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Laura BERTONI

Professore Associato
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

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Pubblicazioni

2023 - Artificial intelligence evaluation of confocal microscope prostate images: our preliminary experience [Articolo su rivista]
Bianchi, G.; Puliatti, S.; Rodriguez, N.; Micali, S.; Bertoni, L.; Reggiani Bonetti, L.; Caramaschi, S.; Bolelli, F.; Pinamonti, M.; Rozze, D.; Grana, C.
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 - Eczema: Features on FCM, Digital H&E, and Corresponding Conventional H&E [Capitolo/Saggio]
Bertoni, L.; Reggiani, C.; Azzoni, P.; Reggiani, Bonetti; Pellacani, G
abstract

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

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2022 - Features of Lichen Planus and Psoriasis on Ex Vivo Confocal Imaging and Histopathologic Correlation [Capitolo/Saggio]
Bertoni, L.; Reggiani, C.; Azzoni, P.; Bonetti, L. R.; Pellacani, G
abstract

2022 - Fluorescence Confocal Microscope for Identification of Discoid Lupus Erythematosus Histologic Features [Capitolo/Saggio]
Bertoni, L.; Reggiani, C.; Azzoni, P.; Caramaschi, S.; Bonetti, L. R.; Pellacani, G
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

2021 - Clinical applications of in vivo and ex vivo confocal microscopy [Articolo su rivista]
Guida, S.; Arginelli, F.; Farnetani, F.; Ciardo, S.; Bertoni, L.; Manfredini, M.; Zerbinati, N.; Longo, C.; Pellacani, G.
abstract

Confocal laser scanning microscopy (CLSM) has been introduced in clinical settings as a tool enabling a quasi-histologic view of a given tissue, without performing a biopsy. It has been applied to many fields of medicine mainly to the skin and to the analysis of skin cancers for both in vivo and ex vivo CLSM. In vivo CLSM involves reflectance mode, which is based on refractive index of cell structures serving as endogenous chromophores, reaching a depth of exploration of 200 µm. It has been proven to increase the diagnostic accuracy of skin cancers, both melanoma and nonmelanoma. While histopathologic examination is the gold standard for diagnosis, in vivo CLSM alone and in addition to dermoscopy, contributes to the reduction of the number of excised lesions to exclude a melanoma, and to improve margin recognition in lentigo maligna, enabling tissue sparing for excisions. Ex vivo CLSM can be performed in reflectance and fluorescent mode. Fluorescence confocal microscopy is applied for “real-time” pathological examination of freshly excised specimens for diagnostic purposes and for the evaluation of margin clearance after excision in Mohs surgery. Further prospective interventional studies using CLSM might contribute to increase the knowledge about its application, reproducing real-life settings.

2021 - Current and future perspectives of digital microscopy with fluorescence confocal microscope for prostate tissue interpretation: A narrative review [Articolo su rivista]
Rocco, B.; Cimadamore, A.; Sarchi, L.; Bonetti, L. R.; Bertoni, L.; Azzoni, P.; Assumma, S.; Turri, F.; Bozzini, G.; Eissa, A.; Micali, S.; Bianchi, G.; Maiorana, A.; Montironi, R.; Pellacani, G.; Sighinolfi, M. C.
abstract

Fluorescence confocal microscopy (FCM) is an optical imaging technique providing digital microscopical images of fresh tissue in a real time fashion, without conventional processing. FCM has been widely applied in several fields of dermatology, including the detection of basal cell carcinoma and of cutaneous inflammatory diseases. The aim of the paper is to provide an overview of FCM applications in the field of prostate tissue interpretation and prostate cancer (PCa) detection. A Literature search (PubMed & Web of Science) was performed to identify articles concerned with the clinical and surgical applications of FCM in prostatic and periprostatic tissues interpretation. Overall, six articles were identified. All articles investigated the level of agreement between FCM and conventional histopathological analysis (hematoxylin-eosin, HE) for the discrimination between normal and PCa tissues. An investigative article on prostate samples retrieved from radical prostatectomy (RP) specimens and an atlas of FCM digital images from the same series were found. Two prospective clinical trials, comparing FCM and HE, pointed out a “substantial” to “almost perfect” discriminative performance of FCM for the diagnosis of PCa on prostate biopsy core. Finally, two studies investigated the intra-operative role of FCM during RP for the control of surgical dissection. In this setting, FCM could be used to analyse samples retrieved from suspicious peri-prostatic areas; FCM has also been tested for an en-face evaluation of flat slices obtained from the systematic sampling of the posterolateral aspects of the prostate, in a NeuroSAFE-like approach. Generally, FCM provides digital microscopical images of fresh tissue in a real time fashion, without requiring conventional processing. Currently, available studies confirmed a high concordance with conventional pathology for the detection of PCa. Further studies are required to validate the technology, to evaluate ISUP score attribution and to implement the fields of application of FCM for the treatment of prostate diseases.

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 - GD2 CAR T cells against human glioblastoma [Articolo su rivista]
Prapa, M.; Chiavelli, C.; Golinelli, G.; Grisendi, G.; Bestagno, M.; Di Tinco, R.; Dall'Ora, M.; Neri, G.; Candini, O.; Spano, C.; Petrachi, T.; Bertoni, L.; Carnevale, G.; Pugliese, G.; Depenni, R.; Feletti, A.; Iaccarino, C.; Pavesi, G.; Dominici, M.
abstract

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF β1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer.

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 - An intraoperative study with ex vivo fluorescence confocal microscopy: diagnostic accuracy of the three visualization modalities [Articolo su rivista]
Reggiani, C.; Pellacani, G.; Reggiani Bonetti, L.; Zanelli, G.; Azzoni, P.; Chester, J.; Kaleci, S.; Ferrari, B.; Bellini, P.; Longo, C.; Bertoni, L.; Magnoni, C.
abstract

Ex-vivo fluorescence confocal microscopy (FCM) is an imaging technique that captures real time digital images of freshly excised tissue with quasi histopathological resolution. FCM reduces time to image acquisition compared to hematoxylin and eosin (H&E) frozen section analysis.

2020 - Digital Biopsy with Fluorescence Confocal Microscope for Effective Real-time Diagnosis of Prostate Cancer: A Prospective, Comparative Study [Articolo su rivista]
Rocco, Bernardo; Sighinolfi, Maria Chiara; Sandri, Marco; Spandri, Valentina; Cimadamore, Alessia; Volavsek, Metka; Mazzucchelli, Roberta; Lopez-Beltran, Antonio; Eissa, Ahmed; Bertoni, Laura; Azzoni, Paola; Reggiani Bonetti, Luca; Maiorana, Antonino; Puliatti, Stefano; Micali, Salvatore; Paterlini, Maurizio; Iseppi, Andrea; Rocco, Francesco; Pellacani, Giovanni; Chester, Johanna; Bianchi, Giampaolo; Montironi, Rodolfo
abstract

A microscopic analysis of tissue is the gold standard for cancer detection. Hematoxylin-eosin (HE) for the reporting of prostate biopsy (PB) is conventionally based on fixation, processing, acquisition of glass slides, and analysis with an analog microscope by a local pathologist. Digitalization and real-time remote access to images could enhance the reporting process, and form the basis of artificial intelligence and machine learning. Fluorescence confocal microscopy (FCM), a novel optical technology, enables immediate digital image acquisition in an almost HE-like resolution without requiring conventional processing.

2020 - Digital frozen section of the prostate surface during radical prostatectomy: a novel approach to evaluate surgical margins [Articolo su rivista]
Rocco, B.; Sighinolfi, M. C.; Cimadamore, A.; Reggiani Bonetti, L.; Bertoni, L.; Puliatti, S.; Eissa, A.; Spandri, V.; Azzoni, P.; Dinneen, E.; Shaw, G.; Nathan, S.; Micali, S.; Bianchi, G.; Maiorana, A.; Pellacani, G.; Montironi, R.
abstract

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 - Ex vivo fluorescence confocal microscopy: prostatic and periprostatic tissues atlas and evaluation of the learning curve [Articolo su rivista]
Bertoni, L.; Puliatti, S.; Reggiani Bonetti, L.; Maiorana, A.; Eissa, A.; Azzoni, P.; Bevilacqua, L.; Spandri, V.; Kaleci, S.; Zoeir, A.; Sighinolfi, M. C.; Micali, S.; Bianchi, G.; Pellacani, G.; Rocco, B.; Montironi, R.
abstract

Ex vivo fluorescence confocal microscopy (FCM) is an optical technology that provides fast H&E-like images of freshly excised tissues, and it has been mainly used for “real-time” pathological examination of dermatological malignancies. It has also shown to be a promising tool for fast pathological examination of prostatic tissues. We aim to create an atlas for FCM images of prostatic and periprostatic tissues to facilitate the interpretation of these images. Furthermore, we aimed to evaluate the learning curve of images interpretation of this new technology. Eighty fresh and unprepared biopsies obtained from radical prostatectomy specimens were evaluated using the FCM VivaScope® 2500 M-G4 (Mavig GmbH, Munich, Germany; Caliber I.D.; Rochester NY, USA) by two pathologists. Images of FCM with the corresponding H&E are illustrated to create the atlas. Furthermore, the two pathologists were asked to re-evaluate the 80 specimens after 90 days interval in order to assess the learning curve of images’ interpretation of FCM. FCM was able to differentiate between different types of prostatic and periprostatic tissues including benign prostatic glands, benign prostatic hyperplasia, high-grade intraepithelial neoplasm, and prostatic adenocarcinoma. As regards the learning curve, FCM demonstrated a short learning curve. We created an atlas that can serve as the base for urologists and pathologists for learning and interpreting FCM images of prostatic and periprostatic tissues. Furthermore, FCM images is easily interpretable; however, further studies are required to explore the potential applications of this new technology in prostate cancer diagnosis and management.

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.

2020 - Positive surgical margin during radical prostatectomy: overview of sampling methods for frozen sections and techniques for the secondary resection of the neurovascular bundles [Articolo su rivista]
Sighinolfi, M. C.; Eissa, A.; Spandri, V.; Puliatti, S.; Micali, S.; Reggiani Bonetti, L.; Bertoni, L.; Bianchi, G.; Rocco, B.
abstract

Objective: The aim of the paper is to provide an overview of intraoperative sampling methods for frozen section (FS) analysis and of surgical techniques for a secondary neurovascular bundle (NVB) resection, as the method of surgical margin (SM) sampling and the management of a positive SM (PSM) at the nerve-sparing (NS) area are under evaluated issues. FS analysis during radical prostatectomy (RP) can help to tailor the plane of dissection based on cancer extension and thus extend the indications for NS surgery. Evidence Acquisition: We performed a PubMed/Medical Literature Analysis and Retrieval System Online (MEDLINE), Web of Science, Cochrane Library, and Elton B. Stephens Co. (EBSCO)host search to include articles published in the last decade, evaluating FS analysis in the NS area and surgical attempts to convert a PSM to a negative status. Evidence Synthesis: Overall, 19 papers met our inclusion criteria. The ways to collect samples for FS analysis included: systematic (analysing the whole posterolateral aspect of the prostate specimen, i.e., neurovascular structure-adjacent frozen-section examination [NeuroSAFE]); magnetic resonance imaging (MRI)-guided (biopsies from MRI-suspicious areas, retrieved by the surgeon in a cognitive way); and random biopsies from the soft periprostatic tissues. Techniques to address a PSM in the NS area included: full resection of the spared NVB, from its caudal to cranial aspect, often including the rectolateral part of the Denonvilliers’ fascia; partial resection of the NVB, in cases where sampling attempts to localise a PSM; incremental approach, meaning a partial or full resection that extends until no prostate tissue is found in the soft periprostatic environment. Conclusions: There is no homogeneity in prostate sampling for FS analysis, although most recent evidence is moving toward a systematic sampling of the entire NS area. The management of a PSM is variable and can be affected by the sampling strategy (difficult localisation of the persisting tumour at the NVB). The difficult identification of the exact soft tissue location contiguous to a PSM could be considered as the critical point of FS analysis and of spared-NVB management.

2020 - Real-time assessment of surgical margins during radical prostatectomy: a novel approach that uses fluorescence confocal microscopy for the evaluation of peri-prostatic soft tissue [Articolo su rivista]
Rocco, B.; Sighinolfi, M. C.; Bertoni, L.; Spandri, V.; Puliatti, S.; Eissa, A.; Reggiani Bonetti, L.; Azzoni, P.; Sandri, M.; De Carne, C.; Turri, F.; Cimadamore, A.; Montironi, R.; Maiorana, A.; Micali, S.; Bianchi, G.; Pellacani, G.
abstract

2020 - “Real-time” Assessment of Surgical Margins During Radical Prostatectomy: State-of-the-Art [Articolo su rivista]
Eissa, A.; Zoeir, A.; Sighinolfi, M. C.; Puliatti, S.; Bevilacqua, L.; Del Prete, C.; Bertoni, L.; Azzoni, P.; Reggiani Bonetti, L.; Micali, S.; Bianchi, G.; Rocco, B.
abstract

Histopathologic examination of the pathologic specimens using hematoxylin & eosin stains represents the backbone of the modern pathology. It is time-consuming; thus, “real-time” assessment of prostatic and periprostatic tissue has gained special interest in the diagnosis and management of prostate cancer. The current study focuses on the review of the different available techniques for “real-time” evaluation of surgical margins during radical prostatectomy (RP). We performed a comprehensive search of the Medline database to identify all the articles discussing “real-time” or intraoperative assessment of surgical margins during RP. Several filters were applied to the search to include only English articles performed on human subjects and published between January 2000 and March 2019. The search revealed several options for pathologic assessment of surgical margins including intraoperative frozen sections, confocal laser endomicroscopy, optical spectroscopy, photodynamic diagnosis, optical coherence tomography, multiphoton microscopy, structured illumination microscopy, 3D augmented reality, and ex vivo fluorescence confocal microscope. Frozen section represents the gold standard technique for real-time pathologic examinations of surgical margins during RP; however, several other options showed promising results in the initial clinical trials, and considering the rapid development in the field of molecular and cellular imaging, some of these options may serve as an alternative to frozen section.

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 - Ex-vivo Fluorescence Confocal Microscopy: The First Application For Real-Time Pathologic Examination of Prostatic Tissue [Articolo su rivista]
Puliatti, Stefano; Bertoni, Laura; Pirola, Giacomo Maria; Azzoni, Paola; Bevilacqua, Luigi; Eissa, Ahmed; Elsherbiny, Ahmed; Sighinolfi, Maria Chiara; Chester, Johanna; Rocco, Bernardo; Micali, Salvatore; Bagni, Ilaria; Reggiani Bonetti, Luca; Maiorana, Antonino; Malvehy, Josep; Longo, Caterina; Montironi, Rodolfo; Bianchi, Giampaolo; Pellacani, Giovanni
abstract

Prostate cancer "real time" intra-operative pathological examination currently utilizes frozen section, despite the many inherent limitations of this procedure. Ex vivo Fluorescence Confocal Microscopy (FCM) is a novel technology, which provides fast microscopic fluorescence and reflectance tissue imaging. FCM has been proven beneficial in colorectal, breast, thyroid and skin lesions. However, it was never applied in urology. The aim of our study is to report the first application of FCM in the urologic field assessing its diagnostic accuracy for non-neoplastic and cancerous prostate tissue (prostatic adenocarcinoma) compared to the gold standard histopathological diagnoses.

2019 - Melanoma types by in vivo reflectance confocal microscopy correlated with protein and molecular genetic alterations: A pilot study [Articolo su rivista]
Beretti, F.; Bertoni, L.; Farnetani, F.; Pellegrini, C.; Gorelli, G.; Cesinaro, A. M.; Reggiani Bonetti, L.; Di Nardo, L.; Kaleci, S.; Chester, J.; Longo, C.; Massi, D.; Fargnoli, M. C.; Pellacani, G.
abstract

Cutaneous melanoma (CM) is one of the most prevalent skin cancers, which lacks both a prognostic marker and a specific and lasting treatment, due to the complexity of the disease and heterogeneity of patients. Reflectance confocal microscopy (RCM) in vivo analysis is a versatile approach offering immediate morphological information, enabling the identification of four primary cutaneous RCM CM types. Whether RCM CM types are associated with a specific protein and molecular genetic profiles at the tissue level remains unclear. The current pilot study was designed to identify potential correlations between RCM CM types and specific biological characteristics, combining immunohistochemistry (IHC) and molecular analyses. Eighty primary CMs evaluated at patient bedside with RCM (type 1 [19, 24%], type 2 [12, 15%], type 3 [7, 9%] and type 4 [42, 52%]) were retrospectively evaluated by IHC stains (CD271, CD20, CD31, cyclin D1), fluorescence in situ hybridization FISH for MYC gain and CDKN2A loss and molecular analysis for somatic mutations (BRAF, NRAS and KIT). RCM CM types correlated with markers of stemness property, density of intra-tumoral lymphocytic B infiltrate and cyclin D1 expression, while no significant association was found with blood vessel density nor molecular findings. RCM CM types show a different marker profile expression, suggestive of a progression and an increase in aggressiveness, according to RCM morphologies.

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.

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 - 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 - DEVELOPMENT OF A NOVEL METHOD FOR AMNIOTIC FLUID STEM CELL STORAGE [Articolo su rivista]
Zavatti, Manuela; Beretti, Francesca; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; Bertoni, Laura; DE POL, Anto; LA SALA, Giovanni Battista; Maraldi, Tullia
abstract

Background - Current procedures for collection of human Amniotic Fluid Stem Cells (hAFSCs) imply that amniotic fluid cells were cultured in flask for two weeks, than can be devoted to research purpose. However, hAFSCs could be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of the study was to verify if a direct freezing of amniotic fluid cells is able to maintain and / or improve the potential of the sub-population of stem cells. Methods - We compared the potential of the hAFSCs depending on the moment in which they are frozen, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis, and differentiation potential of C and D samples were compared. Results - hAFSCs isolated from D samples expressed MSC markers until later passages, had a good proliferation rate, and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, the direct freezing induce a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. Conclusions - This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration affecting stem cell properties. This technique might be a reasonable approach in terms of costs and for the process of accreditation in GMP for a stem cell bank.

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 - Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells [Articolo su rivista]
Zavatti, Manuela; Guida, M; Maraldi, Tullia; Beretti, Francesca; Bertoni, Laura; LA SALA, Giovanni Battista; DE POL, Anto
abstract

Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance osteoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteoblastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects.

2015 - Critical-size bone defect repair using amniotic fluid stem cell/collagen constructs: Effect of oral ferutinin treatment in rats [Articolo su rivista]
Zavatti, Manuela; Bertoni, Laura; Maraldi, Tullia; Resca, Elisa; Beretti, Francesca; Guida, Marianna; La Sala, Giovanni Battista; De Pol, Anto
abstract

Aims: This study aims to evaluate the bone regeneration in a rat calvarias critical size bone defect treated with a construct consisting of collagen type I and human amniotic fluid stem cells (AFSCs) after oral administration of phytoestrogen ferutinin. Main methods: In 12 week old male rats (n = 10), we performed two symmetric full-thickness cranial defects on each parietal region, and a scaffold was implanted into each cranial defect. The rats were divided into four groups: 1) collagen scaffold, 2) collagen scaffold + ferutinin at a dose of 2 mg/kg/5 mL, 3) collagen scaffold + AFSCs, and 4) collagen scaffold + AFSCs + ferutinin. The rats were sacrificed after 4 weeks, and the calvariae were removed, fixed, embedded in paraffin and cut into 7 pm thick sections. Histomorphometric measures, immunohistochemical and immunofluorescence analyses were performed on the paraffin sections. Key findings: The histomorphometric analysis on H&E stained sections showed a significant increase in the regenerated area of the 4th group compared with the other groups. Immunohistochemistly performed with a human anti-mitochondrial antibody showed the presence of AFSCs 4 weeks after the transplant. Immunofluorescence analysis revealed the presence of osteocalcin and estrogen receptors (ER alpha. and GPR30) in all-groups, with a greater expression of all markers in samples where the scaffold was treated with AFSCs and the rats were orally administered ferutinin. Significance: Our results demonstrated that the oral administration of ferutinin is able to improve the bone regeneration of critical-size bone defects in vivo that is obtained with collagen-AFSCs constructs.

2015 - Enrichment in c-Kit improved differentiation potential of amniotic membrane progenitor/stem cells [Articolo su rivista]
Resca, Elisa; Zavatti, Manuela; Maraldi, Tullia; Bertoni, Laura; Beretti, Francesca; Guida, Marianna; La Sala, Giovanni Battista; Guillot, P. V; David, A. L; Sebire, N. J; De Pol, Anto; De Coppi, P.
abstract

Introduction Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. Methods After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. Results Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. Discussion AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC.

2015 - Nuclear Nox4 role in stemness power of human amniotic fluid stem cells [Articolo su rivista]
Maraldi, Tullia; Guida, Marianna; Zavatti, Manuela; Resca, Elisa; Bertoni, Laura; La Sala, Giovanni Battista; De Pol, Anto
abstract

Human amniotic fluid stem cells (AFSC) are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expand in vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS) and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(P)H oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4) depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage.

2014 - Ferutinin dose-dependent effects on uterus and mammary gland in ovariectomized rats [Articolo su rivista]
Ferretti, Marzia; Cavani, Francesco; Manni, Paola; Carnevale, Gianluca; Bertoni, Laura; Zavatti, Manuela; Palumbo, Carla
abstract

The present paper completes our recent study on the effects of phytoestrogen ferutinin in preventing osteoporosis and demonstrating the superior osteoprotective effect of a 2 mg/kg/day dose in ovariectomized (OVX) rats, compared to both estrogens and lower (0.5, 1 mg/kg/day) ferutinin doses. Morphological and morphometrical analyses were performed on the effects of different doses of ferutinin administrated for one month on uterus and on mammary gland of Sprague-Dawley OVX rats, evaluated in comparison with the results for estradiol benzoate. To verify whether ferutinin provides protection against uterine and breast cancer, estimations were made of both the amount of cell proliferation (by Ki-67), and the occurrence of apoptosis (by TUNEL), two processes that in unbalanced ratio form the basis for cancer onset. The results suggest that the effects of ferutinin are dose dependent and that a 2 mg/kg/day dose might offer a better protective action against the onset of both breast and uterine carcinoma compared to ferutinin in lower doses or estradiol benzoate, increasing cellular apoptosis in glandular epithelia.

2014 - Human amniotic fluid stem cells: neural differentiation in vitro and in vivo [Articolo su rivista]
Maraldi, Tullia; Bertoni, Laura; Riccio, Massimo; Zavatti, Manuela; Carnevale, Gianluca; Resca, Elisa; Guida, Marianna; Beretti, Francesca; LA SALA, Giovanni Battista; DE POL, Anto
abstract

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, β-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2 weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.

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 - Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells. [Articolo su rivista]
Zavatti, Manuela; Resca, Elisa; Bertoni, Laura; Maraldi, Tullia; Guida, Marianna; Carnevale, Gianluca; Ferrari, Adriano; DE POL, Anto
abstract

The phytoestrogen Ferutinin plays an important role in prevention of osteoporosis caused by ovariectomy-induced estrogen deficiency in rats, but there is no evidence of its effect on osteoblastic differentiation in vitro. In this study we investigated the effect of Ferutinin on proliferation and osteoblastic differentiation of two different human stem cells populations, one derived from the amniotic fluid (AFSCs) and the other from the dental pulp (DPSCs).AFSCs and DPSCs were cultured in a differentiation medium for 14 or 21days with or without the addition of Ferutinin at a concentration ranging from 10(-11) to 10(-4)M. 17β-Estradiol was used as a positive drug at 10(-8)M. Cell proliferation and expression of specific osteoblast phenotype markers were analyzed.MTT assay revealed that Ferutinin, at concentrations of 10(-8) and 10(-9)M, enhanced proliferation of both AFSCs and DPSCs after 72h of exposure. Moreover, in both stem cell populations, Ferutinin treatment induced greater expression of the osteoblast phenotype markers osteocalcin (OCN), osteopontin (OPN), collagen I, RUNX-2 and osterix (OSX), increased calcium deposition and osteocalcin secretion in the culture medium compared to controls. These effects were more pronounced after 14days of culture in both populations.The enhancing capabilities on proliferation and osteoblastic differentiation displayed by the phytoestrogen Ferutinin make this compound an interesting candidate to promote bone formation in vivo.

2013 - Inhibition of nuclear nox4 activity by plumbagin: effect on proliferative capacity in human amniotic stem cells. [Articolo su rivista]
Guida, Marianna; Maraldi, Tullia; Resca, Elisa; Beretti, Francesca; Zavatti, Manuela; Bertoni, Laura; LA SALA, Giovanni Battista; DE POL, Anto
abstract

Human amniotic fluid stem cells (AFSC) with multilineage differentiation potential are novel source for cell therapy. However, in vitro expansion leads to senescence affecting differentiation and proliferative capacities. Reactive oxygen species (ROS) have been involved in the regulation of stem cell pluripotency, proliferation, and differentiation. Redox-regulated signal transduction is coordinated by spatially controlled production of ROS within subcellular compartments. NAD(P)H oxidase family, in particular Nox4, has been known to produce ROS in the nucleus; however, the mechanisms and the meaning of this function remain largely unknown. In the present study, we show that Nox4 nuclear expression (nNox4) increases during culture passages up to cell cycle arrest and the serum starvation causes the same effect. With the decrease of Nox4 activity, obtained with plumbagin, a decline of nuclear ROS production and of DNA damage occurs. Moreover, plumbagin exposure reduces the binding between nNox4 and nucleoskeleton components, as Matrin 3. The same effect was observed also for the binding with phospho-ERK, although nuclear ERK and P-ERK are unchanged. Taken together, we suggest that nNox4 regulation may have important pathophysiologic effects in stem cell proliferation through modulation of nuclear signaling and DNA damage.

2012 - Effects of different doses of ferutinin on bone formation/resorption in ovariectomized rats. [Articolo su rivista]
Cavani, Francesco; Ferretti, Marzia; Carnevale, Gianluca; Bertoni, Laura; Zavatti, Manuela; Palumbo, Carla
abstract

This study analyzes the effects of different doses of ferutinin on bone loss caused by estrogen deficiency in ovariectomized rats, in comparison with estradiol benzoate. Thirty female Sprague-Dawley rats were ovariectomized and treated for 30 days from the day after ovariectomy. Static/dynamic histomorphometric analyses were performed on trabecular and cortical bone of lumbar vertebrae and femurs. Very low weight increments were recorded only in all F-OVX groups, with respect to the others. Although the great differences in weight, that could imply a decrease of bone mass in F-OVX groups compared to the control ovariectomized group (C-OVX), trabecular bone in lumbar vertebrae did not show significant differences, suggesting that ferutinin, opposing estrogen deficiency, inhibits bone resorption. Newly formed cortical bone was always low in all F-OVX groups and high in C-OVX, suggesting that it is mainly devoted in answering mechanical demands. In contrast, in distal femoral metaphyses, trabecular bone was reduced and the number of osteoclasts was increased in C-OVX with respect to all other groups, suggesting that it is mainly devoted in answering metabolic demands; moreover, ferutinin dose of 2 mg/kg seemed to be more effective than the lower doses used and estrogens, particularly in those skeletal regions with higher metabolic activity. Our results suggest that the role of ferutinin in preventing osteoporosis caused by estrogen deficiency is expressed in decreasing bone erosion; moreover, in all F-OVX groups bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.

2012 - ROLE OF PHYTOESTROGEN FERUTININ IN PREVENTING/RECOVERING BONE LOSS: RESULTS FROM EXPERIMENTAL OVARIECTOMIZED RAT MODEL [Capitolo/Saggio]
Palumbo, Carla; Cavani, Francesco; Bertoni, Laura; Ferretti, Marzia
abstract

In the Chapter 35 of the book are reported observations of recent pubblications on the effect of ferutinin in preventing/recovering severe osteoporosis secondary to ovariectomy in rats. On the basis of the results so far obtained, the authors suggest to enumerate ferutinin among the osteoprotective substances. This fact acquires a more relevant importance in the light of recent tenable evidences reported from various authors concerning the absence of negative side effects by some phytoestrogens (particularly genistein, 8-prenylnaringenin, reveratrol and red clover extract) on the tropism of various organs commonly targeted by estrogens. In conclusion, the results reported not only provide evidence that ferutinin can significantly prevent/recover ovariectomy-induced bone loss in rats, but also that it could protect against the onset of uterus cancer. Although the putative undesired estrogenic-like side effects on uterus of such phytoestrogen have not yet been fully investigated, ferutinin could be an interesting safer alternative new candidate for HRT in treatment of post-menopausal symptoms, since it seems to protect from bone loss induced by ovariectomy (Palumbo et al., 2009; Ferretti et al., 2010) and in part to mime the ovarian endocrine function during menopause.

2012 - Structural and histomorphometric evaluations of ferutinin effects on the uterus of ovariectomized rats during osteoporosis treatment [Articolo su rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Sena, Paola; Carnevale, Gianluca; Zavatti, Manuela; Vittoria Di, Viesti; Zanoli, Paola; Palumbo, Carla
abstract

Aims: The effects of chronic administration of Ferutinin (phytoestrogen found in the plants of genus Ferula),compared with those elicited by estradiol benzoate, were evaluated, following ovariectomy, on the uterus ofovariectomized rats as regard weight, size, structure and histomorphometry.Main methods: The experimental study included 40 female Sprague–Dawley rats, assigned to two different protocols,i.e. preventive and recovering. In the preventive protocol, ferutinin (2 mg/kg/day)was orally administeredfor 30 days, starting from the day after ovariectomy; in the recovering protocol, ferutinin was administered, atthe same dosage, for 30 days starting fromthe 60th day after ovariectomy, when osteoporosiswas clearly established.Its effects were compared with those of estradiol benzoate (1.5 μg per rat twice a week, subcutaneouslyinjected) vs. vehicle-treated ovariectomized controls and vehicle-treated sham-operated controls. Uteri were removed,weighed and analysed under both the structural and histomorphometrical points of view.Key findings: Our data show that ferutinin acts, similarly to estradiol benzoate, on the uterus stimulating endometrialand myometrial hypertrophy; this notwithstanding, the phytoestrogen ferutinin, in contrast to estrogentreatment, appears to increase apoptosis in uterine luminal and glandular epithelia.Significance: Ferutinin, used in osteoporosis treatment primarily for bonemass recovering, seems in linewith aneventual protective function against uterine carcinoma, unlike estrogens so far employed in hormone replacementtherapy (HRT).

2011 - Effect of pulsed electromagnetic fields (PEMFs) on condrogenic phenotype maintenance of MSCs in presence of pro-inflammatory cytochines: preliminary results [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Sena, Paola; Gian Luigi, Sacchetti; Stefania, Setti; Cadossi, Ruggero
abstract

The aim of the present study was to evaluate in vitro the effects of PEMFs on maintenance over time of chondrocyte phenotype of conditioned Mesenchimal Stem Cells (MSCs), in presence of pro-inflammatory cytokines (IL-ß1). MSCs, taken from bone marrow, were pellet-cultured in medium conditioning towards the chondrogenic lineage. Two targets were pursued: the first was to standardize the method to obtain chondrocyte pellets in terms of type/amount of withdrawal, time/degree of differentiation and amount of extracellular matrix production; the second was to extend over time chondrocyte differentiation, checking the phenotype maintenance, after adding pro-inflammatory IL-ß1 cytokine in culture medium with/without the application of PEMFs (device provided by IGEA-Carpi). The pellets obtained were coltured for different times (21, 28, 34 days), verifying the presence of type-II collagen (as index of chondrocyte differentiation) both by means of TEM analysis and immunoreaction. The best differentiation was obtained after 28 days of culture; in such pellets the studies were performed in triplicate for 15 days, identifying four experimental conditions: 1) without IL-b1 and PEMFs; 2) with IL-b1, without PEMFs; 3) without IL-b1 and with PEMFs; 4) with IL-b1 and PEMFs. The parameters of applied PEMFs were 1.7mT and 75Hz, and the time of application was 4 hours/day. Medium was changed every 3-4 days and stored for the evaluations of PGE2 (indicative of inflammation) and proteoglycans (indicative of chondrogenic differentiation). At the end of the experiment, each pellet was fixed with paraformaldehyde 4% and embedded in paraffin; sections (5 µm thick) were obtained and stained with Toluidin Blue in order to evaluate metachromasia. The results indicate that: 1) only the pellets treated with IL-b1 without PEMFs did not show metachromasia, indicanting a chondrocyte de-differentiation towards fibroblastic phenotype; 2) only in pellets treated with IL-b1 and with PEMF application, after about 12 days of treatment the amount of PGE2 in medium decreases (31%) while the proteoglycan production slightly increases (2%).In conclusion, if the results will be confirmed, pulsed electromagnetic fields could be proposed in preventing chondrocyte de-differentiation due to inflammation induced by IL-ß1; this with the final aim to integrate regenerative medicine techniques to apply in the healing of joint cartilage lesions with bio-physic energy devices, in order to obtain a stable-in-time recovery of physiologic function of articular surfaces that suffered a severe injury.

2011 - INTERACTION OF BIOPHYSIC STIMULI ON CONDROGENIC DIFFERENTIATION OF MSCs: PRELIMINARY RESULTS ON THE EVALUATION OF ANTI-INFLAMMATORY EFFECT OF ELECTROMAGNETIC FIELDS [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Taronna, ANGELO PIO; Sena, Paola; Setti, S.; Cadossi, Ruggero
abstract

The aim of the present study is to investigate in vitro chondrocyte-like cells treated with electromagnetic fields to evaluate over time maintenance of chondrocyte phenotype, in presence of pro-inflammatory cytokines (IL-1ß). Mesenchimal Stem Cells taken from bone marrow were cultured (in pellet) in medium conditioning towards the chondrogenic lineage. The targets are firstly to standardize the method to obtain chondrocyte pellets in terms of a) type/amount of withdrawal, b) time/degree of differentiation, and c) amount of extracellular matrix production; secondly, to extend over time chondrocyte differentiation, checking the phenotype maintenance, after adding pro-inflammatory cytokines in culture medium with/without the application of electromagnetic fields (device provided by IGEA-Carpi). The pellets obtained were coltured for different times (21, 28, 34 days), verifying the presence of type 2 collagen (index of chondrocyte differentiation). The best differentiation was obtained after 28 days of culture. In such pellets, after inflammatory induction and application of electromagnetic field (1.7mT, 75Hz) for 15 days, the observations showed that after about 12 days of treatment the amount of PGE2 in medium decreases (31%) while the proteoglycan production slightly increases (2%). In conclusion, electromagnetic fields could be proposed (if the results will be confirmed) in preventing chondrocyte de-differentiation due to inflammation induced by IL-1ß, to integrate regenerative medicine techniques in the healing of cartilage lesions.

2011 - RGB method in immunofluorescence investigations on stem cells [Articolo su rivista]
Riccio, Massimo; E., Resca; Bertoni, Laura; Cavani, Francesco; Sena, Paola; Ferretti, Marzia; Baldini, Andrea; Palumbo, Carla; DE POL, Anto
abstract

Colour is not related to a particular discipline, but it is transversely present in many circles and inalmost all the aspects of life. It has a special value in art, but also as far as other disciplines areconcerned, like the sciences, the colour is at the basis of some of their intrinsic significances and it oftenneeded to allow the interpretation of some of their phenomena as well. As regards the development ofcell biology knowledge, colour acquired more and more importance in revealing the observations of theresearchers. A field in which the methods based on the colours are particularly employed is theimmunofluorescence, used to identify specific proteins in cells and tissues. These techniques combinethe fluorochrome properties with specific molecules, i.e. antibodies, directed against particularsubstances to investigate, for example a specific protein. In single immunofluorescence analysis, thesignal from an excited fluorochrome corresponds to a particular protein. In multiple immunofluorescenceanalysis, two or more signals are simultaneously detected to show the localization of differentproteins on the same sample. The three primary colours red, green and blue were currently assigned tothe signals from immunofluorescence-processed samples and visualized by the RGB method. In thepresent work, different examples of RGB applications in immunocytochemical investigations areshowed: the first concerns the multiple analysis of three markers, localized in different loci of the cellplasma membrane; the second is related to the co-localization of two signals in the same site of specificsubcellular structures. In this case the secondary colours, obtained by overlapping the primary ones,demonstrate the specific co-presence of two proteins in the same site. With the present paper, theauthors wish to underline the relevant role of colours also in those areas in which colours are the meansnot the end.

2010 - Influence of ferutinin on bone metabolism in ovariectomized rats. II: Role in recovering osteoporosis. [Articolo su rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Zavatti, Manuela; Resca, Elisa; Carnevale, Gianluca; Benelli, Augusta; Zanoli, Paola; Palumbo, Carla
abstract

The study investigates the influence of ferutinin (a phytoestrogen extracted from Ferula Hermanis root) on bone metabolism in ovariectomized rats. The study represent the complection of previous investigations (published in 2009, concerning the ferutinine role in preventing osteoporosis due to estrogen deficiency). The present investigation concerns the role of Ferutinine in recovering osteoporosis.

2010 - New aspects of Ferutinin effect in preventing osteoporosis [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Bertoni, Laura; Zavatti, Manuela; Taronna, ANGELO PIO; Carnevale, Gianluca; Benelli, Augusta; Zanoli, Paola; Marotti, Gastone; Palumbo, Carla
abstract

The results of the study suggest that ferutinin role, in preventing osteoporosis due to estrogen deficiency, is expressed in inhibiting osteoclast erosion rather than in enhancing osteoblast deposition (as previously suggested); moreover, in all F-OVX groups the bone turnover is very low and seems correlated to the trivial body weight increase, which, in turn, depends on ferutinin treatment.

2009 - Bone tissue electroporation: a preliminary in vivo study. [Abstract in Rivista]
Cavani, Francesco; Fini, M.; Bertoni, Laura; Ferretti, Marzia
abstract

Goal of the study is to extablish the values of parameners of electroporation above which, in the treated bone regions, cell damage becomes irreversible, leading to cell death.

2009 - Effect of leptin on the development of primary ossification centers in mouse fetuses. [Abstract in Rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Zavatti, Manuela; Benelli, A.; Palumbo, Carla
abstract

Leptin may be considered a significat cartilage/bone growth factor.

2009 - Influence of ferutinin on bone metabolism in ovariectomized rats. I: role in preventing osteoporosis [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Resca, Elisa; Casolari, Barbara; Carnevale, Gianluca; Zavatti, Manuela; C., Montanari; Benelli, Augusta; Zanoli, Paola
abstract

Phytoestrogens play a role in maintaining bone mass in the post-menopausal period for their putative function as osteoprotective agents. The aim of the present study was to investigate the influence of Ferutinin, a phytoestrogen found in the plants of Ferula genus, on bone loss in ovariectomized rats. Such an animal model can simulate the various clinical syndromes deriving from osteoporosis. The effect of the daily oral administration of ferutinin to ovariectomized rats (dosed at 2 mg/kg per day for 30 and 60 days) was compared to that of estradiol benzoate (subcutaneously administered at the dose of 1.5 microg/rat twice a week). After the sacrifice, histomorphometrical analyses were performed on trabecular bone of L4-L5 vertebrae and distal femoral metaphysis, as well as on cortical bone of femoral diaphysis; biochemical parameters (bone mineral components and markers) were also evaluated from the rat serum. The histomorphometrical analyses of trabecular and cortical bone from lumbar vertebrae and femur showed that ferutinin has the same antiosteoporotic effect of estradiol benzoate on bone mass, and in some cases is even stronger. This fact suggests that it could prevent osteoporosis caused by severe estrogen deficiency in ovariectomized rats. The possibility of using ferutinin as an alternative to the commonly employed hormonal replacing therapy in post-menopausal women is discussed.

2009 - Leptin increases growth of primary ossification centers in fetal mice [Articolo su rivista]
Bertoni, Laura; Ferretti, Marzia; Cavani, Francesco; Zavatti, Manuela; Resca, Elisa; Benelli, Augusta; Palumbo, Carla
abstract

The effect of peripheral leptin on fetal primary ossification centers during the early phases of bone histogenesis was investigated by administration of leptin to pregnant mice. Fourteen pregnant mice were divided into two groups. The treated pregnant group was subcutaneously injected in the intrascapular region with supraphysiologic doses (2 mg kg(-1)) of leptin (Vinci Biochem, Firenze, Italy) in a volume of 0.1 mL per 10 g body weight, at the 7th, 9th and 11th day of gestation. The control group was treated with physiological solution in the same manner and same times as the treated group. The new-born mice were killed 1 day after birth and the primary ossification centers were stained with Alizarin Red S after diaphanizing the soft tissues in 1% potassium hydroxide. The development of both endochondral and intramembranous ossification centers was morphometrically analysed in long bones. The results showed that the ossification centers of mice born by mothers treated with leptin grow more rapidly in both length and cross-sectional area compared with mice born by the untreated mothers. As the development of long bones depends on endochondral ossification occurring at proximal and distal epiphyseal plates as well as on intramembranous ossification along the periosteal surface, it appears that leptin activates the differentiation and proliferation of both chondrocytes and osteoblasts. The role of leptin as a growth factor of cartilage and bone is discussed in the light of the data reported in the literature.

2009 - Multipotent stem cells in vitro differentiation on 3-D scaffolds. [Abstract in Rivista]
Palumbo, Carla; Riccio, M.; Resca, E.; Maraldi, Tullia; Bertoni, Laura; Sena, Paola; DE POL, Anto
abstract

Goal of the study is to obtain cell/scaffold complexes to use in regenerative medicine.

2009 - OSTEOGENIC DIFFERENTIATION OF DENTAL PULP STEM CELLS (DPSC) IN 3D-MATRICES TO USE IN REGENERATIVE MEDICINE [Abstract in Rivista]
Resca, Elisa; Riccio, Massimo; Bertoni, Laura; Maraldi, Tullia; Palumbo, Carla; DE POL, Anto
abstract

The investigation concerns the selection of mesenchymal stem cells, derived from adult human dental pulp (DPSC), to commit towards osteogenic differentiation in order to perform new strategies for the regenerative medicine. The final aim of this study is to use DPSC in regenerative medicine approaches for recovering wide gaps of bone tissue due to post-traumatic locomotor apparatus damages.

2008 - Effect of the phytoestrogen ferutinin in preventing and recovering osteoporosis: histomorphometric analysis of bone mass in ovariectomized rats. [Abstract in Rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Resca, Elisa; Carnevale, Gianluca; Zavatti, Manuela; Benelli, A.; Zanoli, P.; Palumbo, Carla
abstract

Ferutinin seems to display the same effects on bone mass obtained with estradiol in OVX rats.

2008 - Influence of density, elasticity, and structure on ultrasound transmission through trabecular bone cylinders [Articolo su rivista]
Cavani, Francesco; Gianluca, Giavaresi; Milena, Fini; Bertoni, Laura; Francesca de, Terlizzi; Reinhard, Barkmann; Canè, Valerio
abstract

The aim of this in vitro study is to evaluate the potentiality of quantitative ultrasound (QUS) to separate information on density, elasticity, and structure on specimens of trabecular bone. Fifteen cylinders of spongy bone extracted from equine vertebrae were progressively demineralized and subjected to QUS, micro computed tomography (mu CT), Dual energy X-ray absorptiometry (DXA) at various mineralization levels. Eventually all cylinders underwent a compression test to calculate the Young's modulus. Correlation analysis shows that speed of sound (SOS) is strictly associated to bone mineral density (BMD), Young's modulus, and all mu CT parameters except for degree of anisotropy (DA). Fast wave amplitude (FWA) is directly correlated with bone surface and total volume ratio (BS/TV) and trabecular separation (Tb Sp), and inversely correlated with trabecular number (Tb N). Because mu CT parameters were strictly correlated to BMD and Young's modulus data, partial correlation analysis was performed between SOS, FWA, and structural and elastic data in order to eliminate the effect of density. SOS was significantly correlated to bone volume and total volume ratio (BV/TV), BS/TV, and Young's modulus, and FWA was significantly correlated to Tb Sp only. These results show that SOS is strongly influenced by volumetric mineral bone density and elastic modulus of the specimen, and FWA is mainly affected by trabecular separation independently on density. Therefore, SOS and FWA are able to provide different and complementary information, at least on trabecular bone samples.

2008 - Sympathectomy alters bone architecture in adult growing rats [Articolo su rivista]
F., Pagani; V., Sibilia; Cavani, Francesco; Ferretti, Marzia; Bertoni, Laura; Palumbo, Carla; N., Lattuada; E., De Luca; A., Rubinacci; F., Guidobono
abstract

Sympathetic nervous system (SNS) fibres and alpha- and beta-receptors are present in bone, indicating that the SNS may participate in bone metabolism. The importance of these observations is controversial because stimulation or inhibition of the SNS has had various effects upon both anabolic and catabolic activity in this tissue. In this study we evaluated the effects of pharmacological sympathectomy, using chronic treatment of maturing male rats with 40 mg of guanethidine/kg i.p., upon various parameters in bone. Double labelling with tetracycline injection was also performed 20 and 2 days before sacrifice. Bone mass, mineral content, density and histomorphometric characteristics in different skeletal regions were determined. Bone metabolic markers included urinary deoxypyridinoline and serum osteocalcin measurements. Guanethidine significantly reduced the accretion of lumbar vertebral bone and of mineral content and density, compared to controls. Femoral bone mineral content and density were also significantly reduced, compared to controls. Histomorphometric analyses indicated these effects were related to a reduction of cortical bone and mineral apposition rate at femoral diaphysials level. Both markers of bone metabolism were reduced in controls as they approached maturity. Guanethidine significantly decreased serum osteocalcin compared to controls, while urinary deoxypyridinoline was unchanged. These data indicate that guanethidine-induced sympathectomy caused a negative balance of bone metabolism, leading to decreased mass by regulating deposition rather than resorption during modeling and remodeling of bone.

2008 - Two peculiar conditions following a coma: A clinical case of heterotopic ossification concomitant with keloid formation [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Pierluigi, Bonucci; Sena, Paola; Bertoni, Laura; Cavani, Francesco; Andrea, Celli; Rovesta, Claudio
abstract

The etiology and formation pattern of heterotopic ossifications (HO) are still unknown. They occur in soft tissues in which bone does not normally form, near one or more proximal joints. In this article, the authors report a peculiar case of a 31-year-old patient affected by scapulo-humeral ankylosis that occurred about 6 months after a coma, in which two unusual concomitant conditions were observed: HO formation in the scapulo-humeral region and the development of keloids during wound repair. The scapulo-humeral ankylosis was resolved surgically with the removal of the HO, which was then studied morphologically to understand its formation pattern. By light microscopy and transmission electron microscopy, it was observed that heterotopic bone displays the normal microscopic structure of primary bone, in which two types of bone tissue were recognized, i.e., woven-fibered bone, deeply located and produced first, and lamellar bone. This suggests that the pattern of HO formation retraces the ontogenetic steps that normally occur during intramembranous ossification. The authors also discuss the peculiar concomitance of HO formation and keloid development, speculating that, although they are different conditions localized in dissimilar regions, they might be hypothetically triggered by a common event, such as the release of factors likely issued during the coma status.

2007 - Adult human dental pulp stem cells (DPSC): preliminary observations for selecting and conditioning DPSC according osteogenic aims. [Abstract in Rivista]
Palumbo, Carla; Riccio, Massimo; Resca, Elisa; Bertoni, Laura; Baldini, Andrea; Strozzi, Antonio; G., Bruzzesi; DE POL, Anto
abstract

The previlinary observations have shown the possibility to obtain in vitro bone formation to apply in regenerative medicine.

2007 - Phytoestrogen effects on bone mass in ovariectomized rats: preliminary histomorphometric analysis. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Cavani, Francesco; Bertoni, Laura; Resca, E.; Carnevale, Gianluca; Zavatti, Manuela; Montanari, C.; Benelli, A.; Zanoli, P.; Marotti, Gastone
abstract

Phytoestrogens ferutinine could prevent in rats the risk of osteoporosis in estrogen deficient conditions and it could enhance the recover of bone mass in osteoporotic OVX rats.

2006 - Different skeletal regional response to continuous brain infusion of leptin in the rat. [Articolo su rivista]
F., Guidobono; F., Pagani; V., Sibila; C., Netti; N., Lattuada; D., Rapetti; E., Mrak; I., Villa; Cavani, Francesco; Bertoni, Laura; Palumbo, Carla; Ferretti, Marzia; Marotti, Gastone; A., Rubinacci
abstract

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 mu g/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydincline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton. (c) 2005 Elsevier Inc. All rights reserved.

2006 - Does Static precede dynamic osteogenesis in endochondral ossification as occurs in intramembranous ossification? [Articolo su rivista]
Ferretti, Marzia; Palumbo, Carla; Bertoni, Laura; Cavani, Francesco; Marotti, Gastone
abstract

Endochondral ossification takes place with calcified cartilage cores providing a rigid scaffold for new bone formation. Intramembranous ossification begins in connective tissue and new bone formed by a process of static ossification (SO) followed by dynamic ossification (DO) as previously described. The aim of the present study was to determine if the process of endochondral ossification is similar to that of intramembranous ossification with both a static and a dynamic phase of osteogenesis. Endochondral ossification centers of the tibiae and humeri of newborn and young growing rabbits were studied by light and transmission electron microscopy. The observations clearly showed that in endochondral ossification, the calcified trabeculae appeared to be lined first by osteoclasts. The osteoclasts were then replaced by flattened cells (likely cells of the reversal phase) and finally by irregularly arranged osteoblastic laminae, typical of DO. This cellular sequence did not include osteoblasts seen in the phase of SO. These findings clearly support our working hypothesis that SO only forms in soft tissues to provide a rigid framework for DO, and that DO requires a rigid mineralized surface. The presence of osteocytes in contact with the calcified cartilage also suggests the existence of stationary osteoblasts in endochondral ossification. Stationary osteoblasts did not appear to be a unique feature of SO. The presence of stationary osteoblasts may appear to provide the initial osteocytes during osteogenesis that may function as mechanosensors throughout the bone tissue. If this is the case, then bone would be capable of sensing mechanical strains from its inception.

2006 - Leptin effect on rat primary ossification centers during bone histogenesis. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Benincasa, Marta; Bertoni, Laura; Cavani, Francesco; Rivasi, M.; Benelli, A.
abstract

During the early phases of endochondral ossification, Leptin positive effects are shown in growith of rat ossification centers.

2006 - Preliminary observations on transplants of vascularized bone scaffolds: an experimental and morphological study. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Bertoni, Laura; Cavani, Francesco; Carbonara, A.; DE SANTIS, Giorgio; Marotti, Gastone
abstract

Bone formation occurring inside and around dead bone implanted scaffolds seems to follow the same sequence of events that are observed during normal intramembranous ossification.

2005 - Endochordral versus intramembranous ossification: analogies and differences. [Abstract in Rivista]
Ferretti, Marzia; Palumbo, Carla; Bertoni, Laura; Marotti, Gastone
abstract

Stationary osteoblasts are not a typical finding of static osteogenesis; they may also exist in the earòly stage of dynamic osteogenesis.

2005 - Heterotopic human bone: mechanism of deposition and structure. [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Rovesta, Claudio
abstract

Heterotopic human bone is a pathologic bone that needs to degenerate rather than to be preserved, and for this reason rapidly osteocyte death occurs.

2003 - Combined skin prick and patch testing enhances identification of peanut-allergic patients with atopic dermatitis [Articolo su rivista]
Seidenari, Stefania; Giusti, Francesca; Bertoni, Laura; L., Mantovani
abstract

Background: Food atopy patch tests (APTs) are considered a useful tool for the diagnosis of food allergy. Hypersensitivity to peanuts has not been investigated by means of APTs so far. Methods: APTs and skin prick tests (SPTs) with peanuts were performed in 136 atopic dermatitis (AD) patients. Relevance of positive and negative responses to these tests was assessed by repeated open challenges with peanuts. Results: Nine percent of our AD patients reacted to the challenge. Positive responses to APTs were recorded in 19\% of the patients, whereas in 12\% positive SPTs were observed. APTs were more frequently positive in subjects with eczematous responses after challenge with respect to those with urticarial reactions. SPT reactivity proved to be higher in patients above 12 years of age, whereas APT positivity was more frequent in children under 6 years. APT sensitivity proved significantly higher than SPT sensitivity, in particular in children under 12 years of age. On the contrary, SPT specificity and positive predictive value were significantly higher with respect to those of APT in the age group of subjects under 6 years of age. Conclusions: Our data suggest that APTs with peanuts may represent a useful integration to standard testing modalities employed for the diagnosis of peanut allergy in AD patients.

2003 - Contact sensitization to disperse dyes in children [Articolo su rivista]
Giusti, Francesca; Massone, Federica; Bertoni, Laura; Pellacani, Giovanni; Seidenari, Stefania
abstract

From January 1996 to December 2000, 1098 children, including 667 subjects with suspected allergic contact dermatitis and 431 patients with atopic dermatitis ( AD), were patch tested with seven disperse dyes: disperse blue 124 (DB124), disperse blue 106 (DB106), disperse red 1 (DR1), disperse yellow 3 (DY3), disperse orange 3 (DO3), p-aminoazobenzene (PAAB), and p-dimethylaminoazobenzene (PDAAB). Of these, 51 patients (4.6\%; 34 girls and 17 boys) proved sensitized to disperse dyes. AD or history of AD was present in 30 patients (59\%). The most common sensitizer was DY3 ( 17 patients), followed by DO3 ( 15 patients), and DB124 ( 14 patients). Among dye-positive patients, about 12\% were sensitized to disperse dyes alone and only 14\% reacted to para-phenylenediamine. In disperse dye-sensitive children not affected by AD, the feet, axillae, and groin appeared to be the most common localizations, whereas in those with AD, involvement of the face and the flexural areas of the limbs was more common. In conclusion, our study showed that in children with suspected contact sensitization, disperse dyes should be regarded as potential triggering allergens.

2003 - Frequency and intensity of responses to mite patch tests are lower in nonatopic subjects with respect to patients with atopic dermatitis [Articolo su rivista]
Seidenari, Stefania; Giusti, Francesca; Pellacani, Giovanni; Bertoni, Laura
abstract

Background: So far the issue of patch tests with mite allergens in subjects not affected by atopic dermatitis (AD) has been poorly investigated. The aim of this study was to assess the frequency and intensity of responses to atopy patch tests with Dermatophagoides in non-AD subjects, and to compare them to the ones observed in AD patients. Methods: Patch tests were performed, employing a mixture of Dermatophagoides pteronyssinus and farinae at two different concentrations (20 and 40% pet.), on 75 non-AD subjects, including 33 patients with allergic rhinitis and 42 healthy volunteers, and on 210 AD patients, as controls. Results: Positive responses to 20% mite patch tests were observed in 17 non-AD subjects (23%) and in 102 AD ones (49%). The former showed a lower intensity of the responses, with a mean score of 1.4 compared with 2.1 in atopics. Conclusions: Although positive responses to mite patch tests are also observable in subjects without AD, their frequency and intensity are significantly lower compared with AD patients.

2002 - Reproducility of APT [Articolo su rivista]
Seidenari, Stefania; Giusti, Francesca; Bertoni, Laura
abstract

Background: The aim of the present study was to evaluate the reproducibility of Atopy Patch Tests (APT) by simultaneously applying 2 identical patch test series in 85 patients affected by atopic dermatitis. Methods: The test substances included 2 commercially available materials (Dermatophagoides mix 20% pet., Alternaria Alternata 2.4% pet.) and 8 self-made food preparations containing egg yolk, egg white, peanuts, cow’s milk, soy milk, wheat flour, rice, and corn meal. Results: The percentage of agreement in the frequency of positive responses varied from 87.1% (corn meal) to 100% (soy milk). The reproducibility was satisfactory for Dermatophagoides mix, Alternaria alternata, egg yolk, and soy milk. Conclusions: For Dermatophagoides and Alternaria Alternata the agreement rate was similar to that of standard patch tests and therefore APTs may be considered sufficiently reproducible to be employed as a diagnostic testing procedure. As regards food materials, such as soy milk, egg yolk, and corn meal, APT reproducibility may be acceptable. For other foodstuffs, preparation methods have to be improved.

2001 - Skin barrier, hydration, and pH of the skin on infants under 2 years of age. [Articolo su rivista]
Giusti, Francesca; A., Martella; Bertoni, Laura; Seidenari, Stefania
abstract

The goal of this study was to instrumentally evaluate the skin of healthy infants and to compare it to adult skin. A total of 70 infants, 45 girls and 25 boys, ages 8-24 months, and 30 healthy women were studied by means of transepidermal water loss (TEWL), capacitance, and pH measurements at two different skin sites, the volar forearm and the buttocks. No significant differences in TEWL were found between infants and adults, either on the buttocks or on the volar forearm. On the contrary, capacitance values were higher in infants. Their skin also appeared less acid than that of adults, with high statistical significance. No TEWL, capacitance, or pH variations were observed in infants according to sex and age. On the basis of the above data, the skin of infants 8-24 months of age shows functional signs of immaturity. This may lead to an increased permeability and a reduced capacity for defense against chemical and microbial aggression.

2000 - Sensitive skin as subclinical expression of contact allergy to nickel sulfate [Articolo su rivista]
M., Francomano; Bertoni, Laura; Seidenari, Stefania
abstract

nd

2000 - Thickness and echogenicity of the skin in children as assessed by 20-MHz ultrasound [Articolo su rivista]
Seidenari, Stefania; G., Giusti; Bertoni, Laura; Magnoni, Cristina; Pellacani, Giovanni
abstract

Background: Skin anatomy and physiology undergo modifications throughout the whole lifespan. in children the skin appears with structural characteristics, as evaluated by histology, which differ from those of adults, especially in the first years of life. Objective: The aim of our study was to evaluate skin thickness and echogenicity at different sites in children by 20-MHz ultrasound. Methods: Eight skin sites of 42 children and 30 young adults were studied with a 20-MHz B scanner. Skin thickness and mean echogenicity were evaluated. The echographic images were processed and segmented by a dedicated program: the 0-30 amplitude interval, which marks the hypoechogenic parts of the tissue, and the 201-255 range, highlighting the hyperreflecting areas, have been selected. Results and Conclusion: Whereas skin thickness shows a gradual increase from birth to adulthood, maturation of the skin leads to variations in the intensity of its echogenicity, depending on the different skin areas. Whereas on the face and the trunk it appears lower in adults with respect to children, a gradual increase can be observed on the limbs with growing age. The distribution of skin reflectivity also greatly varies in different phases of life. Copyright (C) 2000 S. Karger AG, Basel.