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Vallmajo-Martin Q, Kiveliö AS, Metzger S, Milleret V, Lienemann PS, Carrara BM, Millan C, Ghayor C, Ochsenbein-Koelble N, Ehrbar M. Undifferentiated Human Amniotic Fluid Progenitor Cells Promote Bone Regeneration in Vivo. Adv Healthc Mater 2025; 14:e2300843. [PMID: 39930929 DOI: 10.1002/adhm.202300843] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2024] [Revised: 01/22/2025] [Indexed: 04/18/2025]
Abstract
The treatment of large bone defects requires bone tissue substitutes. However, the lack of accessible autologous bone, especially in newborns with spina bifida or cleft palate conditions, severely limits therapeutic options involving bone grafts. Here, an engineering approach to reconstruct bone is presented by combining human amniocentesis-derived amniotic fluid progenitor cells (hAFCs) and a biomimetic, injectable, and fully synthetic poly(ethylene glycol) hydrogel that is crosslinked enzymatically by transglutaminase FXIII (TG-PEG). hAFCs are isolated by their colony-forming capacity, expanded in vitro, and undergo osteogenic, chondrogenic, or adipogenic differentiation under appropriate stimulation. When encapsulated in TG-PEG hydrogels, hAFCs rapidly deposit endogenous extracellular matrix (ECM) in vitro. hAFC-laden TG-PEG hydrogels containing low concentrations of bone morphogenetic protein (BMP-2) promote formation of ectopic bone organoids in vivo in a murine model without requiring prior in vitro differentiation. Strikingly, hAFC-induced constructs form as much bone in this model as adult bone marrow-derived stromal cells (hBMSCs), and significantly more than adipose-derived stromal cells (hASCs). Utilization of autologous hAFCs embedded in TG-PEG hydrogels presents a promising therapeutic strategy for bone replacement, particularly in fetuses and newborns where limited stem cell availability can be overcome through minimally invasive harvest of amniotic fluid.
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Affiliation(s)
- Queralt Vallmajo-Martin
- Department of Obstetrics, University Hospital Zürich (USZ), Zürich, 8091, Switzerland
- Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, 1015, Switzerland
| | - Anna-Sofia Kiveliö
- Department of Obstetrics, University Hospital Zürich (USZ), Zürich, 8091, Switzerland
- Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, 1015, Switzerland
| | - Stéphanie Metzger
- Department of Obstetrics, University Hospital Zürich (USZ), Zürich, 8091, Switzerland
- Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, 1015, Switzerland
| | - Vincent Milleret
- Department of Obstetrics, University Hospital Zürich (USZ), Zürich, 8091, Switzerland
| | - Philipp S Lienemann
- Department of Obstetrics, University Hospital Zürich (USZ), Zürich, 8091, Switzerland
| | - Bianca M Carrara
- Department of Obstetrics, University Hospital Zürich (USZ), Zürich, 8091, Switzerland
| | - Christopher Millan
- Department of Urology, University Hospital Zürich (USZ), Zürich, 8091, Switzerland
| | - Chafik Ghayor
- Center of Dental Medicine, Oral Biotechnology & Bioengineering, University of Zürich (UZH), Zürich, 8006, Switzerland
| | | | - Martin Ehrbar
- Department of Obstetrics, University Hospital Zürich (USZ), Zürich, 8091, Switzerland
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Ouzin M, Kogler G. Mesenchymal Stromal Cells: Heterogeneity and Therapeutical Applications. Cells 2023; 12:2039. [PMID: 37626848 PMCID: PMC10453316 DOI: 10.3390/cells12162039] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2023] [Revised: 08/06/2023] [Accepted: 08/08/2023] [Indexed: 08/27/2023] Open
Abstract
Mesenchymal stromal cells nowadays emerge as a major player in the field of regenerative medicine and translational research. They constitute, with their derived products, the most frequently used cell type in different therapies. However, their heterogeneity, including different subpopulations, the anatomic source of isolation, and high donor-to-donor variability, constitutes a major controversial issue that affects their use in clinical applications. Furthermore, the intrinsic and extrinsic molecular mechanisms underlying their self-renewal and fate specification are still not completely elucidated. This review dissects the different heterogeneity aspects of the tissue source associated with a distinct developmental origin that need to be considered when generating homogenous products before their usage for clinical applications.
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Affiliation(s)
- Meryem Ouzin
- Institute for Transplantation Diagnostics and Cell Therapeutics, University Hospital Düsseldorf, 40225 Düsseldorf, Germany;
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Song MK, Sun HJ, Cho SW. Conditioned medium of amniotic fluid-derived stromal cells exerts a bone anabolic effect by enhancing progenitor population and angiogenesis. J Tissue Eng Regen Med 2022; 16:923-933. [PMID: 35819750 DOI: 10.1002/term.3340] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2021] [Revised: 06/14/2022] [Accepted: 06/29/2022] [Indexed: 11/10/2022]
Abstract
A cell-free approach utilizing the paracrine effects of mesenchymal stromal cells is receiving attention in regenerative medicine. In the present study, we evaluated the effects of a conditioned medium of amniotic fluid-derived stromal cells (AFSC-CM) on bone metabolism. In mice, intraperitoneal injections of AFSC-CM increased bone mass and enhanced bone turnover. The precursor populations of myeloid and mesenchymal lineages, as well as endothelial cells in bone marrow, were also augmented by AFSC-CM administration. In an in vitro culture experiment, AFSC-CM increased osteoclast differentiation of bone marrow-derived macrophages, but had no significant effect on the osteogenic differentiation of preosteoblasts. However, AFSC-CM administration dramatically accelerated the migration and tube formation of endothelial cells, and a cytokine array showed that AFSC-CM contained many angiogenic factors. These results indicate that AFSC-CM exerts a bone anabolic effect by changing the bone marrow microenvironment, including angiogenesis and precursor expansion. Therefore, ameliorating marrow angiogenesis is a potential therapeutic strategy for bone regeneration, for which AFSCs can be a good cellular source.
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Affiliation(s)
- Min-Kyoung Song
- Department of Internal Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, Korea.,Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine, Seoul National University, Daehak-ro, Jongno-gu, Seoul, Korea
| | - Hyun Jin Sun
- Department of Internal Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, Korea
| | - Sun Wook Cho
- Department of Internal Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, Korea.,Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul, Korea
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Basile M, Marchegiani F, Novak S, Kalajzic I, Di Pietro R. Human amniotic fluid stem cells attract osteoprogenitor cells in bone healing. J Cell Physiol 2020; 235:4643-4654. [PMID: 31650536 PMCID: PMC7018542 DOI: 10.1002/jcp.29342] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Accepted: 09/30/2019] [Indexed: 02/05/2023]
Abstract
Current treatments of large bone defects are based on autologous or allogenic bone transplantation. Human amniotic fluid stem cells (hAFSCs) were evaluated for their potential in bone regenerative medicine. In this study, hAFSCs were transduced with lentiviral vector harboring red fluorescent protein to investigate their role in the regeneration of critical-size bone defects in calvarial mouse model. To distinguish donor versus recipient cells, a transgenic mouse model carrying GFP fluorescent reporter was used as recipient to follow the fate of hAFSCs transplanted in vivo into Healos® scaffold. Our results showed that transduced hAFSCs can be tracked in vivo directly at the site of transplantation. The presence of GFP positive cells in the scaffold at 3 and 6 weeks after transplantation indicates that donor hAFSCs can recruit host cells during the repair process. These observations help clarify the role of hAFSCs in bone tissue repair.
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Affiliation(s)
- Mariangela Basile
- Department of Reconstructive Sciences, UConn Health, Farmington, Connecticut
- Department of Medicine and Ageing Sciences, G. d’Annunzio University of Chieti-Pescara, Italy
- StemTeCh Group, CAST, G. d’Annunzio University of Chieti-Pescara, Italy
| | - Francesco Marchegiani
- Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Padua, Italy
| | - Sanja Novak
- Department of Reconstructive Sciences, UConn Health, Farmington, Connecticut
| | - Ivo Kalajzic
- Department of Reconstructive Sciences, UConn Health, Farmington, Connecticut
| | - Roberta Di Pietro
- Department of Medicine and Ageing Sciences, G. d’Annunzio University of Chieti-Pescara, Italy
- StemTeCh Group, CAST, G. d’Annunzio University of Chieti-Pescara, Italy
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Isolation and Molecular Characterization of Amniotic Fluid-Derived Mesenchymal Stem Cells Obtained from Caesarean Sections. Stem Cells Int 2017; 2017:5932706. [PMID: 29225627 PMCID: PMC5684599 DOI: 10.1155/2017/5932706] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2017] [Revised: 09/13/2017] [Accepted: 10/01/2017] [Indexed: 01/12/2023] Open
Abstract
Human amniotic fluid cells are immune-privileged with low immunogenicity and anti-inflammatory properties. They are able to self-renew, are highly proliferative, and have a broad differentiation potential, making them amenable for cell-based therapies. Amniotic fluid (AF) is routinely obtained via amniocentesis and contains heterogeneous populations of foetal-derived progenitor cells including mesenchymal stem cells (MSCs). In this study, we isolated human MSCs from AF (AF-MSCs) obtained during Caesarean sections (C-sections) and characterized them. These AF-MSCs showed typical MSC characteristics such as morphology, in vitro differentiation potential, surface marker expression, and secreted factors. Besides vimentin and the stem cell marker CD133, subpopulations of AF-MSCs expressed pluripotency-associated markers such as SSEA4, c-Kit, TRA-1-60, and TRA-1-81. The secretome and related gene ontology (GO) terms underline their immune modulatory properties. Furthermore, transcriptome analyses revealed similarities with native foetal bone marrow-derived MSCs. Significant KEGG pathways as well as GO terms are mostly related to immune function, embryonic skeletal system, and TGFβ-signalling. An AF-MSC-enriched gene set included putative AF-MSC markers PSG5, EMX-2, and EVR-3. In essence, C-section-derived AF-MSCs can be routinely obtained and are amenable for personalized cell therapies and disease modelling.
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Wang M, Li H, Si J, Dai J, Shi J, Wang X, Guo L, Shen G. Amniotic fluid-derived stem cells mixed with platelet rich plasma for restoration of rat alveolar bone defect. Acta Biochim Biophys Sin (Shanghai) 2017; 49:197-207. [PMID: 28104582 DOI: 10.1093/abbs/gmw133] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2016] [Indexed: 12/12/2022] Open
Abstract
Stem cells isolated from the amniotic fluid have been shown as a promising candidate for cell therapy and tissue engineering. However, the experimental and preclinical applications of amniotic fluid-derived stem cells (AFSCs) in the very field of maxillofacial bone tissue engineering are still limited. In this study, rat AFSCs were successfully harvested and characterized in vitro. The rat AFSCs showed typical fibroblastoid morphology, stable proliferation activity and multi-differentiation potential. Flow-cytometry analysis demonstrated that these cells were positive for CD29, CD44, and CD90, while negative for hematopoietic markers such as CD34 and CD45. The regenerative performance of AFSCs-premixed with platelet rich plasma (PRP) gel in restoration of alveolar bone defect was further investigated using a modified rat maxillary alveolar defect model. Micro-computer tomography and histological examination showed a superior regenerative capacity of AFSCs-premixed with PRP gel at both 4 and 8 weeks after operation comparing with control groups. Moreover, the implanted AFSCs can survive in the defect site and directly participate in the bone tissue regeneration. Taken together, these results indicated the feasibility of an AFSCs-based alveolar bone tissue engineering strategy for alveolar defect restoration.
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Affiliation(s)
- Minjiao Wang
- Department of Oral and Craniomaxillofacial Science, Ninth People's Hospital College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
| | - Hongliang Li
- Department of Oral and Craniomaxillofacial Science, Ninth People's Hospital College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
| | - Jiawen Si
- Department of Oral and Craniomaxillofacial Science, Ninth People's Hospital College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
| | - Jiewen Dai
- Department of Oral and Craniomaxillofacial Science, Ninth People's Hospital College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
| | - Jun Shi
- Department of Oral and Craniomaxillofacial Science, Ninth People's Hospital College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
| | - Xudong Wang
- Department of Oral and Craniomaxillofacial Science, Ninth People's Hospital College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
| | - Lihe Guo
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
| | - Guofang Shen
- Department of Oral and Craniomaxillofacial Science, Ninth People's Hospital College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai 200011, China
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Martinelli D, Pereira RC, Mogni M, Benelli R, Mastrogiacomo M, Coviello D, Cancedda R, Gentili C. A humanized system to expand in vitro amniotic fluid-derived stem cells intended for clinical application. Cytotherapy 2016; 18:438-51. [PMID: 26857233 DOI: 10.1016/j.jcyt.2015.11.020] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2015] [Revised: 11/17/2015] [Accepted: 11/29/2015] [Indexed: 12/16/2022]
Abstract
BACKGROUND AIMS The amniotic fluid is a new source of multipotent stem cells with therapeutic potential for human diseases. In agreement with the regulatory requirement to reduce and possibly to avoid animal-derived reagents in the culture of cells intended for cell therapy, bovine serum, the most common supplement in the culture medium, was replaced by human platelet-derived growth factors. METHODS We tested a new culture medium to expand monolayers of human amniotic fluid stem cells (hAFSC) for clinical use. The AFSC were isolated by c-Kit selection and expanded in media supplemented with either bovine serum or a human platelet lysate (Lyset). RESULTS We compared proliferation kinetics, colony-forming unit percentage, multilineage differentiation, immunophenotypic characterization and inhibition of peripheral blood mononuclear cell proliferation of the two AFSC cell cultures and we found no significant differences. Moreover, the karyotype analysis of the cells expanded in the presence of the platelet lysate did not present cytogenetic abnormalities and in vitro and in vivo studies revealed no cell tumorigenicity. CONCLUSIONS Platelet derivatives represent a rich source of growth factors that can play a safety role in the homeostasis, proliferation and remodeling of tissue healing. We propose human platelet extracts as a preferential alternative to animal serum for the expansion of stem cells for clinical applications.
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Affiliation(s)
- Daniela Martinelli
- Laboratory of Regenerative Medicine, DIMES, IRCCS AOU San Martino-IST Largo Rosanna Benzi 10, University of Genova, Genova, Italy
| | - Rui Cruz Pereira
- Laboratory of Regenerative Medicine, DIMES, IRCCS AOU San Martino-IST Largo Rosanna Benzi 10, University of Genova, Genova, Italy
| | - Massimo Mogni
- Laboratory of Human Genetics, E.O. Ospedali Galliera, Genova, Italy
| | - Roberto Benelli
- Laboratory of Immunology, IRCCS AOU San Martino-IST Largo Rosanna Benzi 10, Genoa, Italy
| | - Maddalena Mastrogiacomo
- Laboratory of Regenerative Medicine, DIMES, IRCCS AOU San Martino-IST Largo Rosanna Benzi 10, University of Genova, Genova, Italy
| | | | - Ranieri Cancedda
- Laboratory of Regenerative Medicine, DIMES, IRCCS AOU San Martino-IST Largo Rosanna Benzi 10, University of Genova, Genova, Italy
| | - Chiara Gentili
- Laboratory of Regenerative Medicine, DIMES, IRCCS AOU San Martino-IST Largo Rosanna Benzi 10, University of Genova, Genova, Italy.
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Dicarlo M, Bianchi N, Ferretti C, Orciani M, Di Primio R, Mattioli-Belmonte M. Evidence Supporting a Paracrine Effect of IGF-1/VEGF on Human Mesenchymal Stromal Cell Commitment. Cells Tissues Organs 2016; 201:333-41. [PMID: 27179123 DOI: 10.1159/000445346] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/09/2016] [Indexed: 11/19/2022] Open
Abstract
Healing of skeletal defects is strictly dependent on osteogenesis and efficient vascularization of engineered scaffolds. Insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) are both involved in these processes. The in vitro administration of IGF-1 in association with VEGF is able to modulate the osteoblastic or endothelial commitment of mesenchymal stromal cells (MSCs) of different origins (e.g. periosteum and skin). In the present study, in order to deepen a possible paracrine effect of IGF-1 and VEGF on periosteum-derived progenitor cells (PDPCs) and skin-derived MSCs (S-MSCs), a Transwell coculture approach was used. We explored the genes involved in endothelial and osteoblastic differentiation, those modulating mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI3K)-AKT signaling pathways as well as genes implicated in stemness (i.e. Sox2, Oct4, and Nanog). Periosteal cells, which are typically committed toward osteoblastogenesis, are driven in the direction of endothelial gene expression when influenced by S-MSCs. The latter, once influenced by PDPCs, lose their endothelial commitment and increase the expression of osteoblast-associated genes. PI3K/AKT and MAPK signaling pathways seem to be markedly involved in this behavior. Our results evidence that paracrine signals between MSCs may differently modulate their commitment in a bone microenvironment, opening stimulating viewpoints for skeletal tissue engineering strategies coupling angiogenesis and osteogenesis processes.
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Affiliation(s)
- Manuela Dicarlo
- Department of Clinical and Molecular Sciences, Universitx00E0; Politecnica delle Marche, Ancona, Italy
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Pipino C, Pandolfi A. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential. World J Stem Cells 2015; 7:681-690. [PMID: 26029340 PMCID: PMC4444609 DOI: 10.4252/wjsc.v7.i4.681] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2014] [Revised: 01/16/2015] [Accepted: 02/11/2015] [Indexed: 02/06/2023] Open
Abstract
In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.
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Ferretti C, Vozzi G, Falconi M, Orciani M, Gesi M, Di Primio R, Mattioli-Belmonte M. Role of IGF1 and IGF1/VEGF on Human Mesenchymal Stromal Cells in Bone Healing: Two Sources and Two Fates. Tissue Eng Part A 2014; 20:2473-82. [DOI: 10.1089/ten.tea.2013.0453] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Affiliation(s)
- Concetta Ferretti
- Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy
| | - Giovanni Vozzi
- Faculty of Engineering, Research Centre “E. Piaggio,” University of Pisa, Pisa, Italy
| | - Mirella Falconi
- Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
| | - Monia Orciani
- Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy
| | - Marco Gesi
- Department of Translational Research and New Technology in Medicine and Surgery, University of Pisa, Pisa, Italy
| | - Roberto Di Primio
- Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy
| | - Monica Mattioli-Belmonte
- Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy
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