Publications by authors named "Ranieri Cancedda"

142 Publications

Platelet Lysate Induces in Human Osteoblasts Resumption of Cell Proliferation and Activation of Pathways Relevant for Revascularization and Regeneration of Damaged Bone.

Int J Mol Sci 2020 Jul 20;21(14). Epub 2020 Jul 20.

Department of Internal Medicine (DIMI), University of Genova, 16132 Genova, Italy.

To understand the regenerative effect of platelet-released molecules in bone repair one should investigate the cascade of events involving the resident osteoblast population during the reconstructive process. Here the in vitro response of human osteoblasts to a platelet lysate (PL) stimulus is reported. Quiescent or very slow dividing osteoblasts showed a burst of proliferation after PL stimulation and returned to a none or very slow dividing condition when the PL was removed. PL stimulated osteoblasts maintained a differentiation capability in vitro and in vivo when tested in absence of PL. Since angiogenesis plays a crucial role in the bone healing process, we investigated in PL stimulated osteoblasts the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) pathways, involved in both angiogenesis and bone regeneration. We observed phosphorylation of STAT3 and a strong induction, nuclear translocation and DNA binding of HIF-1α. In agreement with the induction of HIF-1α an enhanced secretion of vascular endothelial growth factor (VEGF) occurred. The double effect of the PL on quiescent osteoblasts, i.e., resumption of proliferation and activation of pathways promoting both angiogenesis and bone formation, provides a rationale to the application of PL as therapeutic agent in post-traumatic bone repair.
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http://dx.doi.org/10.3390/ijms21145123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403959PMC
July 2020

Progenitor Cells Activated by Platelet Lysate in Human Articular Cartilage as a Tool for Future Cartilage Engineering and Reparative Strategies.

Cells 2020 04 23;9(4). Epub 2020 Apr 23.

Regenerative Medicine Laboratory, Department of Experimental Medicine (DIMES), University of Genova, via Leon Battista Alberti 2, 16132 Genova, Italy.

Regenerative strategies for human articular cartilage are still challenging despite the presence of resident progenitor cell population. Today, many efforts in the field of regenerative medicine focus on the use of platelet derivatives due to their ability to reactivate endogenous mechanisms supporting tissue repair. While their use in orthopedics continues, mechanisms of action and efficacy need further characterization. We describe that the platelet lysate (PL) is able to activate chondro-progenitor cells in a terminally differentiated cartilage tissue. Primary cultures of human articular chondrocytes (ACs) and cartilage explants were set up from donor hip joint biopsies and were treated in vitro with PL. PL recruited a chondro-progenitors (CPCs)-enriched population from ex vivo cartilage culture, that showed high proliferation rate, clonogenicity and nestin expression. CPCs were positive for in vitro tri-lineage differentiation and formed hyaline cartilage-like tissue in vivo without hypertrophic fate. Moreover, the secretory profile of CPCs was analyzed, together with their migratory capabilities. Some CPC-features were also induced in PL-treated ACs compared to fetal bovine serum (FBS)-control ACs. PL treatment of human articular cartilage activates a stem cell niche responsive to injury. These facts can improve the PL therapeutic efficacy in cartilage applications.
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http://dx.doi.org/10.3390/cells9041052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226425PMC
April 2020

Growth Factors Delivery System for Skin Regeneration: An Advanced Wound Dressing.

Pharmaceutics 2020 Feb 3;12(2). Epub 2020 Feb 3.

Department of Internal Medicine (DIMI), University of Genova, viale Benedetto XV 10, 16132 Genova, Italy.

Standard treatments of chronic skin ulcers based on the direct application of dressings still present several limits with regard to a complete tissue regeneration. Innovative strategies in tissue engineering offer materials that can tune cell behavior and promote growth tissue favoring cell recruitment in the early stages of wound healing. A combination of Alginate (Alg), Sericin (SS) with Platelet Lysate (PL), as a freeze-dried sponge, is proposed to generate a bioactive wound dressing to care skin lesions. Biomembranes at different composition were tested for the release of platelet growth factors, cytotoxicity, protective effects against oxidative stress and cell proliferation induction. The highest level of the growth factors release occurred within 48 h, an optimized time to burst a healing process in vivo; the presence of SS differently modulated the release of the factors by interaction with the proteins composing the biomembranes. Any cytotoxicity was registered, whereas a capability to protect cells against oxidative stress and induce proliferation was observed when PL was included in the biomembrane. In a mouse skin lesion model, the biomembranes with PL promoted the healing process, inducing an accelerated and more pronounced burst of inflammation, formation of granulation tissue and new collagen deposition, leading to a more rapid skin regeneration.
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http://dx.doi.org/10.3390/pharmaceutics12020120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076531PMC
February 2020

CFSE: A New Method for Identifying Human Limbal Stem Cells and Following Their Migration in Human Cornea.

In Vivo 2019 Nov-Dec;33(6):1851-1855

Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy.

Aim: To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium.

Materials And Methods: Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined.

Results: CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD).

Conclusion: CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation.
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http://dx.doi.org/10.21873/invivo.11678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899114PMC
March 2020

Platelet Lysate Inhibits NF-κB Activation and Induces Proliferation and an Alert State in Quiescent Human Umbilical Vein Endothelial Cells Retaining Their Differentiation Capability.

Cells 2019 04 9;8(4). Epub 2019 Apr 9.

Department of Experimental Medicine, University of Genova, via Alberti 2, 16132 Genova, Italy.

Injured blood vessel repair and blood circulation re-establishment are crucial events for tissue repair. We investigated in primary cultures of human umbilical vein endothelial cells (HUVEC), the effects of platelet lysate (PL), a cocktail of factors released by activated platelets following blood vessel disruption and involved in the wound-healing process triggering. PL exerted a protective effect on HUVEC in an inflammatory milieu by inhibiting IL-1α-activated NF-κB pathway and by inducing the secretion of PGE₂, a pro-resolving molecule in the wound microenvironment. Moreover, PL enhanced HUVEC proliferation, without affecting their capability of forming tube-like structures on matrigel, and activated resting quiescent cells to re-enter cell cycle. In agreement with these findings, proliferation-related pathways Akt and ERK were activated. The expression of the cell-cycle activator Cyclin D1 was also enhanced, as well as the expression of the High Mobility Group Box-1 (HMGB1), a protein of the alarmin group involved in tissue homeostasis, repair, and remodeling. These in vitro data suggest a possible in vivo contribution of PL to new vessel formation after a wound by activation of cells resident in vessel walls. Our biochemical study provides a rationale for the clinical use of PL in the treatment of wound healing-related pathologies.
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http://dx.doi.org/10.3390/cells8040331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523925PMC
April 2019

Synchrotron radiation techniques boost the research in bone tissue engineering.

Acta Biomater 2019 04 15;89:33-46. Epub 2019 Mar 15.

Institute of Nanotechnology- CNR, Rome Unit, Rome, Italy.

X-ray Synchrotron radiation-based techniques, in particular Micro-tomography and Micro-diffraction, were exploited to investigate the structure of bone deposited in vivo within a porous ceramic scaffold. Bone formation was studied by implanting Mesenchymal Stem Cell (MSC) seeded ceramic scaffolds in a mouse model. Osteoblasts derived from the seeded MSC and from differentiation of cells migrated within the scaffold together with the blood vessels, deposited within the scaffold pores an organic collagenous matrix on which a precursor mineral amorphous liquid-phase, containing Ca++ and PO crystallized filling the gaps between the collagen molecules. Histology offered a valid instrument to investigate the engineered tissue structure, but, unfortunately, limited itself to a macroscopic analysis. The evolution of the X-ray Synchrotron radiation-based techniques and the combination of micro X-ray diffraction with X-ray phase-contrast imaging enabled to study the dynamic of the structural and morphological changes occurring during the new bone deposition, biomineralization and vascularization. In fact, the unique features of Synchrotron radiation, is providing the high spatial resolution probe which is necessary for the study of complex materials presenting heterogeneity from micron-scale to meso- and nano-scale. Indeed, this is the occurrence in the heterogeneous and hierarchical bone tissue where an organic matter, such as the collagenous matrix, interacts with mineral nano-crystals to generate a hybrid multiscale biomaterial with unique physical properties. In this framework, the use of advanced synchrotron radiation techniques allowed to understand and to clarify fundamental aspects of the bone formation process within the bioceramic, i.e. biomineralization and vascularization, including to obtain deeper knowledge on bone deposition, mineralization and reabsorption in different health, aging and pathological conditions. In this review we present an overview of the X-ray Synchrotron radiation techniques and we provide a general outlook of their applications on bone Tissue Engineering, with a focus on our group work. STATEMENT OF SIGNIFICANCE: Synchrotron Radiation techniques for Tissue Engineering In this review we report recent applications of X-ray Synchrotron radiation-based techniques, in particular Microtomography and Microdiffraction, to investigations on the structure of ceramic scaffolds and bone tissue regeneration. Tissue engineering has made significant advances in bone regeneration by proposing the use of mesenchymal stem cells in combination with various types of scaffolds. The efficacy of the biomaterials used to date is not considered optimal in terms of resorbability and bone formation, resulting in a poor vascularization at the implant site. The review largely based on our publications in the last ten years could help the study of the regenerative model proposed. We also believe that the new imaging technologies we describe could be a starting point for the development of additional new techniques with the final aim of transferring them to the clinical practice.
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http://dx.doi.org/10.1016/j.actbio.2019.03.031DOI Listing
April 2019

Beta-tricalcium phosphate ceramic triggers fast and robust bone formation by human mesenchymal stem cells.

J Tissue Eng Regen Med 2019 06 15;13(6):1007-1018. Epub 2019 Apr 15.

Laboratory of Regenerative Medicine, Department of Experimental Medicine (DIMES), University of Genoa, Genoa, Italy.

Due to their osteoconductive and inductive properties, a variety of calcium phosphate (CaP) scaffolds are commonly used in orthopaedics as graft material to heal bone defects. In this study, we have used two CaP scaffolds with different hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ratios (MBCP®; 60/40 and MBCP ®; 20/80) to investigate their intrinsic capacity to favour human bone marrow stem cells (hBMSCs) osteogenic differentiation capacity. We report that MBCP ® showed in in vitro culture model a higher rate of calcium ion release in comparison with MBCP®. In two defined coculture systems, the hBMSC seeded onto MBCP ® presented an increased amount of VEGF secretion, resulting in an enhanced endothelial cell proliferation and capillary formation compared with hBMSC seeded onto MBCP®. When both ceramics combined with hBMSC were implanted in a nude mouse model, we observed a faster osteogenic differentiation and enhancement mature bone deposition sustained by the presence of a vast host vasculature within the MBCP ® ceramics. Bone formation was observed in samples highly positive to the activation of calcium sensing receptor protein (CaSr) on the surface of seeded hBMSC that also shown higher BMP-2 protein expression. With these data we provide valuable insights in the possible mechanisms of ossification and angiogenesis by hBMSC that we believe to be primed by calcium ions released from CaP scaffolds. Evidences could lead to an optimization of ceramic scaffolds to prime bone repair.
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http://dx.doi.org/10.1002/term.2848DOI Listing
June 2019

Platelet Lysate Activates Human Subcutaneous Adipose Tissue Cells by Promoting Cell Proliferation and Their Paracrine Activity Toward Epidermal Keratinocytes.

Front Bioeng Biotechnol 2018 21;6:203. Epub 2018 Dec 21.

Department of Experimental Medicine (DIMES) and Department of Internal Medicine (DIMI), University of Genoa; Biotherapy Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy.

Skin chronic wounds are non-healing ulcerative defects, which arise in association with a morbidity state, such as diabetes and vascular insufficiency or as the consequence of systemic factors including advanced age. Platelet Rich Plasma, a platelet-rich blood fraction, can significantly improve the healing of human skin chronic ulcers. Given that the subcutaneous adipose tissue is located beneath the skin and plays a role in the skin homeostasis, in this study, we investigated the response of human subcutaneous adipose tissue cells to platelet content in a model mimicking the milieu of a deep skin injury. Considering that, at the wound site, plasma turn to serum, platelets are activated and inflammation occurs, human adipose-derived stromal cells (hASC) were cultured with Human Serum (HS) supplemented or not with Platelet Lysate (PL) and/or IL-1α. We observed that HS sustained hASC proliferation more efficiently than FBS and induced a spontaneous adipogenic differentiation in the cells. PL added to HS enhanced hASC proliferation, regardless the presence of IL-1α. In the presence of PL, hASC progressively lessened the adipogenic phenotype, possibly because the proliferation of less committed cells was induced. However, these cells resumed adipogenesis in permissive conditions. Accordingly, PL induced in quiescent cells activation of the proliferation-related pathways ERK, Akt, and STAT-3 and expression of Cyclin D1. Moreover, PL induced an early and transient increase of the pro-inflammatory response triggered by IL-1α, by inducing COX-2 expression and secretion of a large amount of PGE, IL-6, and IL-8. Media conditioned by PL-stimulated hASC exerted a chemotactic activity on human keratinocytes and favored the healing of an scratch wound. In order to bridge the gap between results and possible events, the stimuli were also tested in cultures of human adipose tissue biopsies (hAT). PL induced cell proliferation in hAT and outgrowth of committed progenitor cells able to differentiate in permissive conditions. In conclusion, we report that the adipose tissue responds to the wound microenvironment by activating the proliferation of adipose tissue progenitor cells and promoting the release of factors favoring wound healing.
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http://dx.doi.org/10.3389/fbioe.2018.00203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6308153PMC
December 2018

Circulating healing (CH) cells expressing BST2 are functionally activated by the injury-regulated systemic factor HGFA.

Stem Cell Res Ther 2018 11 8;9(1):300. Epub 2018 Nov 8.

Cellular Oncology Laboratory, Department of Experimental Medicine (DIMES), University of Genova, Largo Rosanna Benzi 10, 16132, Genova, Italy.

Background: Restoration of damaged tissues through the activation of endogenous progenitors is an attractive therapeutic option. A deep evaluation of the intrinsic stem/progenitor cell properties as well as the reciprocal interactions with injured environments is of critical importance.

Methods: Here, we show that bone marrow stromal cell antigen 2 (BST2) allows the isolation of a population of circulating progenitors, the circulating healing (CH) cells, characterized by a distinctive core signature. The bone marrow (BM) origin of BST2 CH cells has been strengthened by the co-expression of leptin receptor, the hallmark of a subpopulation of BM-skeletal stem cells.

Results: BST2 CH cells retained the capacity to (i) respond to injury signals generated by a bone fracture, (ii) modify the expression of cell motility genes following damage, and (iii) react to hepatocyte growth factor-activator (HGFA), an injury-related stimulus sufficient to induce their transition into G, a state in which cells are functionally activated and participate in tissue repair.

Conclusions: Taken together, these results could pave the way for the identification of new strategies to enhance and potentiate endogenous regenerative mechanisms for future therapies.
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http://dx.doi.org/10.1186/s13287-018-1056-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225669PMC
November 2018

Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate.

Acta Biomater 2018 06 17;73:365-376. Epub 2018 Apr 17.

Smart Materials, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genoa, Italy. Electronic address:

Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care.

Statement Of Significance: Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60 °C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and store them, resulting in an easy and fast accessibility and an expanded use of hPL for wound healing.
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http://dx.doi.org/10.1016/j.actbio.2018.04.025DOI Listing
June 2018

High-Resolution X-Ray Tomography: A 3D Exploration Into the Skeletal Architecture in Mouse Models Submitted to Microgravity Constraints.

Front Physiol 2018 6;9:181. Epub 2018 Mar 6.

Dipartimento di Medicina Sperimentale, Universita' di Genova and Ospedale Policlinico San Martino, Genova, Italy.

Bone remodeling process consists in a slow building phase and in faster resorption with the objective to maintain a functional skeleton locomotion to counteract the Earth gravity. Thus, during spaceflights, the skeleton does not act against gravity, with a rapid decrease of bone mass and density, favoring bone fracture. Several studies approached the problem by imaging the bone architecture and density of cosmonauts returned by the different spaceflights. However, the weaknesses of the previously reported studies was two-fold: on the one hand the research suffered the small statistical sample size of almost all human spaceflight studies, on the other the results were not fully reliable, mainly due to the fact that the observed bone structures were small compared with the spatial resolution of the available imaging devices. The recent advances in high-resolution X-ray tomography have stimulated the study of weight-bearing skeletal sites by novel approaches, mainly based on the use of the mouse and its various strains as an animal model, and sometimes taking advantage of the synchrotron radiation support to approach studies of 3D bone architecture and mineralization degree mapping at different hierarchical levels. Here we report the first, to our knowledge, systematic review of the recent advances in studying the skeletal bone architecture by high-resolution X-ray tomography after submission of mice models to microgravity constrains.
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http://dx.doi.org/10.3389/fphys.2018.00181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859385PMC
March 2018

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support.

Front Bioeng Biotechnol 2017 20;5:66. Epub 2017 Nov 20.

Department of Experimental Medicine, University of Genoa, Genoa, Italy.

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.
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http://dx.doi.org/10.3389/fbioe.2017.00066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702080PMC
November 2017

Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1.

J Tissue Eng Regen Med 2018 03 5;12(3):e1691-e1703. Epub 2017 Dec 5.

Biorigen Srl., Largo Rosanna Benzi 10, Genoa, Italy.

The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage.
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http://dx.doi.org/10.1002/term.2595DOI Listing
March 2018

Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment.

Regen Med 2017 07 3;12(5):525-539. Epub 2017 Aug 3.

Department of Experimental Medicine (DIMES), University of Genoa & IRCCS Policlinico San Martino, Genoa, Italy.

Aim: This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation.

Materials & Methods: Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation.

Results & Conclusion: Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.
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http://dx.doi.org/10.2217/rme-2017-0023DOI Listing
July 2017

Learning from Mother Nature: Innovative Tools to Boost Endogenous Repair of Critical or Difficult-to-Heal Large Tissue Defects.

Front Bioeng Biotechnol 2017 28;5:28. Epub 2017 Apr 28.

IRCCS AOU San Martino-IST National Institute of Cancer Research, Genova, Italy.

For repair of chronic or difficult-to-heal tissue lesions and defects, major constraints exist to a broad application of cell therapy and tissue engineering approaches, i.e., transplantation of "" expanded autologous stem/progenitor cells, alone or associated with carrier biomaterials. To enable a large number of patients to benefit, new strategies should be considered. One of the main goals of contemporary regenerative medicine is to develop new regenerative therapies, inspired from Mother Nature. In all injured tissues, when platelets are activated by tissue contact, their released factors promote innate immune cell migration to the wound site. Platelet-derived factors and factors secreted by migrating immune cells create an inflammatory microenvironment, in turn, causing the activation of angiogenesis and vasculogenesis processes. Eventually, repair or regeneration of the injured tissue occurs paracrine signals activating, mobilizing or recruiting to the wound site cells with healing potential, such as stem cells, progenitors, or undifferentiated cells derived from the reprogramming of tissue differentiated cells. This review, largely based on our studies, discusses the identification of new tools, inspired by cellular and molecular mechanisms overseeing physiological tissue healing, that could reactivate dormant endogenous regeneration mechanisms lost during evolution and ontogenesis.
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http://dx.doi.org/10.3389/fbioe.2017.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408079PMC
April 2017

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential.

Stem Cells Transl Med 2017 05 8;6(5):1340-1355. Epub 2017 Mar 8.

Regenerative Medicine Laboratory, Department of Experimental Medicine, University of Genova, Genova, Italy.

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O versus 1% O preconditioning). The capacity of the c-KIT hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355.
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http://dx.doi.org/10.1002/sctm.16-0297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442724PMC
May 2017

Mesenchymal Stem Cell-Derived Extracellular Vesicles as Mediators of Anti-Inflammatory Effects: Endorsement of Macrophage Polarization.

Stem Cells Transl Med 2017 03 31;6(3):1018-1028. Epub 2017 Jan 31.

U.O. Regenerative Medicine, IRCCS AOU San Martino-IST, National Cancer Research Institute, Genova, Italy.

Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived-MSCs to investigate their involvement as modulators of MSC anti-inflammatory effects inducing macrophage polarization. The EV-isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EV and EV ). Both types of EVs were efficiently internalized by responding bone marrow-derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin-induced skeletal muscle damage, EV and EV interacted with macrophages recruited during the initial inflammatory response. In injured and EV-treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206 cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EV administration. Collectively, these data indicate that MSC-EVs possess effective anti-inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. Stem Cells Translational Medicine 2017 Stem Cells Translational Medicine 2017;6:1018-1028.
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http://dx.doi.org/10.1002/sctm.16-0363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442783PMC
March 2017

LCN2 overexpression in bone enhances the hematopoietic compartment via modulation of the bone marrow microenvironment.

J Cell Physiol 2017 Nov 21;232(11):3077-3087. Epub 2017 Feb 21.

Dipartimento di Medicina Sperimentale, Universita' di Genova, Genova, Italy.

Lipocalin-2 (LCN2) is a member of the lipocalin family whose expression is modulated in several conditions, including cell differentiation, innate immunity, stress, and cancer. Although it is known that it is expressed in bone, its function in this tissue remains poorly studied. To this end, we took advantage of transgenic mice lines that expressed LCN2 driven by a bone specific type I collagen (LCN2-Tg). In the bone marrow (BM) of LCN2-Tg mice we observed an increased number of phenotypically long-term hematopoietic stem cells (LT-HSC) that also displayed a higher proliferation rate compared to wild-type controls (Wt). Furthermore, hematopoietic progenitor cells, obtained from LCN2-Tg BM showed an increased clonogenic capacity compared to those obtained from LCN2-Tg spleen, a higher concentration of serum erythropoietin and a higher number of mature erythrocytes in the peripheral blood of old LCN2-Tg animals compared to aged-matched wt. The findings of a combined increase in the BM of the LCN2-Tg mice of SDF-1, SCF, and TIMP-1 levels along with the reduction of both MMP-9 activity and cathepsin K concentration may explain the observed effects on the HSC compartment. This study shows that LCN2 overexpression in bones modifies the BM microenvironment via modulation of the expression of key secreted factors and cytokines, which in turn regulate the HSC niche behavior enhancing both HSC homing in young mice and erythrocytes production in older mice.
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http://dx.doi.org/10.1002/jcp.25755DOI Listing
November 2017

Allogeneic platelet-rich plasma affects monocyte differentiation to dendritic cells causing an anti-inflammatory microenvironment, putatively fostering wound healing.

J Tissue Eng Regen Med 2018 01 11;12(1):30-43. Epub 2017 May 11.

Unit of Molecular Oncology and Angiogenesis, IRCCS San Martino Hospital-IST- National Institute for Cancer Research, Genoa, Italy.

Autologous platelet-rich plasma (PRP) is used clinically to induce repair of different tissues through the release of bioactive molecules. In some patients, the production of efficient autologous PRP is unfeasible due to their compromised health. Allogeneic PRP mismatched for AB0 and Rh antigens was developed. The effect of allogeneic PRP on immune response should be defined to use it in clinical practice avoiding side effects. Thus, whether PRP affects the differentiation of peripheral blood monocytes to dendritic cells upon stimulation with granulocyte monocyte colony stimulating factor and interleukin-4 was investigated. Indeed, these cells are the main players of immune response and tissue repair. PRP inhibited the differentiation of monocytes to CD1a dendritic cells and favoured the expansion of phagocytic CD163 CD206 fibrocyte-like cells. These cells produced interleukin-10 and prostaglandin-E , but not interferon-γ, upon stimulation with lipopolysaccharides. Moreover, they promoted the expansion of regulatory CD4 CD25 FoxP3 T cells upon allostimulation or antigen specific priming. Finally, the conditioned medium harvested from monocytes differentiated with PRP triggered a strong chemotactic effect on mesenchymal cells in both scratch and transwell migration assays. These results strongly suggest that allogeneic PRP can foster the differentiation of monocytes to a regulatory anti-inflammatory population, possibly favouring wound healing. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2361DOI Listing
January 2018

Platelet-rich plasma-based bioactive membrane as a new advanced wound care tool.

J Tissue Eng Regen Med 2018 01 18;12(1):e82-e96. Epub 2017 Apr 18.

Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy.

Chronic skin ulcers, consequence of diabetes and other pathological conditions, heavily compromise the patient life quality and represent a high and constantly growing cost for National Health Services. Autologous platelet-rich plasma (PRP), has been proposed to treat these lesions. The absence of guidelines for the PRP production and the need of a fresh preparation for each treatment lead us to develop a protocol for the production of an allogenic PRP-based bioactive membrane (BAM), standardized for platelet concentration and growth factor release. This work compares BAMs obtained starting from two different platelet concentrations. There was no direct correlation between the amount of growth factors released by BAM in vitro and the initial platelet count. However, different release kinetics were noticed for different growth factors, suggesting that they were differently retained by the two BAMs. The angiogenic potential of both BAMs was determined by Luminex Angiogenesis Assay. The biological activity of the factors released by the two BAMs was confirmed by cell proliferation and migration. A diabetic mouse chronic ulcer model was used to define the best PRP therapeutic dose in vivo. Both BAMs induced wound healing by increasing the thickness of the regenerated epidermis and the vessel number. However, a too high platelet concentration resulted in a slowdown of the membrane resorption that interfered with the skin healing. Overall, the results indicate that the BAMs could represent a natural and effective wound healing tool for the treatment of skin ulcers. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2357DOI Listing
January 2018

Human Articular Chondrocytes Regulate Immune Response by Affecting Directly T Cell Proliferation and Indirectly Inhibiting Monocyte Differentiation to Professional Antigen-Presenting Cells.

Front Immunol 2016 24;7:415. Epub 2016 Oct 24.

Molecular Oncology and Angiogenesis Unit, Department of Integrated Oncological Therapies, IRCCS AOU San Martino IST , Genova , Italy.

Autologous chondrocyte implantation is the current gold standard cell therapy for cartilage lesions. However, in some instances, the heavily compromised health of the patient can either impair or limit the recovery of the autologous chondrocytes and a satisfactory outcome of the implant. Allogeneic human articular chondrocytes (hAC) could be a good alternative, but the possible immunological incompatibility between recipient and hAC donor should be considered. Herein, we report that allogeneic hAC inhibited T lymphocyte response to antigen-dependent and -independent proliferative stimuli. This effect was maximal when T cells and hAC were in contact and it was not relieved by the addition of exogenous lymphocyte growth factor interleukin (IL)-2. More important, hAC impaired the differentiation of peripheral blood monocytes induced with granulocyte monocyte colony-stimulating factor and IL-4 (Mo) to professional antigen-presenting cells, such as dendritic cells (DC). Indeed, a marked inhibition of the onset of the CD1a expression and an ineffective downregulation of CD14 antigens was observed in Mo-hAC co-cultures. Furthermore, compared to immature or mature DC, Mo from Mo-hAC co-cultures did not trigger an efficacious allo-response. The prostaglandin (PG) E present in the Mo-hAC co-culture conditioned media is a putative candidate of the hAC-mediated inhibition of Mo maturation. Altogether, these findings indicate that allogeneic hAC inhibit, rather than trigger, immune response and strongly suggest that an efficient chondrocyte implantation could be possible also in an allogeneic setting.
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http://dx.doi.org/10.3389/fimmu.2016.00415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075572PMC
October 2016

The human amniotic fluid stem cell secretome effectively counteracts doxorubicin-induced cardiotoxicity.

Sci Rep 2016 07 21;6:29994. Epub 2016 Jul 21.

Regenerative Medicine Laboratory, Department of Experimental Medicine, University of Genova, Genova, Italy.

The anthracycline doxorubicin (Dox) is widely used in oncology, but it may cause a cardiomyopathy with bleak prognosis that cannot be effectively prevented. The secretome of human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to significantly reduce ischemic cardiac damage. Here it is shown that, following hypoxic preconditioning, hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with mouse neonatal ventricular cardiomyocytes (mNVCM) reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is associated with decreased DNA damage, nuclear translocation of NF-kB, and upregulation of the NF-kB controlled genes, Il6 and Cxcl1, promoting mNVCM survival. Furthermore, hAFS-CM induces expression of the efflux transporter, Abcb1b, and Dox extrusion from mNVCM. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation of Il6, Cxcl1 and Abcb1b, and prevention of Dox-initiated senescence and apoptosis in response to hAFS-CM. These results support the concept that hAFS are a valuable source of cardioprotective factors and lay the foundations for the development of a stem cell-based paracrine treatment of chemotherapy-related cardiotoxicity.
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http://dx.doi.org/10.1038/srep29994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956770PMC
July 2016

A humanized system to expand in vitro amniotic fluid-derived stem cells intended for clinical application.

Cytotherapy 2016 Mar;18(3):438-51

Laboratory of Regenerative Medicine, DIMES, IRCCS AOU San Martino-IST Largo Rosanna Benzi 10, University of Genova, Genova, Italy. Electronic address:

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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http://dx.doi.org/10.1016/j.jcyt.2015.11.020DOI Listing
March 2016

Combined platelet and plasma derivatives enhance proliferation of stem/progenitor cells maintaining their differentiation potential.

Cytotherapy 2015 Dec;17(12):1793-806

DIMES, University of Genoa, Genoa, Italy; IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy. Electronic address:

Background Aims: Platelet derivatives have been proposed as alternatives to animal sera given that for cell therapy applications, the use of fetal bovine/calf serum (FBS/FCS) is subjected to severe limitations for safety and ethical concerns. We developed a cell culture medium additive obtained by the combination of two blood-derived standardized components.

Methods: A platelet lysate (PL) and a platelet-poor plasma (PPP) were produced in a lyophilized form. Each component was characterized for its growth factor content (platelet-derived growth factor-BB/vascular endothelial growth factor). PL and PPP were used as single components or in combination in different ratio at cumulative 5% final concentration in the culture medium.

Results: The single components were less effective than the component combination. In primary cell cultures (bone marrow stromal cells, adipose derived adult stem cells, osteoblasts, chondrocytes, umbilical cord-derived mesenchymal stromal cells, lymphocytes), the PL/PPP supplement promoted an increased cell proliferation in respect to the standard FCS culture in a dose-dependent manner, maintaining the cell functionality, clonogenicity, phenotype and differentiative properties throughout the culture. At a different component ratio, the supplement was also used to support proliferation of a cell line (U-937).

Conclusions: The PL/PPP supplement is an efficient cell culture medium additive that can replace FCS to promote cell proliferation. It can outdo FCS, especially when adopted in primary cultures from tissue biopsies. Moreover, the dual component nature of the supplement allows the researcher to determine the more appropriate ratio of the two components for the nutritional and functional requirements of the cell type of interest.
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http://dx.doi.org/10.1016/j.jcyt.2015.09.004DOI Listing
December 2015

Identification of a New Cell Population Constitutively Circulating in Healthy Conditions and Endowed with a Homing Ability Toward Injured Sites.

Sci Rep 2015 Nov 12;5:16574. Epub 2015 Nov 12.

Department of Experimental Medicine (DIMES), University of Genova, Genova, 16132, Italy.

Stem and progenitor cells are the critical units for tissue maintenance, regeneration, and repair. The activation of regenerative events in response to tissue injury has been correlated with mobilization of tissue-resident progenitor cells, which is functional to the wound healing process. However, until now there has been no evidence for the presence of cells with a healing capacity circulating in healthy conditions. We identified a rare cell population present in the peripheral blood of healthy mice that actively participates in tissue repair. These Circulating cells, with a Homing ability and involved in the Healing process (CH cells), were identified by an innovative flowcytometry strategy as small cells not expressing CD45 and lineage markers. Their transcriptome profile revealed that CH cells are unique and present a high expression of key pluripotency- and epiblast-associated genes. More importantly, CH-labeled cells derived from healthy Red Fluorescent Protein (RFP)-transgenic mice and systemically injected into syngeneic fractured wild-type mice migrated and engrafted in wounded tissues, ultimately differentiating into tissue-specific cells. Accordingly, the number of CH cells in the peripheral blood rapidly decreased following femoral fracture. These findings uncover the existence of constitutively circulating cells that may represent novel, accessible, and versatile effectors of therapeutic tissue regeneration.
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http://dx.doi.org/10.1038/srep16574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642305PMC
November 2015

High-Resolution X-Ray Techniques as New Tool to Investigate the 3D Vascularization of Engineered-Bone Tissue.

Front Bioeng Biotechnol 2015 7;3:133. Epub 2015 Sep 7.

Dipartimento di Medicina Sperimentale dell'Università di Genova, AOU San Martino-IST , Genova , Italy.

The understanding of structure-function relationships in normal and pathologic mammalian tissues is at the basis of a tissue engineering (TE) approach for the development of biological substitutes to restore or improve tissue function. In this framework, it is interesting to investigate engineered bone tissue, formed when porous ceramic constructs are loaded with bone marrow stromal cells (BMSC) and implanted in vivo. To monitor the relation between bone formation and vascularization, it is important to achieve a detailed imaging and a quantitative description of the complete three-dimensional vascular network in such constructs. Here, we used synchrotron X-ray phase-contrast micro-tomography to visualize and analyze the three-dimensional micro-vascular networks in bone-engineered constructs, in an ectopic bone formation mouse-model. We compared samples seeded and not seeded with BMSC, as well as samples differently stained or unstained. Thanks to the high quality of the images, we investigated the 3D distribution of both vessels and collagen matrix and we obtained quantitative information for all different samples. We propose our approach as a tool for quantitative studies of angiogenesis in TE and for any pre-clinical investigation where a quantitative analysis of the vascular network is required.
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http://dx.doi.org/10.3389/fbioe.2015.00133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561513PMC
October 2015

Effects of long time exposure to simulated micro- and hypergravity on skeletal architecture.

J Mech Behav Biomed Mater 2015 Nov 23;51:1-12. Epub 2015 Jun 23.

Dipartimento di Medicina Sperimentale, Universita' di Genova & IRCCS AOU San Martino-IST, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy. Electronic address:

This manuscript reports the structural alterations occurring in mice skeleton as a consequence of the longest-term exposition (90 days) to simulated microgravity (hindlimb unloading) and hypergravity (2g) ever tested. Bone microstructural features were investigated by means of standard Cone Beam X-ray micro-CT, Synchrotron Radiation micro-CT and histology. Morphometric analysis confirmed deleterious bone architectural changes in lack of mechanical loading with a decrease of bone volume and density, while bone structure alterations caused by hypergravity were less evident. In the femurs from hypergravity-exposed mice, the head/neck cortical thickness increment was the main finding. In addition, in these mice the rate of larger trabeculae (60-75 μm) was significantly increased. Interestingly, the metaphyseal plate presented a significant adaptation to gravity changes. Mineralization of cartilage and bone deposition was increased in the 2g mice, whereas an enlargement of the growth plate cartilage was observed in the hindlimb unloaded group. Indeed, the presented data confirm and reinforce the detrimental effects on bone observed in real space microgravity and reveal region-specific effects on long bones. Finally these data could represent the starting point for further long-term experimentations that can deeply investigate the bone adaptation mechanisms to different mechanical force environments.
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http://dx.doi.org/10.1016/j.jmbbm.2015.06.014DOI Listing
November 2015

Skin physiology in microgravity: a 3-month stay aboard ISS induces dermal atrophy and affects cutaneous muscle and hair follicles cycling in mice.

NPJ Microgravity 2015 27;1:15002. Epub 2015 May 27.

Laboratory of Connective Tissues Biology, GIGA-Research, University of Liège, Sart Tilman, Belgium.

Aims: The Mice Drawer System (MDS) Tissue Sharing program was the longest rodent space mission ever performed. It provided 20 research teams with organs and tissues collected from mice having spent 3 months on the International Space Station (ISS). Our participation to this experiment aimed at investigating the impact of such prolonged exposure to extreme space conditions on mouse skin physiology.

Methods: Mice were maintained in the MDS for 91 days aboard ISS (space group (S)). Skin specimens were collected shortly after landing for morphometric, biochemical, and transcriptomic analyses. An exact replicate of the experiment in the MDS was performed on ground (ground group (G)).

Results: A significant reduction of dermal thickness (-15%, =0.05) was observed in S mice accompanied by an increased newly synthetized procollagen (+42%, =0.03), likely reflecting an increased collagen turnover. Transcriptomic data suggested that the dermal atrophy might be related to an early degradation of defective newly formed procollagen molecules. Interestingly, numerous hair follicles in growing anagen phase were observed in the three S mice, validated by a high expression of specific hair follicles genes, while only one mouse in the G controls showed growing hairs. By microarray analysis of whole thickness skin, we observed a significant modulation of 434 genes in S versus G mice. A large proportion of the upregulated transcripts encoded proteins related to striated muscle homeostasis.

Conclusions: These data suggest that a prolonged exposure to space conditions may induce skin atrophy, deregulate hair follicle cycle, and markedly affect the transcriptomic repertoire of the cutaneous striated muscle panniculus carnosus.
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http://dx.doi.org/10.1038/npjmgrav.2015.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515501PMC
May 2015

Transplanted Umbilical Cord Mesenchymal Stem Cells Modify the In Vivo Microenvironment Enhancing Angiogenesis and Leading to Bone Regeneration.

Stem Cells Dev 2015 Jul 18;24(13):1570-81. Epub 2015 Mar 18.

1 Department of Experimental Medicine (DIMES), University of Genoa , Genoa, Italy .

Umbilical cord mesenchymal stem cells (UC-MSCs) show properties similar to bone marrow mesenchymal stem cells (BM-MSCs), although controversial data exist regarding their osteogenic potential. We prepared clinical-grade UC-MSCs from Wharton's Jelly and we investigated if UC-MSCs could be used as substitutes for BM-MSCs in muscoloskeletal regeneration as a more readily available and functional source of MSCs. UC-MSCs were loaded onto scaffolds and implanted subcutaneously (ectopically) and in critical-sized calvarial defects (orthotopically) in mice. For live cell-tracking experiments, UC-MSCs were first transduced with the luciferase gene. Angiogenic properties of UC-MSCs were tested using the mouse metatarsal angiogenesis assay. Cell secretomes were screened for the presence of various cytokines using an array assay. Analysis of implanted scaffolds showed that UC-MSCs, contrary to BM-MSCs, remained detectable in the implants for 3 weeks at most and did not induce bone formation in an ectopic location. Instead, they induced a significant increase of blood vessel ingrowth. In agreement with these observations, UC-MSC-conditioned medium presented a distinct and stronger proinflammatory/chemotactic cytokine profile than BM-MSCs and a significantly enhanced angiogenic activity. When UC-MSCs were orthotopically transplanted in a calvarial defect, they promoted increased bone formation as well as BM-MSCs. However, at variance with BM-MSCs, the new bone was deposited through the activity of stimulated host cells, highlighting the importance of the microenvironment on determining cell commitment and response. Therefore, we propose, as therapy for bone lesions, the use of allogeneic UC-MSCs by not depositing bone matrix directly, but acting through the activation of endogenous repair mechanisms.
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http://dx.doi.org/10.1089/scd.2014.0490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4499786PMC
July 2015

A modified rabbit ulna defect model for evaluating periosteal substitutes in bone engineering: a pilot study.

Front Bioeng Biotechnol 2014 6;2:80. Epub 2015 Jan 6.

DIMES, University of Genova , Genova , Italy ; IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro , Genova , Italy.

The present work defines a modified critical size rabbit ulna defect model for bone regeneration in which a non-resorbable barrier membrane was used to separate the radius from the ulna to create a valid model for evaluation of tissue-engineered periosteal substitutes. Eight rabbits divided into two groups were used. Critical defects (15 mm) were made in the ulna completely eliminating periosteum. For group I, defects were filled with a nanohydroxyapatite poly(ester urethane) scaffold soaked in PBS and left as such (group Ia) or wrapped with a tissue-engineered periosteal substitute (group Ib). For group II, an expanded-polytetrafluoroethylene (e-PTFE) (GORE-TEX(®)) membrane was inserted around the radius then the defects received either scaffold alone (group IIa) or scaffold wrapped with periosteal substitute (group IIb). Animals were euthanized after 12-16 weeks, and bone regeneration was evaluated by radiography, computed microtomography (μCT), and histology. In the first group, we observed formation of radio-ulnar synostosis irrespective of the treatment. This was completely eliminated upon placement of the e-PTFE (GORE-TEX(®)) membrane in the second group of animals. In conclusion, modification of the model using a non-resorbable e-PTFE membrane to isolate the ulna from the radius was a valuable addition allowing for objective evaluation of the tissue-engineered periosteal substitute.
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http://dx.doi.org/10.3389/fbioe.2014.00080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285175PMC
January 2015