped cells and smaller cells with irregular shape surrounded by extracellular matrix were present in the center. By RT-PCR, EBs-hGMSCs expressed specific transcription markers related to the three germ layers as MAP-2, PAX-6 (ectoderm), MSX-1, Flk-1 (mesoderm), GATA-4, and GATA-6 (endoderm). Moreover, in EB-hGMSCs the overexpression of DNMT1 and ACH3 other than the down regulation of p21 was detectable. Immunofluorescence staining also showed a positivity for specific etodermal and mesodermal markers. In conclusion, 5-Aza was able to induce the direct conversion of adult hGMSCs into cells of three embryonic lineages: endoderm, ectoderm, and mesoderm, suggesting their possible application in autologous cell therapy for clinical organ repair.

An intriguing relation between periodontal and cardiovascular diseases

Periodontitis is a chronic inflammatory condition promoted by bacterial colonization of the gingiva that causes alveolar bone and tooth loss. Under these conditions, bacteria, as well as bacterial products and inflammatory mediators can move from the gingival pocket to the well vascularised periodontal tissues and into the circulation. In fact, recently a possible connection between oral infection and cardiovascular disease was suggested (1). One of the major pathogens involved in the progression of periodontal disease is the Porphyromonas gingivalis (LPS-G). The aim of the study was to induce endothelial differentiation in human periodontal ligaments stem cells (hPDLSCs) (2) on decellularized pig heart valve as scaffold and evaluate the role of LPS-G on cell cultures in terms of reactive oxygen species (ROS) production and NFKB pathway. Many studies have shown that ROS provoked oxidative stress plays a critical role in the development of cardiovascular disease. Excessive generation of ROS can cause cellular dysfunction and injury by directly oxidizing and damaging proteins, DNA and lipids, which ultimately result in cell death. To induce endothelial differentiation, human periodontal ligament stem cells (hPDLSCs) were cultured with endothelial growth medium (EGM-2MV) supplemented with vascular endothelial growth factor (VEGF) and seeded on decellularized pig heart valve. Valve leaflets were incubated for 30 min with 10 μM DCFH-DA at 37°C in humidified incubator. At the end of loading, the valve leaflets were observed using multiphoton microscope. Results of time lapse experiments revealed that after treatment, with 2,5 mg/ml LPS-G, a rapid sustained increase in ROS generation was observed mainly in differentiated hPDLSCs. Comparing the average response of undifferentiated and differentiated hPDLSCs is clearly evident that the latter showed an six fold increase in ROS production after LPS-G exposure, while on undifferentiated appeared to be negligible. Moreover, in endothelial differentiated cells the NFKB nuclear translocation in presence of LPS-G was evident. A reasonable conclusion could be that the treatment of periodontal disease not only improve dental but presumably also cardiovascular health.

Bone regeneration process driven to human periodontal ligament stem cells cultured onto corticocancellous scaffold

The aim of our research was to develop tissue-engineerized constructs composed by porcine cortico-cancellous scaffold (Osteobiol Dual Block) (DB) and xenofree ex vivo culture of human Periodontal Ligament Stem Cells (hPDLSCs) induced to osteogenic differentiation. hPDLSCs placed in xeno-free media formulation mantained the stem cells features, the expression of stemness and pluripotency markers, and the capacity to differentiate in different mesenchymal cell lines (1). Micrographs performed by transmission electron microscopy suggested that after one week of culture, both uninduced and osteogenic induced cells joined and grew on DB secreting extracellular matrix, hierarchically assembled in fibrils in osteogenic differentiation induced samples (2). Quantitative RT-PCR (qRT-PCR) of 92 osteogenesis-related assays of hPDLSCs seeded on the DB showed the upregulation of key genes involved in the osteogenic differentiation pathway such as RUNX2, collagens and SMAD. hPDLSCs induced to osteogenic differentiation in presence of DB expressed osteogenic- related transcripts such as BMP1-4-6, RUNX-2, collagens, MSX1-2, TGFβ3 and SMAD. Functional study revealed a significant increased response of calcium transients, in presence of the 3D-DB both in undifferentiated and differentiated cells stimulated with calcitonin and parathormone, suggesting that the biomaterial could drive the osteogenic differentiation process of hPDLSCs. These data were confirmed from the increase of gene expression of L-type voltage-dependent Ca2+ (VDCCL), subunits α1C and α2D1 in undifferentiated cells in presence of DB. Our results propose to consider DB a biocompatible, osteoinductive and osteoconductive biomaterial making it promising tools to regulate cell activities in biological environments and for a potential use for the development of new custom made tissue-engineering.

Development of Xeno-free culture system for human Periodontal Ligament Stem Cells

The opportunity of transplanting adult stem cells into damaged organs has opened new prospectives for the treatment of several human pathologies. Aim of this study was to develop a culture system for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free media formulation ensuring the maintenance of the stem cells features comprising: the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype and genomic stability, essential elements for conforming to translation to cell therapy1. Somatic stem cells were isolated from the human periodontium using a minimally invasive periodontal access flap surgery. Expanded hPDLSCs in a xeno-free culture showed the morphological features of stem cells, expressed the markers associated with pluripotency, and a normal karyotype. Under appropriate culture conditions, hPDLSCs presented adipogenic and osteogenic potential; indeed, a very high accumulation of lipid droplets was evident in the cytoplasm of adipogenic induced cells, and indisputable evidence of osteogenic differentiation, investigated by transmission electron microscopy, and analyzed for gene expression analysis has been shown2. Our results prove that the novel xeno-free culture method might provide the basis for GMP culture of autologous stem cells, readily accessible from human periodontium, and can be a resource to facilitate their use in human clinical studies for potential therapeutic regeneration.

In vitro cardiomyogenic differentiation of human dental pulp stem cells. The role of 5-azacytidine

Cellular cardiomyoplasty has been introduced as a potential therapy for treating heart failure and has generated significant interest in identifying various cell types capable of restoring the injured myocardium. Autologous human dental pulp stem cells (hDPSCs) show the ability to differentiate into various cell type such as osteoblasts1, chondroblasts, and adipoblasts. Aim of this study is to investigate the aptitude of hDPSCs to differentiate towards a cardiomyogenic phenotype in vitro. In this study we developed an efficient protocol for the generation of functional cardiomyocytes. Second passaged cells were treated with 5μM of 5-azacytidine2, a well-known demethylating agent, for 48h. Initial exposition of cultured hDPSCs to 5-azacytidine leads to development of embryoid bodies (EBs) expressing specific transcripts markers of the three germ layers as : ectoderm (MAP2), mesoderm (MSX1), and endoderm (PAX6) and various precardiac markers including MYH6, MESP. EBs subsequently plated onto gelatin-coated tissue culture dishes express cardiac markers as Nkx2.5 and connexin 43 determinated by PCR and immunofluorescence analysis at confocal microscopy, indicating that EBs can differentiate into functional cardiomyocytes. These results demonstrate that hDPSCs can be an easily source of stem cells able to undergo versus the cardiomyiogenic lineage.