[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Nov 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Oct 2015 · Frontiers in Bioengineering and Biotechnology
[Show abstract][Hide abstract] ABSTRACT: A deeper comprehension of the biomineralization (BM) process is at the basis of tissue engineering and regenerative medicine developments. Several in-vivo and in-vitro studies were dedicated to this purpose via the application of 2D and 3D diagnostic techniques. Here, we develop a new methodology, based on different complementary experimental techniques (X-ray phase contrast tomography, micro-X-ray diffraction and micro-X-ray fluorescence scanning technique) coupled to new analytical tools. A qualitative and quantitative structural investigation, from the atomic to the micrometric length scale, is obtained for engineered bone tissues. The high spatial resolution achieved by X-ray scanning techniques allows us to monitor the bone formation at the first-formed mineral deposit at the organic–mineral interface within a porous scaffold. This work aims at providing a full comprehension of the morphology and functionality of the biomineralization process, which is of key importance for developing new drugs for preventing and healing bone diseases and for the development of bio-inspired materials.
No preview · Article · Jul 2015 · Nuclear Instruments and Methods in Physics Research Section B Beam Interactions with Materials and Atoms
[Show abstract][Hide abstract] ABSTRACT: 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%, P=0.05) was observed in S mice accompanied by an increased newly synthetized procollagen (+42%, P=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.
[Show abstract][Hide abstract] ABSTRACT: Umbilical Cord Mesenchymal Stem Cells (UC-MSC) show properties similar to Bone Marrow Mesenchymal Stem Cells (BM-MSC), though controversial data exists regarding their osteogenic potential. We prepared clinical-grade UC-MSC from Wharton's Jelly and we investigated if UC-MSC could be used as substitutes for BM-MSC in muscoloskeletal regeneration as a more readily available and functional source of MSCs. UC-MSC were loaded onto scaffolds and implanted subcutaneously (ectopically) and in critical-size calvarial defects (orthotopically) in mice. For live cell-tracking experiments, UC-MSC were first transduced with the luciferase gene. Angiogenic properties of UC-MSC 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-MSC, contrary to BM-MSC, 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 pro-inflammatory/chemotactic cytokine profile than BM-MSC and a significantly enhanced angiogenic activity. When UC-MSC were orthotopically transplanted in a calvarial defect, they promoted increased bone formation as well as BM-MSC. However, at variance with BM-MSC, 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-MSC not depositing directly bone matrix, but acting through the activation of endogenous repair mechanisms.
Full-text · Article · Feb 2015 · Stem Cells and Development
[Show abstract][Hide abstract] ABSTRACT: Large bone defects (critical-size defects) caused by disease, trauma, or tumor resection are not healed by the intrinsic regenerative capacity of bone and remain one of the more difficult regenerative therapies to successfully achieve. During the last two decades, major progresses have been made with regard to the in vitro fabrication of bone substitutes starting from suitable scaffolds, mainly resorbable porous ceramics, seeded with autologous (from the same patient) osteogenic cells, such as bone marrow-derived mesenchymal stem cells. The use of these ex vivo tissue engineered constructs is highly limited because of the difficult logistic of collecting the cells from the patients, expanding them in culture, and returning them to the surgical theater and because of the high cost of the culture procedure requiring the use of GMP facilities in order to cope with the strict rules defined by the European and National Regulatory Agencies. As a result, the classical tissue engineering approach involving ex vivo expanded patient cells should be confined only to extreme life or organ-saving situations.The rapidly developing knowledge about the pathways occurring during the healing process provides information about the natural response of the body to injury. This suggests a novel approach based on the activation of the endogenous regenerative capacity of the tissue itself. The endogenous regenerative potential correlates with the presence in the tissues of a population of stem/progenitor cells, responding to exogenous signal to generate a progeny of differentiated cells repairing or rebuilding the tissue. Differently from the "traditional" tissue engineering scheme, an endogenous regeneration strategy aims at the stimulation of the intrinsic potential of a tissue to heal or regenerate by using "off-the-shelf" standardized bioactive biomaterials without cells.In an effort to mimic the natural microenvironment during wound healing, the integration of different growth factors and cytokines into the scaffolds has been proposed. More recently, the idea of integrating a physiological cocktail of factors into the scaffold to lead tissue regeneration and to reduce morbidity and recovery time took place. In the arena of sustained multiple growth factor delivery, platelet-rich plasma (PRP) is a valid contender. It can deliver a high concentration of multiple growth factors in the "right" composition and in the "right" relative concentrations using a low cost strategy.
[Show abstract][Hide abstract] ABSTRACT: Background
Metformin is a widely used oral hypoglycemizing agent recently proposed as potential anti-cancer drug. In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential.
Antiproliferative and cytotoxic effects of metformin were tested in human SKNBE2 and SH-SY5Y neuroblastoma cell lines and in SKNBE2 cells in which differentiation is induced by retinoic acid treatment or stable overexpression of NDM29 non-coding RNA, both conditions characterized by a neuron-like differentiated phenotype.
We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged. Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29.
These data suggest the efficacy of metformin as neuroblastoma anticancer agent, and support the requirement of further studies on the possible role of the differentiation status on the antiproliferative effects of this drug.
Full-text · Article · Jul 2014 · Cancer Cell International
[Show abstract][Hide abstract] ABSTRACT: The therapeutic use of stem cells is a very promising strategy in the area of regenerative medicine. The stem cell regenerative paradigm has been mostly based on the assumption that progenitor cells play a critical role in tissue repair by their plasticity and differentiation potential. However, recent works suggest that the mechanism underlying the benefits of stem cell transplantation might relate to a paracrine modulatory effect rather than the replacement of affected cells at the site of injury. Preclinical and clinical skeletal studies, conducted in animal and adult series, support the use of mesenchymal stem cells (MSCs) for bone healing in critical clinical situations. These results have led to an increasing number of papers reporting the use of MSCs in adult clinical trials, whereas only few papers reported the use of these cells in pediatric skeletal disorders, probably because of unknown long-term results and long-life consequences of cellular therapy. The exponential growth of knowledge in adult MSCs could be translated and applied to pediatric disorders. Pediatric osteoarticular diseases have an enormous potential to be treated by MSCs, as severe congenital bone or local cartilage defects, not responding to conventional surgery treatment, might be successfully treated by cellular therapy. Translating basic stem cell research into routine therapies is a complex multistep process which entails the managing of the expected therapeutic benefits with the potential risks in correlation within the existing regulations. Here, we reported the state of art on the use of MSC in skeletal pediatric disorders.
No preview · Article · Jun 2014 · European Journal of Pediatric Surgery
[Show abstract][Hide abstract] ABSTRACT: Wound healing is achieved through distinct programmed phases: hemostasis, inflammation, mesenchymal cell proliferation and migration, and tissue remodeling. At the injury site, clot formation and platelet degranulation release cytokines, growth factors and actively participating in the healing process and regulating the migration of inflammatory cells, such as neutrophils, macrophages, and lymphocytes. We previously demonstrated that, in an inflammatory environment, PGE2 secreted by mesenchymal stem cells (MSCs) promoted the macrophage switch from a pro-inflammatory to a pro-resolving phenotype. Using an in vitro model, we here evaluated the role carried out by the two main players of the wound healing process, the platelet degranulation content mimicked by the platelet lysate (PL) and the inflammatory stimulus, on the modulation of mouse bone marrow-derived MSC paracrine activity. We demonstrated that, in MSCs, PL induced NF-kB activation, expression of COX-2, mPGE synthase and PGE2 production; in an inflammatory microenvironment, PL increased the inflammatory response and promoted the secretion of the pro-inflammatory cytokine IL-6. We assayed on mouse primary macrophages the paracrine activity of MSCs exposed to the different microenvironments and we observed that PL-treated MSC conditioned medium maintained macrophages in a pro-inflammatory state. The involved factors were GM-CSF induced by PL in MSCs and TNF-α induced by PL-MSC conditioned medium in macrophages. Our findings indicate that PL triggers an inflammatory response in MSCs and induces the secretion of factors maintaining macrophages in a pro-inflammatory state thus enhancing the initial inflammatory response to the injury, a key element in the activation of wound healing.
No preview · Article · Apr 2014 · Stem cells and development
[Show abstract][Hide abstract] ABSTRACT: The effects of 3 months of spaceflight (SF), hindlimb suspension, or exposure to 2G on the characteristics of neck muscle in mice were studied. Three 8-week-old male C57BL/10J wild-type mice were exposed to microgravity on the International Space Station in mouse drawer system (MDS) project, although only one mouse returned to the Earth alive. Housing of mice in a small MDS cage (11.6 × 9.8-cm and 8.4-cm height) and/or in a regular vivarium cage was also performed as the ground controls. Furthermore, ground-based hindlimb suspension and 2G exposure by using animal centrifuge (n = 5 each group) were performed. SF-related shift of fiber phenotype from type I to II and atrophy of type I fibers were noted. Shift of fiber phenotype was related to downregulation of mitochondrial proteins and upregulation of glycolytic proteins, suggesting a shift from oxidative to glycolytic metabolism. The responses of proteins related to calcium handling, myofibrillar structure, and heat stress were also closely related to the shift of muscular properties toward fast-twitch type. Surprisingly, responses of proteins to 2G exposure and hindlimb suspension were similar to SF, although the shift of fiber types and atrophy were not statistically significant. These phenomena may be related to the behavior of mice that the relaxed posture without lifting their head up was maintained after about 2 weeks. It was suggested that inhibition of normal muscular activities associated with gravitational unloading causes significant changes in the protein expression related to metabolic and/or morphological properties in mouse neck muscle.
[Show abstract][Hide abstract] ABSTRACT: Computed x-ray phase contrast micro-tomography is the most valuable tool for a three dimensional (3D) and non destructive analysis of the tissue engineered bone morphology. We used a Talbot interferometer installed at SYRMEP beamline of the ELETTRA synchrotron (Trieste, Italy) for a precise 3D reconstruction of both bone and soft connective tissue, regenerated in vivo within a porous scaffold. For the first time the x-ray tomographic reconstructions have been combined with x-ray scanning micro-diffraction measurement on the same sample, in order to give an exhaustive identification of the different tissues participating to the biomineralization process. As a result, we were able to investigate in detail the different densities in the tissues, distinguishing the 3D organization of the amorphous calcium phosphate from the collagen matrix. Our experimental approach allows for a deeper understanding of the role of collagen matrix in the organic-mineral transition, which is a crucial issue for the development of new bio-inspired composites.
Full-text · Article · Jan 2014 · Physics in Medicine and Biology