BackgroundTissue engineering appears to be an attractive alternative to the traditional approach in the treatment of fracture non-unions. Mesenchymal stromal cells (MSCs) are considered an appealing cell source for clinical intervention. However, ex vivo cell expansion and differentiation towards the osteogenic lineage, together with the design of a suitable scaffold have yet to be optimized. Major concerns exist about the safety of MSC-based therapies, including possible abnormal overgrowth and potential cancer evolution.AimsWe examined the long-term efficacy and safety of ex vivo expanded bone marrow MSCs, embedded in autologous fibrin clots, for the healing of atrophic pseudarthrosis of the upper limb. Our research work relied on three main issues: use of an entirely autologous context (cells, serum for ex vivo cell culture, scaffold components), reduced ex vivo cell expansion, and short-term MSC osteoinduction before implantation.Methods and FindingsBone marrow MSCs isolated from 8 patients were expanded ex vivo until passage 1 and short-term osteo-differentiated in autologous-based culture conditions. Tissue-engineered constructs designed to embed MSCs in autologous fibrin clots were locally implanted with bone grafts, calibrating their number on the extension of bone damage. Radiographic healing was evaluated with short- and long-term follow-ups (range averages: 6.7 and 76.0 months, respectively). All patients recovered limb function, with no evidence of tissue overgrowth or tumor formation.ConclusionsOur study indicates that highly autologous treatment can be effective and safe in the long-term healing of bone non-unions. This tissue engineering approach resulted in successful clinical and functional outcomes for all patients.

Current evidence suggests a complex interaction between adipokines and microRNA (miRNA) in osteoarthritis (OA) pathogenesis. The present study explored the role of miR-34a and miR-181a in regulating apoptosis and oxidative stress induced by visfatin in human OA chondrocytes. Chondrocytes were transfected with miR-34a and miR-181a inhibitors and stimulated with visfatin for 24 h, in the presence of nuclear factor (NF)-κB inhibitor (BAY-11-7082, 2 h pre-incubation). Apoptosis and reactive oxygen species (ROS) production were detected by cytometry, miRNA, antioxidant enzymes, nuclear factor erythroid (NRF)2 and B-cell lymphoma (BCL)2 expressions by quantitative real time polymerase chain reaction (real time PCR) and western blot. P50 NF-κB subunit was measured by immunofluorescence. Visfatin significantly induced apoptosis and superoxide anion production, increased miR-34a, miR-181a, superoxide dismutase (SOD)-2, catalase (CAT), NRF2 and decreased BCL2 gene and protein expression in OA chondrocytes. All the visfatin-caused effects were suppressed by using miR-34a and miR-181a inhibitors. Pre-incubation with BAY-11-7082 counteracted visfatin-induced expression of miRNA, BCL2, SOD-2, CAT and NRF2. Inhibition of miR-34a and miR-181a significantly reduced the activation of p50 NF-κB. Visfatin confirms its ability to induce apoptosis and oxidative stress in human OA chondrocytes; these effects appeared mediated by miR-34a and miR-181a via NF-κB pathway. We highlight the relevance of visfatin as potential therapeutic target for OA treatment.

Synovial membrane inflammation actively participate to structural damage during osteoarthritis (OA). Adipokines, miRNA, and oxidative stress contribute to synovitis and cartilage destruction in OA. We investigated the relationship between visfatin, resistin and miRNA in oxidative stress regulation, in human OA synovial fibroblasts. Cultured cells were treated with visfatin and resistin. After 24 h, we evaluated various pro-inflammatory cytokines, metalloproteinases (MMPs), type II collagen (Col2a1), miR-34a, miR-146a, miR-181a, antioxidant enzymes, and B-cell lymphoma (BCL)2 by qRT-PCR, apoptosis and mitochondrial superoxide production by cytometry, p50 nuclear factor (NF)-κB by immunofluorescence. Synoviocytes were transfected with miRNA inhibitors and oxidative stress evaluation after adipokines stimulus was performed. The implication of NF-κB pathway was assessed by the use of a NF-κB inhibitor (BAY-11-7082). Visfatin and resistin significantly up-regulated gene expression of interleukin (IL)-1β, IL-6, IL-17, tumor necrosis factor (TNF)-α, MMP-1, MMP-13 and reduced Col2a1. Furthermore, adipokines induced apoptosis and superoxide production, the transcriptional levels of BCL2, superoxide dismutase (SOD)-2, catalase (CAT), nuclear factor erythroid 2 like 2 (NRF2), miR-34a, miR-146a, and miR-181a. MiRNA inhibitors counteracted adipokines modulation of oxidative stress. Visfatin and resistin effects were suppressed by BAY-11-7082. Our data suggest that miRNA may represent possible mediators of oxidative stress induced by visfatin and resistin via NF-κB pathway in human OA synoviocytes.

Growing evidence indicates the important role of adipokines and microRNA (miRNA) in osteoarthritis (OA) pathogenesis. The purpose of the present study was to investigate the effect of visfatin and resistin on some miRNA (34a, 140, 146a, 155, 181a, let-7e), metalloproteinases (MMPs), and collagen type II alpha 1 chain (Col2a1) in human OA chondrocytes and in the T/C-28a2 cell line. The implication of nuclear factor (NF)-κB in response to adipokines was also assessed. Chondrocytes were stimulated with visfatin (5 or 10 μg/mL) and resistin (50 or 100 ng/mL) with or without NF-κB inhibitor (BAY-11-7082, 1 μM) for 24 h. Viability and apoptosis were detected by MMT and cytometry, miRNA, MMP-1, MMP-13, and Col2a1 by qRT-PCR and NF-κB activation by immunofluorescence. Visfatin and resistin significantly reduced viability, induced apoptosis, increased miR-34a, miR-155, miR-181a, and miR-let7e, and reduced miR-140 and miR-146a gene expression in OA chondrocytes. MMP-1, MMP-13, and Col2a1 were significantly modulated by treatment of OA chondrocytes with adipokines. Visfatin and resistin significantly increased NF-κB activation, while the co-treatment with BAY11-7082 did not change MMPs or Col2a1 levels beyond that caused by single treatment. Visfatin and resistin regulate the expression levels of some miRNA involved in OA pathogenesis and exert catabolic functions in chondrocytes via the NF-κB pathway. These data confirm the complex relationship between adipokines and miRNA.

BackgroundWe recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation.Methodology/Principal FindingsMPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15.Conclusions/SignificanceMPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested from human bone marrow.