[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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Cell International 07/2014; 14:59. · 1.99 Impact Factor
[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.
European Journal of Pediatric Surgery 06/2014; 24(03). · 0.98 Impact Factor
[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.
Stem cells and development 04/2014; · 4.15 Impact Factor
[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.
Physics in Medicine and Biology 01/2014; 59(1):189-201. · 2.92 Impact Factor
[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: 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.
Frontiers in Bioengineering and Biotechnology 01/2014; 2:80.
[Show abstract][Hide abstract] ABSTRACT: Studies about cartilage repair in the hip and infant chondroctyes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts.
Hip cartilage was obtained from five human donors (age 1-10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De- and re-differentiation of chondrocytes was assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice.
The donor tissue was heterogenous showing differentiated articular cartilage and non-differentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutanuous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen.
These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP.
Osteoarthritis and Cartilage 10/2013; · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair.
[Show abstract][Hide abstract] ABSTRACT: Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca(2+) concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.
PLoS ONE 08/2013; 8(8):e72028. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of platelet rich plasma (PRP) in bone repair remains highly controversial. In this work, we evaluated the effect of lyophilized PRP on bone regeneration when associated with a silicon stabilized hydroxyapatite tricalcium phosphate scaffold in a rabbit calvarial defect (Skelite™). Critical defects were created in the calvaria of twenty-four rabbits. The periosteum was removed and the defects were either left empty or filled with allogeneic PRP gel; Skelite particles; Skelite and PRP gel. Four animals were killed after 4 weeks, 10 animals after 8 and 10 after 16 weeks. Specimens were processed for X-ray microtomography (μCT) and for resin embedded histology. μCT analysis revealed significant osteoid-like matrix and new bone deposition in PRP + Skelite group at both 8 and 16 weeks in respect to Skelite alone. Histologically, PRP + Skelite defects were highly cellular with more abundant osteoid deposition and more regular collagen fibres. Moreover, in vitro migration assays confirmed the chemotactic effect of PRP to endothelial and osteoprogenitor cells. We conclude that the addition of PRP influenced the local tissue microenvironment by providing key cryptic factors for regeneration, thereby enhancing progenitor cell recruitment, collagen and bone matrix deposition, and by creating a bridging interface between the scaffold and bone.
Journal of cranio-maxillo-facial surgery: official publication of the European Association for Cranio-Maxillo-Facial Surgery 08/2013; 42(5). · 1.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently reported that mouse bone marrow stromal cells (BMSCs), also known as bone marrow-derived mesenchymal stem cells (MSCs), seeded onto a scaffold and implanted in vivo, leaded to an ectopic bone deposition by host cells. This MSCs capacity was critically dependent on their commitment level, being present only in MSCs cultured in presence of Fibroblast Growth Factor-2 (FGF-2). Taking advantage of a chimeric mouse model, in this study we show that seeded MSCs trigger a cascade of events resulting in the mobilization of macrophages, the induction of their functional switch from a pro-inflammatory to a pro-resolving phenotype, and the subsequent formation of a bone regenerative niche through the recruitment, within the first two weeks of implantation, of endothelial progenitors and of cells with an osteogenic potential (CD146+CD105+), both of them derived from the bone marrow. Moreover, we demonstrated that, in an inflammatory environment, MSCs secrete a large amount of PGE2 playing a key role in the macrophage phenotype switch.
Stem cells and development 08/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Serum of animal origin and in particular fetal bovine serum is the most commonly utilized cell culture medium additive for in vitro cell growth and differentiation. However, several major concerns have been raised by the scientific community regarding the use of animal sera for human cell-based culture applications. Among the possible alternatives to the animal serum, platelet-derived compounds have been proposed since more than 10 years. Nevertheless, the high degree of variability between the different platelet preparations, and the lack of standardized manufacturing and quality control procedures, made difficult to reach a consensus on the applicability of this novel cell culture medium supplement. In this study, we describe the preparation of a standardized platelet-rich plasma (PRP) derivative obtained starting from human-certified buffy coat samples with a defined platelet concentration and following protocols including also freeze-drying, gamma irradiation and biological activity testing prior the product release as cell culture medium additive. Biological activity testing of the different preparations was done by determining the capability of the different PRP preparations to sustain human bone marrow mesenchymal stem cell (MSC) clone formation and proliferation. Taking advantage of a developed MSC in vitro clonogenicity test, we also determined biological activity and stability of the freeze-dried gamma-sterilized PRP preparations after their storage for different times and at different temperatures. The PRP effects on cell proliferation were determined both on primary cell cultures established from different tissues and on a cell line. Results were compared with those obtained in "traditional" parallel control cultures performed in the presence of bovine serum [10% fetal calf serum (FCS)]. Compared to FCS, the PRP addition to the culture medium increased the MSC colony number and average size. In primary cell cultures and in cell line cultures, the PRP promoted cell proliferation also in conditions where the FCS had not a proliferation stimulating effect due to either the nature of the cells and the tissue of origin (such as human articular chondrocytes from elderly patients) or to the critical low density cell seeding (such as for HeLa cells). In summary, the standardized PRP formulation would provide an "off-the-shelf" product to be used for the selection and expansion of several cell types also in critical cell culture conditions.
[Show abstract][Hide abstract] ABSTRACT: FE65 proteins constitute a family of adaptors which modulates the processing of amyloid precursor protein (APP) and the consequent amyloid β production. Thus, they have been involved in the complex and partially unknown cascade of reactions at the base of Alzheimer's disease (AD) etiology. However, FE65 and FE65-like (APBB2) proteins may be linked to neurodegeneration through the regulation of cell cycle in post-mitotic neurons. In this work we disclose novel molecular mechanisms by which APBB2 can modulate APP processing. We show that APBB2 mRNA splicing, driven by the over-expression of a novel non-coding (nc) RNA named 45A, allow the generation of alternative protein forms endowed with differential effects on Aβ production, cell cycle control, and DNA damage response. 45A overexpression also favors cell transformation and tumorigenesis leading to a marked increase of malignancy of neuroblastoma cells. Therefore, our results highlight a novel regulatory pathway of considerable interest linking APP processing with cell cycle regulation and DNA-surveillance systems, that may represent a molecular mechanism to induce neurodegeneration in post-mitotic neurons.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 06/2013; 1833(6):1511-1526. · 5.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract In the Regenerative Medicine, platelet by-products containing factors physiologically involved in wound healing, have been successfully used in the form of Platelet Rich Plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects. Measurement of endocannabinoids and related compounds in PRP showed a significant amount of anandamide, 2-arachidonoylglycerol, palmitoylethanolamide and oleoylethanolamide. Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that, under inflammatory conditions, PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue. Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated a potent anti-nociceptive effect against both early and late nocifensive responses. This effect was observed following intra-paw injection of: 1) total PRP; 2) lipids extracted from PRP; 3) an endocannabinoid-enriched lipid fraction of PRP. In all conditions, antagonists of endocannabinoid CB1 and CB2 receptors, if injected in the paw, abrogated the anti-nociceptive effects strongly suggesting for this preparation a peripheral mechanism of action. In conclusion, we showed that PRP and PRP lipid extract exert a potent anti-nociceptive activity linked, at least in part, to their endocannabinoids and related compounds content, and to their capability of elevating the levels of these lipid mediators in cells.
Tissue Engineering Part A 04/2013; · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: The aim of our study was to analyse the clinical and histological outcome after the treatment of focal cartilage defects in non-degenerative and degenerative knees with bone marrow stimulation and subsequent covering with a cell-free resorbable polyglycolic acid-hyaluronan (PGA-HA) implant immersed with autologous platelet-rich plasma (PRP). METHODS: Fifty-two patients (mean age 44 years) with focal chondral defects in radiologically confirmed non-degenerative or degenerative knees were subjected to subchondral drilling arthroscopically. Subsequently, defects were covered with the PGA-HA implant immersed with autologous PRP. At 2-year follow-up, the patients' situation was assessed using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and compared to the pre-operative situation and 3-12-month follow-up. Biopsies (n = 4) were harvested at 18-24 months after implantation and were analysed by histology and collagen type II immune staining. RESULTS: At 1- and 2-year follow-up, the KOOS showed clinically meaningful and significant (p < 0.05) improvement in all subcategories compared to baseline and to 3-month follow-up. There were no differences in KOOS data obtained after 2 years compared to 1 year after the treatment. Histological analysis of the biopsy tissue showed hyaline-like to hyaline cartilage repair tissue that was rich in cells with a chondrocyte morphology, proteoglycans and type II collagen. CONCLUSIONS: Covering of focal cartilage defects with the PGA-HA implant and PRP after bone marrow stimulation improves the patients' situation and has the potential to regenerate hyaline-like cartilage. LEVEL OF EVIDENCE: Case series, Level IV.
Knee Surgery Sports Traumatology Arthroscopy 04/2013; · 2.68 Impact Factor