Cell labelling with superparamagnetic iron oxide has no effect on chondrocyte behaviour

SummaryBackgroundTissue engineering and regenerative medicine are two rapidly advancing fields of research offering potential for effective treatment of cartilage lesions. Today, chondrocytes are the cell type of choice for use in cartilage repair approaches such as autologous chondrocyte implantation. To verify the safety and efficacy of such approaches it is necessary to determine the fate of these transplanted cells. One way of doing this is prelabelling cells before implantation and tracking them using imaging techniques. The use of superparamagnetic iron oxide (SPIO) for tracking of cells with magnetic resonance imaging (MRI) is ideal for this purpose. It is non-radioactive, does not require viral transfection and is already approved for clinical use as a contrast agent.ObjectiveThe purpose of this study was to assess the effect of SPIO labelling on adult human chondrocyte behaviour.MethodsCells were culture expanded and dedifferentiated for two passages and then labelled with SPIO. Effect on cell proliferation was tested. Furthermore, cells were cultured for 21 days in alginate beads in redifferentiation medium. Following this period, cells were analysed for expression of cartilage-related genes, proteoglycan production and collagen protein expression.ResultsSPIO labelling did not significantly affect any of these parameters relative to unlabelled controls. We also demonstrated SPIO retention within the cells for the full duration of the experiment.ConclusionsThis paper demonstrates for the first time the effects of SPIO labelling on chondrocyte behaviour, illustrating its potential for in vivo tracking of implanted chondrocytes

Targeting the Mitotic Checkpoint to Kill Tumor Cells

One of the most common hallmarks of cancer cells is aneuploidy or an abnormal number of chromosomes. This abnormal chromosome content is a consequence of chromosome missegregation during mitosis, a defect that is seen more frequently in tumor cell divisions as in normal cell divisions. In fact, a large fraction of human tumors display a chromosome instable phenotype, meaning that they very frequently missegregate chromosomes. This can cause variegated aneuploidy within the tumor tissue. It has been argued that this hallmark of cancer could be exploited in anti-cancer therapies. Here we test this hypothesis by inactivation of the mitotic checkpoint through RNAi-mediated depletion of an essential checkpoint component, Mps1. The mitotic checkpoint delays segregation of chromosomes during mitosis until all chromosomes are properly attached to the mitotic spindle. Its inactivation will therefore lead to increased segregation errors. Indeed, we show that this can lead to increased cell death in tumor cells. We demonstrate that increased cell death is associated with a dramatic increase in segregation errors. This suggests that inhibition of the mitotic checkpoint might represent a useful anti-cancer strategy

Properties of commonly used calcium phosphate cements in trauma and orthopaedic surgery

Introduction Half of the population sustains at least one fracture during their lifetime, and the majority of these fractures heal successfully. Successful fracture healing requires the following five elements; (i) osteogenic cells (e.g., osteoblasts), (ii) osteoinductive stimuli (e.g., bone morphogenetic proteins); (iii) an osteoconductive matrix; (iv) adequate blood and nutrient supply, and (v) sufficient mechanical support. One or more elements can be compromised due to the existence of a bone defect. Bone defects are treated with bone grafts in order to avoid insufficient fracture healing. Insufficient fracture healing is encountered in 5–10% of the fractures, resulting in delayed union, malunion, or non-union

Square root singularity in the viscosity of neutral colloidal suspensions at large frequencies

The asymptotic frequency $\omega$, dependence of the dynamic viscosity of neutral hard sphere colloidal suspensions is shown to be of the form $\eta_0 A(\phi) (\omega \tau_P)^{-1/2}$, where $A(\phi)$ has been determined as a function of the volume fraction $\phi$, for all concentrations in the fluid range, $\eta_0$ is the solvent viscosity and $\tau_P$ the P\'{e}clet time. For a soft potential it is shown that, to leading order steepness, the asymptotic behavior is the same as that for the hard sphere potential and a condition for the cross-over behavior to $1/\omega \tau_P$ is given. Our result for the hard sphere potential generalizes a result of Cichocki and Felderhof obtained at low concentrations and agrees well with the experiments of van der Werff et al, if the usual Stokes-Einstein diffusion coefficient $D_0$ in the Smoluchowski operator is consistently replaced by the short-time self diffusion coefficient $D_s(\phi)$ for non-dilute colloidal suspensions.Comment: 18 pages LaTeX, 1 postscript figur

Chromosomal Instability by Inefficient Mps1 Auto-Activation Due to a Weakened Mitotic Checkpoint and Lagging Chromosomes

BACKGROUND: Chromosomal instability (CIN), a feature widely shared by cells from solid tumors, is caused by occasional chromosome missegregations during cell division. Two of the causes of CIN are weakened mitotic checkpoint signaling and persistent merotelic attachments that result in lagging chromosomes during anaphase. PRINCIPAL FINDINGS: Here we identify an autophosphorylation event on Mps1 that is required to prevent these two causes of CIN. Mps1 is phosphorylated in mitotic cells on at least 7 residues, 4 of which by autophosphorylation. One of these, T676, resides in the activation loop of the kinase domain and a mutant that cannot be phosphorylated on T676 is less active than wild-type Mps1 but is not kinase-dead. Strikingly, cells in which endogenous Mps1 was replaced with this mutant are viable but missegregate chromosomes frequently. Anaphase is initiated in the presence of misaligned and lagging chromosomes, indicative of a weakened checkpoint and persistent merotelic attachments, respectively. CONCLUSIONS/SIGNIFICANCE: We propose that full activity of Mps1 is essential for maintaining chromosomal stability by allowing resolution of merotelic attachments and to ensure that single kinetochores achieve the strength of checkpoint signaling sufficient to prevent premature anaphase onset and chromosomal instability. To our knowledge, phosphorylation of T676 on Mps1 is the first post-translational modification in human cells of which the absence causes checkpoint weakening and CIN without affecting cell viability

Monocyte subsets in blood correlate with obesity related response of macrophages to biomaterials in vitro

Macrophages play a key role in the foreign body response. In this study it was investigated whether obesity affects th

Recellularization of auricular cartilage via elastase-generated channels

Decellularized tissue matrices are promising substrates for tissue generation by stem cells to replace poorly regenerating tissues such as cartilage. However, the dense matrix of decellularized cartilage impedes colonisation by stem cells. Here, we show that digestion of elastin fibre bundles traversing auricular cartilage creates channels through which cells can migrate into the matrix. Human chondrocytes and bone marrow-derived mesenchymal stromal cells efficiently colonise elastin-treated scaffolds through these channels, restoring a glycosaminoglycan-rich matrix and improving mechanical properties while maintaining size and shape of the restored tiss

Silencing of anti-chondrogenic microRNA-221 in human mesenchymal stem cells promotes cartilage repair in vivo

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation towards the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the anti-chondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs towards chondrogenic differentiation in the absence of TGF-β. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a pro-chondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. This article is protected by copyright. All rights reserved

MSC encapsulation in alginate microcapsules prolongs survival after intra-articular injection, a longitudinal in vivo cell and bead integrity tracking study

Mesenchymal stem cells (MSC) are promising candidates for use as a biological therapeutic. Since locally injected MSC disappear within a few weeks, we hypothesize that efficacy of MSC can be enhanced by prolonging their presence. Previously, encapsulation in alginate was suggested as a suitable approach for this purpose. We found no differences between the two alginate types, alginate high in mannuronic acid (High M) and alginate high in guluronic acid (High G), regarding MSC viability, MSC immunomodulatory capability, or retention of capsule integrity after subcutaneous implantation in immune competent rats. High G proved to be more suitable for production of injectable beads. Firefly luciferase-expressing rat MSC were used to track MSC viability. Encapsulation in high G alginate prolonged the presence of metabolically active allogenic MSC in immune competent rats with monoiodoacetate-induced osteoarthritis for at least 8 weeks. Encapsulation of human MSC for local treatment by intra-articular injection did not significantly influence the effect on pain, synovial inflammation, or cartilage damage in this disease model. MSC encapsulation in alginate allows for an injectable approach which prolongs the presence of viable cells subcutaneously or in an osteoarthritic joint. Further fine tuning of alginate formulation and effective dosage for might be required in order to improve therapeutic efficacy depending on the target disease. [Figure not available: see fulltext.]