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ALESSANDRA PISCIOTTA
Personale tecnico amministrativo
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa
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Pubblicazioni
2024
- A comprehensive review on the role of mesenchymal stromal/stem cells in the management of rheumatoid arthritis
[Articolo su rivista]
Pignatti, Elisa; Maccaferri, Monia; Pisciotta, Alessandra; Carnevale, Gianluca; Salvarani, Carlo
abstract
Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease with systemic manifestations. Although the success of immune modulatory drug therapy is considerable, about 40% of patients do not respond to treatment. Mesenchymal stromal/stem cells (MSCs) have been demonstrated to have therapeutic potential for inflammatory diseases. Areas covered: This review provides an update on RA disease and on pre-clinical and clinical studies using MSCs from bone marrow, umbilical cord, adipose tissue, and dental pulp, to regulate the immune response. Moreover, the clinical use, safety, limitations, and future perspective of MSCs in RA are discussed. Using the PubMed database and ClincalTrials.gov, peer-reviewed full-text papers, abstracts and clinical trials were identified from 1985 through to April 2023. Expert opinion: MSCs demonstrated a satisfactory safety profile and potential for clinical efficacy. However, it is mandatory to deepen the investigations on how MSCs affect the proinflammatory deregulated RA patients' cells. MSCs are potentially good candidates for severe RA patients not responding to conventional therapies but a long-term follow-up after stem cells treatment and standardized protocols are needed. Future research should focus on well-designed multicenter randomized clinical trials with adequate sample sizes and properly selected patients satisfying RA criteria for a valid efficacy evaluation.
2023
- Assessing the Ability of Human Dental Pulp Stem Cells to Modulate the Macrophages Phenotype.
[Poster]
Maccaferri, M; Pisciotta, A; Carnevale, G; Salvarani, C; Pignatti, E.
abstract
2023
- Checkpoint immunitari sulle cellule staminali della polpa dentale umana modulati in vitro da linfociti e monociti da pazienti con artrite reumatoide
[Poster]
Rossi, Alessandro; Bonacini, Martina; Ferrigno, Ilaria; Carnevale, Gianluca; Pisciotta, Alessandra; DI TINCO, Rosanna; Pignatti, Elisa; Catanoso, Mariagrazia; Crescentini, Filippo; Citriniti, Giorgia; Magnani, Luca; Caruso, Andrea; Germanò, Giuseppe; Brandolino, Fabio; Chiapparoli, Ilaria; Dardani, Lucia; Cocchiara, Emanuele; Zerbini, Alessandro; Salvarani, Carlo; Croci, Stefania
abstract
2023
- EFFETTO DEL MICROAMBIENTE INFIAMMATORIO INDOTTO DAI MONOCITI SUI FIBROBLASTI E AZIONE MODULATORIA DELLE CELLULE STAMINALI DELLA POLPA DENTALE UMANA
[Poster]
Maccaferri, Monia; Corbelli, Tommaso; Lazzari, Giorgia; Pisciotta, Alessandra; Carnevale, Gianluca; Salvarani, Carlo; Pignatti, Elisa
abstract
2023
- Effect of the Inflammatory Microenvironment Induced by Monocytes on Fibroblasts and Modulatory Action of Human Dental Pulp Stem Cells.
[Poster]
Maccaferri, M; Pisciotta, A; Carnevale, G; Salvarani, C; Pignatti, E.
abstract
2023
- Flow-dependent shear stress affects the biological properties of pericyte-like cells isolated from human dental pulp
[Articolo su rivista]
Bertani, Giulia; Di Tinco, Rosanna; Bertoni, Laura; Orlandi, Giulia; Pisciotta, Alessandra; Rosa, Roberto; Rigamonti, Luca; Signore, Michele; Bertacchini, Jessika; Sena, Paola; De Biasi, Sara; Villa, Erica; Carnevale, Gianluca
abstract
Background: Human dental pulp stem cells represent a mesenchymal stem cell niche localized in the perivascular area of dental pulp and are characterized by low immunogenicity and immunomodulatory/anti-inflammatory properties. Pericytes, mural cells surrounding the endothelium of small vessels, regulate numerous functions including vessel growth, stabilization and permeability. It is well established that pericytes have a tight cross talk with endothelial cells in neoangiogenesis and vessel stabilization, which are regulated by different factors, i.e., microenvironment and flow-dependent shear stress. The aim of this study was to evaluate the effects of a pulsatile unidirectional flow in the presence or not of an inflammatory microenvironment on the biological properties of pericyte-like cells isolated from human dental pulp (hDPSCs). Methods: Human DPSCs were cultured under both static and dynamic conditions with or without pre-activated peripheral blood mononuclear cells (PBMCs). Pulsatile unidirectional flow shear stress was generated by using a specific peristaltic pump. The angiogenic potential and inflammatory properties of hDPSCs were evaluated through reverse phase protein microarrays (RPPA), confocal immunofluorescence and western blot analyses. Results: Our data showed that hDPSCs expressed the typical endothelial markers, which were up-regulated after endothelial induction, and were able to form tube-like structures. RPPA analyses revealed that these properties were modulated when a pulsatile unidirectional flow shear stress was applied to hDPSCs. Stem cells also revealed a downregulation of the immune-modulatory molecule PD-L1, in parallel with an up-regulation of the pro-inflammatory molecule NF-kB. Immune-modulatory properties of hDPSCs were also reduced after culture under flow-dependent shear stress and exposure to an inflammatory microenvironment. This evidence was strengthened by the detection of up-regulated levels of expression of pro-inflammatory cytokines in PBMCs. Conclusions: In conclusion, the application of a pulsatile unidirectional flow shear stress induced a modulation of immunomodulatory/inflammatory properties of dental pulp pericyte-like cells.
2023
- Food Supplements for Skin Health: In Vitro Efficacy of a Combination of Rhodiola rosea, Tribulus terrestris, Moringa oleifera and Undaria pinnatifida on UV-Induced Damage
[Articolo su rivista]
Paganelli, Alessia; Pisciotta, Alessandra; Bertani, Giulia; DI TINCO, Rosanna; Tagliaferri, Nadia; Orlandi, Giulia; Azzoni, Paola; Bertoni, Laura
abstract
An increasing number of people seek treatment for aging-related conditions. Plant-derived nutraceuticals are currently of great interest in the setting of dermo-cosmetic studies for their preventive role in photoaging. We conducted an in vitro study on the possible preventive properties against photoaging of a commercially available product (Venerinase®). A mixture of Rhodiola rosea, Tribulus terrestris, Moringa oleifera, Undaria pinnatifida, folic acid and vitamin B12 (Venerinase®) was tested for its potential anti-aging effects on the skin in vitro. Conventional histology, immunofluorescence and real time PCR were employed in the research protocol. The tested product was proven to prevent UV-induced morphological changes both in keratinocytes and fibroblasts. Moreover, senescence-related and proinflammatory pathways commonly triggered by UV exposure were demonstrated to be inhibited by Venerinase® pretreatment. Our results support the potential clinical benefits of oral supplements for the treatment and/or prevention of cutaneous photodamage.
2023
- Human dental pulp stem cells (hDPSCs) promote the lipofibroblast transition in the early stage of a fibro-inflammatory process
[Articolo su rivista]
Pisciotta, Alessandra; DI TINCO, Rosanna; Bertani, Giulia; Orlandi, Giulia; Bertoni, Laura; Pignatti, Elisa; Orciani, Monia; Sena, Paola; Bertacchini, Jessika; Salvarani, Carlo; Carnevale, Gianluca
abstract
Introduction: In autoimmune diseases, particularly in systemic sclerosis and chronic periaortitis, a strict correlation between chronic inflammation and fibrosis exists. Since the currently used drugs prove mostly effective in suppressing inflammation, a better comprehension of the molecular mechanisms exerted by cell types implicated in fibro-inflammation is needed to develop novel therapeutic strategies. Mesenchymal stromal/stem cells (MSCs) are being matter of deep investigation to unveil their role in the evolution of fibrogenetic process. Several findings pointed out the controversial implication of MSCs in these events, with reports lining at a beneficial effect exerted by external MSCs and others highlighting a direct contribution of resident MSCs in fibrosis progression. Human dental pulp stem cells (hDPSCs) have demonstrated to hold promise as potential therapeutic tools due to their immunomodulatory properties, which strongly support their contribution to tissue regeneration.
Methods: Our present study evaluated hDPSCs response to a fibro-inflammatory microenvironment, mimicked in vitro by a transwell co-culture system with human dermal fibroblasts, at early and late culture passages, in presence of TGF-β1, a master promoter of fibrogenesis.
Results and Discussion: We observed that hDPSCs, exposed to acute fibro-inflammatory stimuli, promote a myofibroblast-to-lipofibroblast transition, likely based on BMP2 dependent pathways. Conversely, when a chronic fibro-inflammatory microenvironment is generated, hDPSCs reduce their anti-fibrotic effect and acquire a pro-fibrotic phenotype. These data provide the basis for further investigations on the response of hDPSCs to varying fibro-inflammatory conditions.
2023
- Wound Healing after Acellular Dermal Substitute Positioning in Dermato-Oncological Surgery: A Prospective Comparative Study
[Articolo su rivista]
Paganelli, Alessia; Naselli, Andrea Giovanni; Bertoni, Laura; Rossi, Elena; Azzoni, Paola; Pisciotta, Alessandra; Cesinaro, Anna Maria; Benassi, Luisa; Kaleci, Shaniko; Garbarino, Federico; Ferrari, Barbara; Fiorentini, Chiara; Reggiani, Camilla; Magnoni, Cristina
abstract
Background: MatriDerm and Integra are both widely used collagenic acellular dermal matrices (ADMs) in the surgical setting, with similar characteristics in terms of healing time and clinical indication. The aim of the present study is to compare the two ADMs in terms of clinical and histological results in the setting of dermato-oncological surgery. Methods: Ten consecutive patients with medical indications to undergo surgical excision of skin cancers were treated with a 2-step procedure at our Dermatologic Surgery Unit. Immediately after tumor removal, both ADMs were positioned on the wound bed, one adjacent to the other. Closure through split-thickness skin grafting was performed after approximately 3 weeks. Conventional histology, immunostaining and ELISA assay were performed on cutaneous samples at different timepoints. Results: No significant differences were detected in terms of either final clinical outcomes or in extracellular matrix content of the neoformed dermis. However, Matriderm was observed to induce scar retraction more frequently. In contrast, Integra was shown to carry higher infectious risk and to be more slowly reabsorbed into the wound bed. Sometimes foreign body-like granulomatous reactions were also observed, especially in Integra samples. Conclusions: Even in the presence of subtle differences between the ADMs, comparable global outcomes were demonstrated after dermato-oncological surgery.
2022
- Characterization of Dental Pulp Stem Cells Response to Bone Substitutes Biomaterials in Dentistry
[Articolo su rivista]
Di Tinco, R.; Consolo, U.; Pisciotta, A.; Orlandi, G.; Bertani, G.; Nasi, M.; Bertacchini, J.; Carnevale, G.
abstract
Bone substitute biomaterials (BSBs) represent a promising alternative to bone autografts, due to their biocompatibility, osteoconduction, slow resorption rates, and the ability to define and maintain volume for bone gain in dentistry. Many biomaterials are tailored to provide structural and biological support for bone regeneration, and allow the migration of bone-forming cells into the bone defect. Neural crest-derived stem cells isolated from human dental pulp (hDPSCs) represent a suitable stem cell source to study the biological effects of BSBs on osteoprogenitor cells involved in the physiological bone regenerative processes. This study aimed to evaluate how three different BSBs affect the stem cell properties, osteogenic differentiation, and inflammatory properties of hDPSCs. Our data highlight that BSBs do not alter cell proliferation and stemness markers expression, nor induce any inflammatory responses. Bone metabolism data show that hDPSCs exposed to the three BSBs distinctively secrete the factors supporting osteoblast activity and osteoclast activity. Our data indicate that (i) hDPSCs are a suitable stem cell source to study the effects of BSBs, and that (ii) the formulation of BSBs may condition the biological properties of stem cells, suggesting their versatile suitability to different dentistry applications.
2022
- Effects of Energy Drink Acute Assumption in Gastrointestinal Tract of Rats
[Articolo su rivista]
Nasi, Milena; De Gaetano, Anna; Carnevale, Gianluca; Bertoni, Laura; Selleri, Valentina; Zanini, Giada; Pisciotta, Alessandra; Caramaschi, Stefania; Reggiani Bonetti, Luca; Farinetti, Alberto; Cossarizza, Andrea; Pinti, Marcello; Manenti, Antonio; Mattioli, Anna Vittoria
abstract
...
2022
- MODULAZIONE DEI MONOCITI NEI PROCESSI INFIAMMATORI CON IL CONTRIBUTO DELLE CELLULE STAMINALI DELLA POLPA DENTALE UMANA (hDPSC)
[Poster]
Pignatti, E.; Maccaferri, M.; Pisciotta, A.; Di Tinco, R.; Bertani, G.; Bertoni, L.; Croci, S.; Bonacini, M.; Carnevale, G.; Salvarani, C.
abstract
2022
- PEDOT: PSS promotes neurogenic commitment of neural crest-derived stem cells
[Articolo su rivista]
Pisciotta, A.; Lunghi, A.; Bertani, G.; Di Tinco, R.; Bertoni, L.; Orlandi, G.; Biscarini, F.; Bianchi, M.; Carnevale, G.
abstract
: Poly (3,4-ethylendioxythiophene) polystyrene sulphonate (PEDOT:PSS) is the workhorse of organic bioelectronics and is steadily gaining interest also in tissue engineering due to the opportunity to endow traditional biomaterials for scaffolds with conductive properties. Biomaterials capable of promoting neural stem cell differentiation by application of suitable electrical stimulation protocols are highly desirable in neural tissue engineering. In this study, we evaluated the adhesion, proliferation, maintenance of neural crest stemness markers and neurogenic commitment of neural crest-derived human dental pulp stem cells (hDPSCs) cultured on PEDOT:PSS nanostructured thin films deposited either by spin coating (SC-PEDOT) or by electropolymerization (ED-PEDOT). In addition, we evaluated the immunomodulatory properties of hDPSCs on PEDOT:PSS by investigating the expression and maintenance of the Fas ligand (FasL). We found that both SC-PEDOT and ED-PEDOT thin films supported hDPSCs adhesion and proliferation; however, the number of cells on the ED-PEDOT after 1 week of culture was significantly higher than that on SC-PEDOT. To be noted, both PEDOT:PSS films did not affect the stemness phenotype of hDPSCs, as indicated by the maintenance of the neural crest markers Nestin and SOX10. Interestingly, neurogenic induction was clearly promoted on ED-PEDOT, as indicated by the strong expression of MAP-2 and β -Tubulin-III as well as evident cytoskeletal reorganisation and appreciable morphology shift towards a neuronal-like shape. In addition, strong FasL expression was detected on both undifferentiated or undergoing neurogenic commitment hDPSCs, suggesting that ED-PEDOT supports the expression and maintenance of FasL under both expansion and differentiation conditions.
2022
- RUOLO DEI MACROFAGI IN CONDIZIONI PRO- ED ANTI-INFIAMMATORIE E MECCANISMI DI REGOLAZIONE INDOTTI DALLE hDPSCs
[Poster]
Maccaferri, M.; Pisciotta, A.; Di Tinco, R.; Bertani, G.; Bertoni, L.; Carnevale, G.; Salvarani, C.; Pignatti, E.
abstract
2022
- Use of confocal microscopy imaging for in vitro assessment of adipose-derived mesenchymal stromal cells seeding on acellular dermal matrices: 3D reconstruction based on collagen autofluorescence
[Articolo su rivista]
Paganelli, A.; Tarentini, E.; Benassi, L.; Scelfo, D.; Pisciotta, A.; Rossi, E.; Magnoni, C.
abstract
Background: Both mesenchymal stromal cells (MSCs) and acellular dermal matrices (ADMs) represent fascinating therapeutic tools in the wound healing scenario. Strategies aimed at combining these two treatment modalities are currently under investigation. Moreover, scarcity of quantitative, nondestructive techniques for quality assessment of engineered tissues poses great limitations in regenerative medicine and collagen autofluorescence-based imaging techniques are acquiring great importance in this setting. Objective: Our goals were to assess the in vitro interactions between ADSCs and ADMs and to analyze extracellular-matrix production. Methods: Adipose-derived MSCs (ADSC) were plated on 8-mm punch biopsies of a commercially available ADM (Integra®). Conventional histology with hematoxylin-eosin staining, environmental scanning electron microscopy, and confocal-laser scanning microscopy were used to obtain imaging of ADSC-seeded ADMs. Collagen production by ADSCs was quantified by mean fluorescence intensity (MFI), expressed in terms of positive pixels/field, obtained through ImageJ software processing of three-dimensional projections from confocal scanning images. Control conditions included: fibroblast-seeded ADM, ADSC- and fibroblast-induced scaffolds, and Integra® alone. Results: ADSCs were efficiently seeded on Integra® and were perfectly incorporated in the pores of the scaffold. Collagen production was revealed to be significantly higher when ADSCs were seeded on ADM rather than in all other control conditions. Collagen autofluorescence was efficiently used as a surrogate marker of ECM production. Conclusions: Combined therapies based on MSCs and collagenic ADMs are promising therapeutic options for chronic wounds. Not only ADSCs can be efficiently seeded on ADMs, but ADMs also seem to potentiate their regenerative properties, as highlightable from fluorescence confocal imaging.
2021
- Evaluation of antimicrobial effect of air-polishing treatments and their influence on human dental pulp stem cells seeded on titanium disks
[Articolo su rivista]
Di Tinco, R.; Bertani, G.; Pisciotta, A.; Bertoni, L.; Bertacchini, J.; Colombari, B.; Conserva, E.; Blasi, E.; Consolo, U.; Carnevale, G.
abstract
Dental implants are one of the most frequently used treatment options for tooth replacement, and titanium is the metal of choice due to its demonstrated superiority in resisting corrosion, lack of allergic reactions and mechanical strength. Surface roughness of titanium implants favors the osseointegration process; nevertheless, its topography may provide a suitable substrate for bacterial biofilm deposition, causing peri-implantitis and leading to implant failure. Subgingival prophylaxis treatments with cleansing powders aimed to remove the bacterial accumulation are under investigation. Two different air-polishing powders-glycine and tagatose-were assayed for their cleaning and antimicrobial potential against a Pseudomonas biofilm and for their effects on human dental pulp stem cells (hDPSCs), seeded on sandblasted titanium disks. Immunofluorescence analyses were carried out to evaluate cell adhesion, proliferation, stemness and osteogenic differentiation. The results demonstrate that both the powders have a great in vitro cleaning potential in the early period and do not show any negative effects during hDPSCs osteogenic differentiation process, suggesting their suitability for enhancing the biocompatibility of titanium implants. Our data suggest that the evaluated cleansing systems reduce microbial contamination and allow us to propose tagatose as an adequate alternative to the gold standard glycine for the air-polishing prophylaxis treatment.
2021
- Human Dental Pulp Stem Cells Modulate Cytokine Production in vitro by Peripheral Blood Mononuclear Cells From Coronavirus Disease 2019 Patients
[Articolo su rivista]
Croci, S.; Bonacini, M.; Dolci, G.; Massari, M.; Facciolongo, N.; Pignatti, E.; Pisciotta, A.; Carnevale, G.; Negro, A.; Cassone, G.; Muratore, F.; Belloni, L.; Zerbini, A.; Salvarani, C.
abstract
A subset of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) developed a condition of hyper-inflammation, which can cause multi-organ damage and the more severe forms of coronavirus disease 2019 (COVID-19). Mesenchymal stem cells (MSCs) can promote tissue regeneration and modulate immune responses and, thus, have the rational requirements to be used to counteract SARS-CoV-2-induced pneumonia and hyper-inflammation. The aim of the present study was to gain insight into possible mechanisms of action of MSCs obtained from human dental pulp [dental pulp stem cells (DPSCs)] in COVID-19 patients. We investigated the concentrations of 18 cytokines in supernatants of peripheral blood mononuclear cells (PBMCs) obtained from COVID-19 patients cultured in vitro alone and in contact with DPSCs. The modulation of cytokines in PBMCs was confirmed by real-time PCR. IL-6 was the sole cytokine detected in supernatants of DPSCs. In resting conditions, co-culture increased IL-1β, IL-2, IL-5, IL-6, IL-10, IL-18, TNFα, and granulocyte macrophage colony-stimulating factor (GM-CSF) levels. When PBMCs were activated with anti-CD3/CD28 antibody-coated beads, co-culture increased IL-6 and GM-CSF, whereas it decreased IFNγ, TNFα, IL-2, IL-5, IL-9, IL-10, IL-12 (p70), IL-17A, IL-18, IL-21, IL-23, and IL-27 levels. Concentrations of IL-1β, IL-4, IL-13, and IL-22 were not affected. The comparison of cytokine concentrations in supernatants of PBMCs from COVID-19 patients vs. healthy subjects revealed lower concentrations of IL-10 and higher concentrations of IL-18 in supernatants of CD3/CD28-activated PBMCs from COVID-19 patients. Results are explorative but indicate that DPSCs can modulate the production of cytokines deregulated in COVID-19 patients, supporting their potential use in COVID-19.
2021
- Immunomodulating Profile of Dental Mesenchymal Stromal Cells: A Comprehensive Overview
[Articolo su rivista]
Paganelli, Alessia; Trubiani, Oriana; Diomede, Francesca; Pisciotta, Alessandra; Paganelli, Roberto
abstract
: Dental mesenchymal stromal cells (MSCs) are multipotent cells present in dental tissues, characterized by plastic adherence in culture and specific surface markers (CD105, CD73, CD90, STRO-1, CD106, and CD146), common to all other MSC subtypes. Dental pulp, periodontal ligament, apical papilla, human exfoliated deciduous teeth, alveolar bone, dental follicle, tooth germ, and gingiva are all different sources for isolation and expansion of MSCs. Dental MSCs have regenerative and immunomodulatory properties; they are scarcely immunogenic but actively modulate T cell reactivity. in vitro studies and animal models of autoimmune diseases have provided evidence for the suppressive effects of dental MSCs on peripheral blood mononuclear cell proliferation, clearance of apoptotic cells, and promotion of a shift in the Treg/Th17 cell ratio. Appropriately stimulated MSCs produce anti-inflammatory mediators, such as transforming growth factor-β (TGF-β), prostaglandin E2, and interleukin (IL)-10. A particular mechanism through which MSCs exert their immunomodulatory action is via the production of extracellular vesicles containing such anti-inflammatory mediators. Recent studies demonstrated MSC-mediated inhibitory effects both on monocytes and activated macrophages, promoting their polarization to an anti-inflammatory M2-phenotype. A growing number of trials focusing on MSCs to treat autoimmune and inflammatory conditions are ongoing, but very few use dental tissue as a cellular source. Recent results suggest that dental MSCs are a promising therapeutic tool for immune-mediated disorders. However, the exact mechanisms responsible for dental MSC-mediated immunosuppression remain to be clarified, and impairment of dental MSCs immunosuppressive function in inflammatory conditions and aging must be assessed before considering autologous MSCs or their secreted vesicles for therapeutic purposes.
2021
- Role of PD-L1 in licensing immunoregulatory function of dental pulp mesenchymal stem cells
[Articolo su rivista]
Di Tinco, R.; Bertani, G.; Pisciotta, A.; Bertoni, L.; Pignatti, E.; Maccaferri, M.; Bertacchini, J.; Sena, P.; Vallarola, A.; Tupler, R.; Croci, S.; Bonacini, M.; Salvarani, C.; Carnevale, G.
abstract
Background: Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. Methods: Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. Results: Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. Conclusions: Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties.
2020
- Effects of a Novel Bioactive Glass Composition on Biological Properties of Human Dental Pulp Stem Cells
[Articolo su rivista]
Di Tinco, Rosanna; Sergi, Rachele; Bertani, Giulia; Pisciotta, Alessandra; Bellucci, Devis; Carnevale, Gianluca; Cannillo, Valeria; Bertoni, Laura
abstract
2020
- Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs)
[Articolo su rivista]
Pisciotta, Alessandra; Bertani, Giulia; Bertoni, Laura; Di Tinco, Rosanna; De Biasi, Sara; Vallarola, Antonio; Pignatti, Elisa; Tupler, Rossella; Salvarani, Carlo; de Pol, Anto; Carnevale, Gianluca
abstract
2020
- Neural crest derived stem cells from dental pulp and tooth-Associated stem cells for peripheral nerve regeneration
[Articolo su rivista]
Pisciotta, A.; Bertoni, L.; Vallarola, A.; Bertani, G.; Mecugni, D.; Carnevale, G.
abstract
The peripheral nerve injuries, representing some of the most common types of traumatic lesions affecting the nervous system, are highly invalidating for the patients besides being a huge social burden. Although peripheral nervous system owns a higher regenerative capacity than does central nervous system, mostly depending on Schwann cells intervention in injury repair, several factors determine the extent of functional outcome after healing. Based on the injury type, different therapeutic approaches have been investigated so far. Nerve grafting and Schwann cell transplantation have represented the gold standard treatment for peripheral nerve injuries, however these approaches own limitations, such as scarce donor nerve availability and donor site morbidity. Cell based therapies might provide a suitable tool for peripheral nerve regeneration, in fact, the ability of different stem cell types to differentiate towards Schwann cells in combination with the use of different scaffolds have been widely investigated in animal models of peripheral nerve injuries in the last decade. Dental pulp is a promising cell source for regenerative medicine, because of the ease of isolation procedures, stem cell proliferation and multipotency abilities, which are due to the embryological origin from neural crest. In this article we review the literature concerning the application of tooth derived stem cell populations combined with different conduits to peripheral nerve injuries animal models, highlighting their regenerative contribution exerted through either glial differentiation and neuroprotective/neurotrophic effects on the host tissue.
2019
- Evaluation of Biological Response of STRO-1/c-Kit Enriched Human Dental Pulp Stem Cells to Titanium Surfaces Treated with Two Different Cleaning Systems.
[Articolo su rivista]
Conserva, E; Pisciotta, A; Bertoni, L; Bertani, Giulia; Meto, A; Colombari, B; Blasi, E; Bellini, P; de Pol, A; Consolo, U; Carnevale, G.
abstract
Peri-implantitis-an infection caused by bacterial deposition of biofilm-is a common complication in dentistry which may lead to implant loss. Several decontamination procedures have been investigated to identify the optimal approach being capable to remove the bacterial biofilm without modifying the implant surface properties. Our study evaluated whether two different systems-Ni-Ti Brushes (Brush) and Air-Polishing with 40 µm bicarbonate powder (Bic40)-might alter the physical/chemical features of two different titanium surfaces-machined (MCH) and Ca++ nanostructured (NCA)-and whether these decontamination systems may affect the biological properties of human STRO-1+/c-Kit+ dental pulp stem cells (hDPSCs) as well as the bacterial ability to produce biofilm. Cell morphology, proliferation and stemness markers were analysed in hDPSCs grown on both surfaces, before and after the decontamination treatments. Our findings highlighted that Bic40 treatment either maintained the surface characteristics of both implants and allowed hDPSCs to proliferate and preserve their stemness properties. Moreover, Bic40 treatment proved effective in removing bacterial biofilm from both titanium surfaces and consistently limited the biofilm re-growth. In conclusion, our data suggest that Bic40 treatment may operatively clean smooth and rough surfaces without altering their properties and, consequently, offer favourable conditions for reparative cells to hold their biological properties.
2019
- In vitro Engineering of a Skin Substitute Based on Adipose-Derived Stem Cells
[Articolo su rivista]
Paganelli, A.; Benassi, L.; Pastar, I.; Pellegrini, M.; Azzoni, P.; Vaschieri, C.; Pisciotta, A.; Carnevale, G.; Pellacani, G.; Magnoni, C.
abstract
In the field of wound healing, stem cell-based strategies are gaining importance for their regenerative potential. Adipose-derived stem cells (ADSCs) are a particular subset of mesenchymal stem cells present in the stromal-vascular fraction of the adipose tissue, today considered very attractive for their relative abundance and accessibility in the human body. However, ADSCs are still not routinely used in normal clinical practice. Several studies have also reported ADSC transplantation in association with biomaterials in an attempt to enhance the local retention and growth rate of the cells. The aim of our study was to evaluate the ability of ADSCs to build a dermal scaffold to be potentially used as a dermal substitute in the field of wound healing, with optimal biocompatibility and mechanical properties. ADSCs were defined as CD90-, CD73-, and CD105-positive cells. ADSCs turned out to be capable of secreting all the main components of the extracellular matrix (ECM) upon stimulation, thus efficiently producing a collagen and fibronectin-containing dermal matrix. We also checked whether the ADSC-produced dermal scaffold could be seeded with keratinocytes. The scaffolding material directly produced by ADSCs has several advantages when compared to the commercially available ones: it is easily obtained from the patients and it is 100% biocompatible and supports cell-ECM interaction. Moreover, it represents a possible powerful therapeutic tool for patients with chronic ulcers since it appears to be potentially grafted with keratinocytes layers, thus bypassing the classical two-step grafting procedure.
2019
- Poorly differentiated clusters (PDC) in colorectal cancer: Does their localization in tumor matter?
[Articolo su rivista]
Bertoni, Laura; Barresi, Valeria; REGGIANI BONETTI, Luca; Caramaschi, Stefania; Mangogna, Alessandro; Lionti, Simona; Azzoni, Paola; Carnevale, Gianluca; Pisciotta, Alessandra; Salviato, Tiziana
abstract
2019
- Regenerative potential of human dental pulp stem cells in the treatment of stress urinary incontinence: In vitro and in vivo study
[Articolo su rivista]
Zordani, Alessio; Pisciotta, Alessandra; Bertoni, Laura; Bertani, Giulia; Vallarola, Antonio; Giuliani, Daniela; Puliatti, Stefano; Mecugni, Daniela; Bianchi, Giampaolo; De Pol, Anto; Carnevale, Gianluca
abstract
OBJECTIVES:
To evaluate the regenerative potential of human dental pulp stem cells (hDPSCs) in an animal model of stress urinary incontinence (SUI). SUI, an involuntary leakage of urine, is due to physical stress involving an increase in bladder pressure and a damage of external urethral sphincter affecting muscles and nerves. Conventional therapies can only relieve the symptoms. Human DPSCs are characterized by peculiar stemness and immunomodulatory properties and might provide an alternative tool for SUI therapy.
MATERIALS AND METHODS:
In vitro phase: hDPSCs were induced towards the myogenic commitment following a 24 hours pre-conditioning with 5-aza-2'-deoxycytidine (5-Aza), then differentiation was evaluated. In vivo phase: pudendal nerve was transected in female rats to induce stress urinary incontinence; then, pre-differentiated hDPSCs were injected in the striated urethral sphincter. Four weeks later, urethral sphincter regeneration was assayed through histological, functional and immunohistochemical analyses.
RESULTS:
Human DPSCs were able to commit towards myogenic lineage in vitro and, four weeks after cell injection, hDPSCs engrafted in the external urethral sphincter whose thickness was almost recovered, committed towards myogenic lineage in vivo, promoted vascularization and an appreciable recovery of the continence. Moreover, hDPSCs were detected within the nerve, suggesting their participation in repair of transected nerve.
CONCLUSIONS:
These promising data and further investigations on immunomodulatory abilities of hDPSCs would allow to make them a potential tool for alternative therapies of SUI.
2019
- Titanium Surface Properties Influence the Biological Activity and FasL Expression of Craniofacial Stromal Cells.
[Articolo su rivista]
Conserva, E; Pisciotta, A; Borghi, F; Nasi, M; Pecorini, Simone; Bertoni, L; de Pol, A; Consolo, U; Carnevale, G.
abstract
Mesenchymal stromal cells (MSCs) can be easily isolated form craniofacial bones during routine dentistry procedures. Due to their embryological origin from neural crest, they represent a suitable cell population to study cell-biomaterial interaction in the craniofacial field, including osteoinductive/osteointegrative processes. The biological and immunomodulatory properties of MSCs may be influenced by chemistry and topography of implant surfaces. We investigated if and how three different titanium surfaces, machined (MCH), sandblasted with resorbable blasting medium (RBM), and Ca++-nanostructured (NCA), may affect biological activity, osseointegration, and immunomodulatory properties of craniofacial MSCs. Cell proliferation, morphology, osteogenic markers, and FasL were evaluated on MSCs isolated from the mandibular bone after seeding on these three different surfaces. No statistically significant differences in cell proliferation were observed whereas different morphologies and growth patterns were detected for each type of surface. No difference in the expression of osteogenic markers was revealed. Interestingly, FasL expression, involved in the immunomodulatory activity of stem cells, was influenced by surface properties. Particularly, immunofluorescence analysis indicated that FasL expression increased on MCH surface compared to the others confirming the suggested role of FasL in promoting osteogenic differentiation. Titanium surface treatments and topography might reflect different biological behaviours of craniofacial MSCs and influence their osseointegration/immunomodulation properties.
2019
- Use of high fidelity simulation: a two-year training project experience for third year students in Nursing Course Degree of Reggio Emilia
[Relazione in Atti di Convegno]
Mecugni, Daniela; Curia, Giulia; Pisciotta, Alessandra; Amaducci, Giovanna
abstract
Nursing students at the end of their studies are supposed to own skills that allow them, in a short time, to act effectively and safely. Therefore, it is of primary importance that during the training period they have the opportunity, under protected conditions, to practice the management of scenarios realistically representative of the clinical setting. High-fidelity simulation workshops, aimed to provide adequate skills in the management of vital criticality, represent innovative and exciting learning tools, thanks to teaching method and forefront technology involved. The laboratory activities included allow the students to completely descend in simulated scenarios either by the use of computerized interactive cases and by computerized manikins. In our study both these types of simulation laboratories were proposed to 3rd year Nursing students attending the academic years 2015/2016 and 2016/2017. The survey administered to both groups at the end of the workshop revealed a highly positive feedback towards this innovative teaching approach that allowed the students to understand the correlation between pseudorealistic simulations and theoretical notions learned during lectures. The experience built through this two-year study allowed us to lay the bases for further studies on this topic.
2018
- Anterior Capsule of the Lens: Comparison of Morphological Properties and Apoptosis Induction following FLACS and Standard Phacoemulsification Surgery
[Articolo su rivista]
Pisciotta, Alessandra; De Maria, Michele; Verdina, Tommaso; Fornasari, Elisa; De Pol, Anto; Cavallini, Gian Maria
abstract
Purpose. Comparative evaluation of morphological features of anterior capsules and apoptosis induction in epithelial cells after femtosecond laser-assisted cataract surgery (FLACS) and standard phacoemulsification surgery. Methods. Group 1: 30 FLACS anterior capsulotomies and Group 2: 30 manual anterior continuous curvilinear capsulorhexes. All patients were operated on by the same experienced surgeon. Morphological features of the anterior capsules and apoptosis induction in epithelial cells were evaluated. Results. All patients revealed a significant mean best-corrected visual acuity (BCVA) improvement 3 months after surgery, and no major intraoperative nor postoperative complications occurred. The capsular epithelium appeared to be preserved in both groups. Scanning electron microscopy analysis revealed irregular saw-tooth shaped edges in capsules from Group 1 whereas capsules from Group 2 showed regular and smooth edges. A statistically significant higher expression of the downstream apoptotic effector cleaved caspase 3 was observed in Group 1. Conclusions. The saw-tooth appearance was likely due to the progressive sequence of laser pulses on the capsule. The low energy/high frequency properties of the laser pulse, combined with an overlapped pulse pattern, resulted in highly continuous morphology of capsule edges. The higher apoptosis induction in FLACS group might be due to photodisruption-dependent plasma generation and formation of cavitation bubbles.
2018
- Ex vivo fluorescence confocal microscopy for intraoperative, real-time diagnosis of cutaneous inflammatory diseases: A preliminary study
[Articolo su rivista]
Bertoni, L; Azzoni, P; Reggiani, C; Pisciotta, A; Carnevale, G; Chester, J; Kaleci, S; Reggiani Bonetti, L; Cesinaro, Am; Longo, C; Pellacani, G.
abstract
Ex vivo fluorescence confocal microscopy (FCM) is an innovative imaging tool that can be used intraoperatively to obtain real-time images of untreated excised tissue with almost histologic resolution. As inflammatory diseases often share overlapping clinical features, histopathology evaluation is required for dubious cases, delaying definitive diagnoses, and therefore therapy. This study identifies key-features at ex vivo FCM for differential diagnoses of cutaneous inflammatory diseases, in particular, psoriasis, eczema, lichen planus and discoid lupus erythematosus. Retrospective ex vivo FCM and histological evaluations with relevant diagnoses were correlated with prospectively reported histopathologic diagnoses, to evaluate agreement and the level of expertise required for correct diagnoses. We demonstrated that ex vivo FCM enabled the distinction of the main inflammatory features in most cases, providing a substantial concordance to histopathologic diagnoses. Moreover, ex vivo FCM and histological evaluations reached a substantial agreement with histopathologic diagnoses both for all raters and for each operator. After a yet to be defined learning curve, these preliminary results suggest that dermatologists may be able to satisfactorily interpret ex vivo FCM images for correct real-time diagnoses. Despite some limitations mainly related to the equipment of FCM with a single objective lens, our study suggests that ex vivo FCM seems a promising tool in assisting diagnoses of cutaneous inflammatory lesions, with a level of accuracy quite close to that offered by histopathology. This is the first study to investigate ex vivo FCM application in cutaneous inflammatory lesions, and to evaluate the diagnostic capability of this technology.
2018
- Human dental pulp stem cells expressing STRO-1, c-kit and CD34 markers in peripheral nerve regeneration
[Articolo su rivista]
Carnevale, Gianluca; Pisciotta, Alessandra; Riccio, Massimo; Bertoni, Laura; DE BIASI, Sara; Gibellini, Lara; Zordani, Alessio; Cavallini, Gian Maria; LA SALA, Giovanni Battista; Bruzzesi, Giacomo; Ferrari, Adriano; Cossarizza, Andrea; DE POL, Anto
abstract
Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The application of stem cells able to differentiate in Schwann cell-like cells in vitro and in vivo, could represent an attractive therapeutic approach for the treatment of nerve injuries. Further, stem cells sources sharing the same embryological origin as Schwann cells might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal subpopulation of human STRO-1(+) /c-Kit(+) /CD34(+) DPSCs, expressing P75(NTR) , nestin and SOX-10, to differentiate into Schwann cell-like cells in vitro and to promote axonal regeneration in vivo, which led to functional recovery as measured by sustained gait improvement, in animal rat model of peripheral nerve injury. Transplanted human dental pulp stem cells (hDPSCs) engrafted into sciatic nerve defect, as revealed by the positive staining against human nuclei, showed the expression of typical Schwann cells markers, S100b and, noteworthy, a significant number of myelinated axons was detected. Moreover, hDPSCs promoted axonal regeneration from proximal to distal stumps 1 month after transplantation. This study demonstrates that STRO-1(+) /c-Kit(+) /CD34(+) hDPSCs, associated with neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues. Copyright © 2016 John Wiley & Sons, Ltd.
2018
- LonP1 Differently Modulates Mitochondrial Function and Bioenergetics of Primary Versus Metastatic Colon Cancer Cells
[Articolo su rivista]
Gibellini, L; Losi, L; De Biasi, S; Nasi, M; Lo Tartaro, D; Pecorini, S; Patergnani, S; Pinton, P; De Gaetano, A; Carnevale, G; Pisciotta, A; Mariani, F; Roncucci, L; Iannone, A; Cossarizza, A; Pinti, M.
abstract
Mitochondrial Lon protease (LonP1) is a multi-function enzyme that regulates mitochondrial functions in several human malignancies, including colorectal cancer (CRC). The mechanism(s) by which LonP1 contributes to colorectal carcinogenesis is not fully understood. We found that silencing LonP1 leads to severe mitochondrial impairment and apoptosis in colon cancer cells. Here, we investigate the role of LonP1 in mitochondrial functions, metabolism, and epithelial-mesenchymal transition (EMT) in colon tumor cells and in metastasis. LonP1 was almost absent in normal mucosa, gradually increased from aberrant crypt foci to adenoma, and was most abundant in CRC. Moreover, LonP1 was preferentially upregulated in colorectal samples with mutated p53 or nuclear β-catenin, and its overexpression led to increased levels of β-catenin and decreased levels of E-cadherin, key proteins in EMT, in vitro. LonP1 upregulation also induced opposite changes in oxidative phosphorylation, glycolysis, and pentose pathway in SW480 primary colon tumor cells when compared to SW620 metastatic colon cancer cells. In conclusion, basal LonP1 expression is essential for normal mitochondrial function, and increased LonP1 levels in SW480 and SW620 cells induce a metabolic shift toward glycolysis, leading to EMT.
2018
- Use of a 3D floating sphere culture system to maintain the neural crest-related properties of human dental pulp stem cells
[Articolo su rivista]
Pisciotta, Alessandra; Bertoni, Laura; Riccio, Massimo; Mapelli, Jonathan; Bigiani, Albertino; Noce, Marcella La; Orciani, Monia; de Pol, Anto; Carnevale, Gianluca
abstract
Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this study was to evaluate the ability of three dimensional floating spheres to preserve embryological and biological properties of this sub-population. In addition, the expression of the inwardly rectifying potassium channel Kir4.1, Fas and FasL was investigated in 3D-sphere derived hDPSCs. Our data showed that 3D sphere-derived hDPSCs maintained their fibroblast-like morphology, preserved stemness markers expression and proliferative capability. The expression of neural crest markers and Kir4.1 was observed in undifferentiated hDPSCs, furthermore this culture system also preserved hDPSCs differentiation potential. The expression of Fas and FasL was observed in undifferentiated hDPSCs derived from sphere culture and, noteworthy, FasL was maintained even after the neurogenic commitment was reached, with a significantly higher expression compared to osteogenic and myogenic commitments. These data demonstrate that 3D sphere culture provides a favorable micro-environment for neural crest-derived hDPSCs to preserve their biological properties.
2017
- Activation of Fas/FasL pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells
[Articolo su rivista]
CARNEVALE, Gianluca; Carpino, Guido; Cardinale, Vincenzo; PISCIOTTA, ALESSANDRA; RICCIO, Massimo; Bertoni, Laura; GIBELLINI, Lara; DE BIASI, SARA; Nevi, Lorenzo; Costantini, Daniele; Overi, Diletta; COSSARIZZA, Andrea; DE POL, Anto; Gaudio, Eugenio; Alvaro, Domenico
abstract
Intrahepatic cholangiocarcinoma (iCCA) represents a heterogeneous group of malignancies emerging from the biliary tree, often in the context of chronic bile ducts inflammation. The immunological features of iCCA cells and their capability to control the lymphocytes response have not yet been investigated. The aims of the present study were to evaluate the interaction between iCCA cells and human peripheral blood mononuclear cells (PBMCs) and the role of Fas/FasL in modulating T-cells and NK-cells response after direct co-culture. iCCA cells express high levels of Fas and FasL that increase after co-culture with PBMCs inducing apoptosis in CD4(+), CD8(+) T-cells and in CD56(+) NK-cells. In vitro, c-FLIP is expressed in iCCA cells and the co-culture with PBMCs induces an increase of c-FLIP in both iCCA cells and biliary tree stem cells. This c-FLIP increase does not trigger the caspase cascade, thus hindering apoptotis of iCCA cells which, instead, underwent proliferation. The increased expression of Fas, FasL and c-FLIP is confirmed in situ, in human CCA and in primary sclerosing cholangitis. In conclusion our data indicated that iCCA cells have immune-modulatory properties by which they induce apoptosis of T and NK cells, via Fas/FasL pathway, and escape inflammatory response by up-regulating c-FLIP system.
2017
- Corrigendum to: Osteogenic differentiation of hDPSCs on biogenic bone apatite thin films (Stem Cells International (2017) 2017 (3579283) DOI: 10.1155/2017/3579283)
[Articolo su rivista]
Bianchi, Michele; Pisciotta, Alessandra; Bertoni, Laura; Berni, Matteo; Gambardella, Alessandro; Visani, Andrea; Russo, Alessandro; DE POL, Anto; Carnevale, Gianluca
abstract
In the article titled "Osteogenic differentiation of hDPSCs on biogenic bone apatite thin films" [1], the second affiliation was incorrect. The corrected affiliations are shown above.
2017
- Osteogenic Differentiation of hDPSCs on Biogenic Bone Apatite Thin Films
[Articolo su rivista]
Bianchi, Michele; Pisciotta, Alessandra; Bertoni, Laura; Berni, Matteo; Gambardella, Alessandro; Visani, Andrea; Russo, Alessandro; DE POL, Anto; Carnevale, Gianluca
abstract
A previous study reported the structural characterization of biogenic apatite (BAp) thin films realized by a pulsed electron deposition system by ablation of deproteinized bovine bone. Thin films annealed at 400 degrees C exhibited composition and crystallinity degree very close to those of biogenic apatite; this affinity is crucial for obtaining faster osseointegration compared to conventional, thick hydroxyapatite (HA) coatings, for both orthopedics and dentistry. Here, we investigated the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (hDPCS) on as-deposited and heat-treated BAp and stoichiometric HA. First, we showed that heat-treated BAp films can significantly promote hDPSC adhesion and proliferation. Moreover, hDPSCs, while initially maintaining the typical fibroblast-like morphology and stemness surface markers, later started expressing osteogenic markers such as Runx-2 and OSX. Noteworthy, when cultured in an osteogenic medium on annealed BAp films, hDPSCs were also able to reach a more mature and terminal commitment, with respect to HA and as-deposited films. Our findings suggest that annealed BAp films not only preserve the typical biological properties of stemness of, hDPSCs but also improve their ability of osteogenic commitment.
2016
- Optimized Cryopreservation and Banking of Human Bone-Marrow Fragments and Stem Cells
[Articolo su rivista]
Carnevale, Gianluca; Pisciotta, Alessandra; Riccio, Massimo; De Biasi, Sara; Gibellini, Lara; Ferrari, Adriano; La Sala, Giovanni Battista; Bruzzesi, Giacomo; Cossarizza, Andrea; De Pol, Anto
abstract
Adult mesenchymal stem cells are a promising source for cell therapies and tissue engineering applications. Current procedures for banking of human bone-marrow mesenchymal stem cells (hBM-MSCs) require cell isolation and expansion, and thus the use of large amounts of animal sera. However, animal-derived culture supplements have the potential to trigger infections and severe immune reactions. The aim of this study was to investigate an optimized method for cryopreservation of human bone-marrow fragments for application in cell banking procedures where stem-cell expansion and use are not immediately needed. Whole trabecular fragments enclosing the bone marrow were stored in liquid nitrogen for 1 year in a cryoprotective solution containing a low concentration of dimethyl sulfoxide and a high concentration of human serum (HuS). After thawing, the isolation, colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, cell senescence, apoptosis, and multi-lineage differentiation potential of hBM-MSCs were tested in media containing HuS compared with hBM-MSCs isolated from fresh fragments. Human BM-MSCs isolated from cryopreserved fragments expressed MSC markers until later passages, had a good proliferation rate, and exhibited the capacity to differentiate toward osteogenic, adipogenic, and myogenic lineages similar to hBM-MSCs isolated from fresh fragments. Moreover, the cryopreservation method did not induce cell senescence or cell death. These results imply that minimal processing may be adequate for the banking of tissue samples with no requirement for the immediate isolation and use of hBM-MSCs, thus limiting cost and the risk of contamination, and facilitating banking for clinical use. Furthermore, the use of HuS for cryopreservation and expansion/differentiation has the potential for clinical application in compliance with good manufacturing practice standards.
2015
- Different origin of adipogenic stem cells influences the response to antiretroviral drugs
[Articolo su rivista]
Gibellini, Lara; DE BIASI, Sara; Nasi, Milena; Carnevale, Gianluca; Pisciotta, Alessandra; Bianchini, Elena; Bartolomeo, Regina; Polo, Miriam; DE POL, Anto; Pinti, Marcello; Cossarizza, Andrea
abstract
Lipodystrophy (LD) is a main side effect of antiretroviral therapy for HIV infection, and can be provoked by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). LD exists in different forms, characterized by fat loss, accumulation, or both, but its pathogenesis is still unclear. In particular, few data exist concerning the effects of antiretroviral drugs on adipocyte differentiation. Adipose tissue can arise either from mesenchymal stem cells (MSCs), that include bone marrow-derived MSCs (hBM-MSCs), or from ectodermal stem cells, that include dental pulp stem cells (hDPSCs). To analyze whether the embryonal origin of adipocytes might impact the occurrence of different phenotypes in LD, we quantified the effects of several antiretroviral drugs on the adipogenic differentiation of hBM-MSCs and hDPSCs. hBM-MSCs and hDPSCs were isolated from healthy donors. Cells were treated with 10 and 50μM stavudine (d4T), efavirenz (EFV), atazanavir (ATV), ritonavir (RTV), and ATV-boosted RTV. Viability and adipogenesis were evaluated by staining with propidium iodide, oil red, and adipoRed; mRNA levels of genes involved in adipocyte differentiation, i.e. CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ), and in adipocyte functions, i.e. fatty acid synthase (FASN), fatty acid binding protein-4 (FABP4), perilipin-1 (PLIN1) and 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2), were quantified by real time PCR. We found that ATV, RTV, EFV, and ATV-boosted RTV, but not d4T, caused massive cell death in both cell types. EFV and d4T affected the accumulation of lipid droplets and induced changes in mRNA levels of genes involved in adipocyte functions in hBM-MSCs, while RTV and ATV had little effects. All drugs stimulated the accumulation of lipid droplets in hDPSCs. Thus, the adipogenic differentiation of human stem cells can be influenced by antiretroviral drugs, and depends, at least in part, on their embryonal origin.
2015
- Human dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations
[Articolo su rivista]
Pisciotta, Alessandra; Carnevale, Gianluca; Meloni, Simona; Riccio, Massimo; De Biasi, Sara; Gibellini, Lara; Ferrari, Adriano; Bruzzesi, Giacomo; De Pol, Anto
abstract
Human dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency.
2015
- Neural crest derived niche of human dental pulp stem cells promotes peripheral nerve regeneration and remyelination in animal model of critical sized sciatic nerve injury
[Articolo su rivista]
Carnevale, Gianluca; Pisciotta, Alessandra; DE BIASI, Sara; Gibellini, Lara; Cossarizza, Andrea; Bruzzesi, Giacomo; Ferrari, Adriano; DE POL, Anto
abstract
ABSTRACT
Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The use of stem cells, easily accessible, capable of rapid expansion in culture as well as fully integrate into the host tissue and capable to differentiate in myelinating cells of the peripheral nervous system, represent an attractive therapeutic approach for the treatment of nerve injuries. Farther, stem cells sources sharing the same embryological origin of Schwann cells, might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal sub-population of STRO-1+/c-Kit+/CD34+ hDPSCs (1, 2), most of which being positive for neural crest (P75NTR) and neural progenitor cells (nestin) markers, to differentiate into Schwann cells-like cells in vitro and to promote axonal regeneration in vivo. As a matter of fact, following culture in appropriate induction medium, STRO-1+/c-Kit+/CD34+ hDPSCs were able to commit towards Schwann cells express- ing P75NTR, GFAP and S100b. After transplantation in animal model of sciatic nerve defect, hDPSCs promoted axonal regeneration from proximal to distal stumps, providing guidance to newly formed myelinated nerve fibers, which led to functional recovery as measured by sustained gait improvement. Particularly, transplanted hDP- SCs engrafted into critical sized sciatic nerve defect, as revealed by the positive stain- ing against human nuclei, showed the expression of typical Schwann cells markers, S100b and GFAP. In conclusion this study demonstrates that STRO-1+/c-Kit+/CD34+ hDPSCs, associated to neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues.
2015
- Stem cells isolated from human dental pulp and amniotic fluid improve skeletal muscle histopathology in mdx/SCID mice
[Articolo su rivista]
Pisciotta, Alessandra; Riccio, Massimo; Carnevale, Gianluca; Lu, Aiping; DE BIASI, Sara; Gibellini, Lara; LA SALA, Giovanni Battista; Bruzzesi, Giacomo; Ferrari, Adriano; Huard, Johnny; DE POL, Anto
abstract
Introduction: Duchenne muscular dystrophy (DMD), caused by a lack of the functional structural protein dystrophin, leads to severe muscle degeneration where the patients are typically wheelchair-bound and die in their mid-twenties from cardiac or respiratory failure or both. The aim of this study was to investigate the potential of human dental pulp stem cells (hDPSCs) and human amniotic fluid stem cells (hAFSCs) to differentiate toward a skeletal myogenic lineage using several different protocols in order to determine the optimal conditions for achieving myogenic commitment and to subsequently evaluate their contribution in the improvement of the pathological features associated with dystrophic skeletal muscle when intramuscularly injected into mdx/SCID mice, an immune-compromised animal model of DMD. Methods: Human DPSCs and AFSCs were differentiated toward myogenic lineage in vitro through the direct co-culture with a myogenic cell line (C2C12 cells) and through a preliminary demethylation treatment with 5-Aza-2′-deoxycytidine (5-Aza), respectively. The commitment and differentiation of both hDPSCs and hAFSCs were evaluated by immunofluorescence and Western blot analysis. Subsequently, hDPSCs and hAFSCs, preliminarily demethylated and pre-differentiated toward a myogenic lineage for 2 weeks, were injected into the dystrophic gastrocnemius muscles of mdx/SCID mice. After 1, 2, and 4 weeks, the gastrocnemius muscles were taken for immunofluorescence and histological analyses. Results: Both populations of cells engrafted within the host muscle of mdx/SCID mice and through a paracrine effect promoted angiogenesis and reduced fibrosis, which eventually led to an improvement of the histopathology of the dystrophic muscle. Conclusion: This study shows that hAFSCs and hDPSCs represent potential sources of stem cells for translational strategies to improve the histopathology and potentially alleviate the muscle weakness in patients with DMD.
2014
- HUMAN BILIARY TREE STEM/PROGENITOR CELLS (hbTSCS) FROM PERIBILIARY GLANDS (PBGS) OF ADULT LIVER DISPLAY IMMUNOMODULATORY PROPERTIES THROUGH Fas/Fas LIGAND INDUCED T-CELL LYMPHOCYTE APOPTOSIS
[Abstract in Rivista]
Carnevale, G; Riccio, M; Cardinale, V; Gibelini, L; De Biasi, S; Pisciotta, A; Carpino, G; Gentile, R; Berloco, Pb; Brunelli, R; Bastianelli, C; Cossarizza, A; Gaudio, E; Alvaro, D; De Pol, A
abstract
Background and Aims: hBTSCs have the potential for regenerative
medicine in liver and pancreas diseases. T-cell control was recently
demonstrated for mesenchymal stem cells. The aims of this study
were to evaluate Fas-L expression within the stem cell niches of
adult biliary tree (PBGs), and to study the interaction between
hBTSCs and human lymphocytes.
Methods: HLA antigens, Fas and Fas-L expression were evaluated by
immunofluorescence and western blotting (WB) in cells of human
biliary tree in comparison with fibroblast cells, dental pulp stem
cells and bone marrow mesenchymal stem cells. The influence of
hBTSCs on lymphocytes’ activation and apoptosis were assessed by
co-culturing experiments.
Results: Adult hBTSCs expressed both class I and class
II HLA antigens, whereas fetal hBTSCs only class I HLA
antigens. 10 to 30% of the hBTSCs in PBGs were positive
for Fas-L. Fas-L+ cells were mostly located at the bottom
of PBGs and co-expressed EpCAM (Epithelial-Cell-AdhesionMolecule)
and proliferation marker (PCNA:Proliferating-CellNuclear-Antigen).
Mature cells at the bile duct surface epithelium
(mature cholangiocytes) were almost all negative for Fas-L. In
culture experiments confocal microscopy demonstrated that Fas-L
expression was restricted to EpCAM+/LGR5+ (a marker associated
with endodermal stem cells) hBTSCs. WB confirmed that hBTSCs
constitutively expressed high level of Fas-L which increased after
co-culture with T-cells. FACS analysis reveled that activated CD4+
and CD8+ T-cells co-cultured with hBTSCs underwent to a massiveinduction of apoptosis. Fas receptor appeared over-expressed in
T-cells co-cultured with hBTSCs respect to resting T-cells.
Conclusions: Our data demonstrated that hBTSCs can induce
“premature” apoptosis in T-cells trough the activation of Fas/Fas-L
pathway
2014
- Human biliary tree stem/progenitor cells (hBTSCs) from peribiliary glands (PBGs) of adult liver display immunomodulatory properties through Fas/Fas ligand induced T-cell lymphocyte apoptosis
[Abstract in Rivista]
Carnevale, G.; Riccio, M.; Cardinale, V.; Gibelini, L.; De Biasi, S.; Pisciotta, A.; Carpino, G.; Gentile, R.; Berloco, P. B.; Brunelli, R.; Bastianelli, C.; Cossarizza, A.; Gaudio, E.; Alvaro, D.; De Pol, A.
abstract
Background and aim: hBTSCs have been retrieved in peribiliary glands
(PBGs) of adult and fetal biliary tree, and have the potential for regenerative
medicine in liver, biliary tree, and pancreas diseases. The ability of stem cells
to control T-cells’ immune responses was recently demonstrated by human
mesenchymal stem cells. The aims of the present study were to evaluate Fas-L
expression within the stem cell niches of adult biliary tree, and to study the in
vitro interaction between hBTSCs and human lymphocytes.
Material and methods: HLA antigens, Fas and Fas-L expression were evaluated
in situ and in vitro by immunofluorescence and Western blots in cells
of the human biliary tree in comparison with fibroblast cells, dental pulp
stem cells and bone marrow mesechymal stem cells. Co-cultures of hBTSCs
with human leucocytes were used to analyze the influence of hBTSCs on
lymphocytes’ activation and apoptosis.
Results: Adult hBTSCs expressed both class I and class II HLA antigens,
whereas fetal hBTSCs had class I HLA antigens only. In PBG niche 10-30%
BTSCs were positive for Fas-L. Fas-L positive cells were mostly located at the
bottom of PBGs and co-expressed EpCAM (epithelial cell adhesion molecule)
and a marker of proliferation (PCNA: Proliferating Cell Nuclear Antigen).
Conversely, mature cells at the surface epithelium and cholangiocytes of large
intrahepatic ducts were almost all negative for Fas-L. In culture experiments
confocal microscopy demonstrated that Fas-L expression was restricted to
EpCAM+/LGR5+(a marker associated with endodermal stem cells) cells.
Western blot data confirmed that hBTSCs constitutively expressed high level
of Fas-L that increased after co-culture with T-cells. FACS analysis of T-cells
co-cultured with hBTSCs indicated that hBTSCs were able to induce apoptosis
in activated CD4+ and CD8+ T-cell populations. Moreover, Fas receptor
appears to be more expressed in T-cells co-cultured with hBTSCs than in
resting T-cells.
Conclusions: In conclusion our data suggest that hBTSCs could modulate
the T-cells response through the production of Fas-L, which influences the
lymphocyte Fas/Fas-L pathway by inducing “premature” apoptosis in CD4+
and CD8+ T-cells.
2014
- The Fas/Fas ligand apoptosis pathway underlies immunomodulatory properties of human biliary tree stem/progenitor cells
[Articolo su rivista]
Riccio, Massimo; Carnevale, Gianluca; Cardinale, Vincenzo; Gibellini, Lara; DE BIASI, Sara; Pisciotta, Alessandra; Carpino, Guido; Gentile, Raffaele; Berloco, Pasquale B; Brunelli, Roberto; Bastianelli, Carlo; Napoletano, Chiara; Cantafora, Alfredo; Cossarizza, Andrea; Gaudio, Eugenio; Alvaro, Domenico; DE POL, Anto
abstract
Human biliary tree stem/progenitor cells (hBTSCs) are multipotent epithelial stem cells, easily obtained from the biliary tree, with the potential for regenerative medicine in liver, biliary tree, and pancreas diseases. Recent reports indicate that human mesenchymal stem cells are able to modulate the T cell immune response. However, no information exists on the capabilities of hBTSCs to control the allogeneic response. The aims of this study were to evaluate FasL expression in hBTSCs, to study the in vitro interaction between hBTSCs and human lymphocytes, and the role of Fas/FasL modulation in inducing T cell apoptosis in hBTSCs/T cell co-cultures.
2013
- Enrichment in c-Kit(+) enhances mesodermal and neural differentiation of human chorionic placental cells
[Articolo su rivista]
Resca, Elisa; Zavatti, Manuela; Bertoni, Laura; Maraldi, Tullia; DE BIASI, Sara; Pisciotta, Alessandra; A., Nicoli; LA SALA, Giovanni Battista; P. V., Guillot; A. L., David; N. J., Sebire; P. D., Coppi; DE POL, Anto
abstract
OBJECTIVE: Human term placenta (HTP) has attracted increasing attention as an alternative source of stem cells for regenerative medicine since the amniochorionic membrane harbors stem cells populations that are easily accessible, abundantly available without ethical objections. In the chorionic side of HTP we found a progenitor perivascular "niche" in which rare cells co-express Oct-4 and c-Kit. We investigated the stem cell characteristics and differentiation potential of a chorionic derived population enriched in c-Kit(+) cells and compared this to the unenriched population. STUDY DESIGN: Cells, isolated from the chorion of HTP, were expanded and enriched in c-Kit(+) cells (Chorionic Stem Cells-CSC). Histological staining, immunofluorescence, Western blot and flow cytometry were used to verify the stem cells characteristics of the populations and to compare the differentiation capability towards mesodermal and neural lineages in vitro. RESULTS: The expression of the pluripotent marker Oct-4 was greater in the CSCs compared to the unselected cells (Chorionic Cell-CC) but both Oct-4 and c-Kit expression decreased during passages. After differentiation, CSC displayed stronger chondrogenic and osteogenic potential and a greater adipogenic forming capacity compared to unselected ones. CSC differentiated better into immature oligodendrocytes while CC showed a neuronal progenitor differentiation potential. Moreover, both populations were able to differentiate in hepatogenic lineage. CONCLUSION: CSC display improved Oct-4 expression and a high differentiation potential into mesodermal lineages and oligodendrocytes.
2013
- Human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization.
[Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Pisciotta, Alessandra; Zavatti, Manuela; Carnevale, Gianluca; Beretti, Francesca; LA SALA, Giovanni Battista; A., Motta; DE POL, Anto
abstract
INTRODUCTION: The main aim of this study is to evaluate potential human stem cells, such as dental pulp stem cells (DPSC) and amniotic fluid stem cells (AFSC), combined with collagen scaffold, to reconstruct critical size cranial bone defects in animal model. METHODS: We performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. After 4 and 8 weeks cranial tissue samples were taken for histological and immunofluorescence analysis. RESULTS: We observed a new bone formation in all the samples but the most relevant difference in defect correction were shown by stem cell-collagen samples at 4 weeks after implant, suggesting a faster regeneration ability of the combined constructs. The presence of human cells in the newly-formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Furthermore, human cells were found to be an essential part of new vessel formation in the scaffold. CONCLUSIONS: All these data confirmed the strong potential of bioengineered constructs of stem cell-collagen scaffold for correcting large cranial defects in animal model and highlighting the role of stem cells in neo vascularization during skeletal defect reconstruction.
2013
- In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp.
[Articolo su rivista]
Carnevale, Gianluca; Riccio, Massimo; Pisciotta, Alessandra; Beretti, Francesca; Maraldi, Tullia; Zavatti, Manuela; Cavallini, Gian Maria; LA SALA, Giovanni Battista; Ferrari, Adriano; DE POL, Anto
abstract
AIM: To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS: Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic β-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS: Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION: The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration.
2012
- Fibroin scaffold repairs critical-size bone defects in vivo supported by human amniotic fluid and dental pulp stem cells.
[Articolo su rivista]
Riccio, Massimo; Maraldi, Tullia; Pisciotta, Alessandra; LA SALA, Giovanni Battista; Ferrari, Adriano; G., Bruzzesi; A., Motta; C., Migliaresi; DE POL, Anto
abstract
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.
2012
- Human serum promotes osteogenic differentiation of Human Dental Pulp Stem Cells in vitro and in vivo.
[Articolo su rivista]
Pisciotta, Alessandra; Riccio, Massimo; Carnevale, Gianluca; Beretti, Francesca; Gibellini, Lara; Maraldi, Tullia; Cavallini, Gian Maria; Ferrari, Adriano; Bruzzesi, G; DE POL, Anto
abstract
Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.
2011
- Human amniotic fluid stem cells seeded in fibroin scaffold produce in vivo mineralized matrix.
[Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Resca, Elisa; Pisciotta, Alessandra; LA SALA, Giovanni Battista; Ferrari, Adriano; Bruzzesi, G; Motta, A; Migliaresi, C; Marzona, Laura; DE POL, Anto
abstract
This study investigated the potential of amniotic fluid stem cells (AFSCs) to synthesize mineralized extracellular matrix (ECM) within different porous scaffolds of collagen, poly-D,L-lactic acid (PDLLA), and silk fibroin. The AFSCs were initially differentiated by using an osteogenic medium in two-dimensional culture, and expression of specific bone proteins and the physiologic mineral production by the AFSCs were analyzed. In particular, during differentiation process, AFSCs expressed proteins like Runt-related transcription factor 2 (Runx2), Osterix, Osteopontin, and Osteocalcin with a sequential expression, analogous to those occurring during osteoblast differentiation, and produced extracellular calcium stores. AFSCs were then cultured on three-dimensional (3D) scaffolds and evaluated for their ability to differentiate into osteoblastic cells in vivo. Stem cells were cultured in vitro for 1 week in collagen, fibroin, and PDLLA scaffolds. The effect of predifferentiation of the stem cells in scaffolds on the subsequent bone formation in vivo was determined in a rat subcutaneous model. With the addition of a third dimension, osteogenic differentiation and mineralized ECM production by AFSCs were significantly higher. This study demonstrated the strong potential of AFSCs to produce 3D mineralized bioengineered constructs in vivo and suggests that fibroin may be an effective scaffold material for functional repair of critical size bone defects.
2010
- Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures
[Articolo su rivista]
Riccio, Massimo; Resca, Elisa; Maraldi, Tullia; Pisciotta, Alessandra; Ferrari, Adriano; Bruzzesi, G; DE POL, Anto
abstract
The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During the differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, MatrigelTM and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in MatrigelTM DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.