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Heslington, United Kingdom

Saleh F.A.,University of York | Whyte M.,Smith and Nephew Research Center | Genever P.G.,University of York
European Cells and Materials | Year: 2011

An increasing body of data suggest that mesenchymal stem cells (MSCs) reside in a perivascular niche. To more closely mimic this in vivo microenvironment and for better understanding of its complexity, and the factors that regulate the MSC activity, human umbilical vein endothelial cells (HUVECs) were co-cultured with human bone marrow MSCs - using a novel three-dimensional (3D) spheroid co-culture system. Using confocal microscopy of fluorescently labelled cells, we observed HUVECs and MSCs to self-assemble and form organised structures with segregated cell-type partitioning. Under osteogenic conditions, the rate and extent of differentiation in MSC/ HUVEC spheroids was significantly elevated compared to 3D co-cultures of MSCs and human dermal fibroblast controls as shown by alkaline phosphatase staining. Conversely, HUVECs inhibited adipogenic differentiation and the proliferation of MSCs in 3D co-cultures indicating that HUVECs suppressed MSC cycling and selectively promoted osteogenic differentiation in 3D. We have also shown that HUVECs enhanced activation of endogenous Wnt signalling and bone morphogenetic protein (BMP) signalling as shown by increased levels of active nuclear β-catenin and pSmad 1/5/8 immunopositivity respectively. These data suggest strongly that endothelial cells regulate the MSC activity in simulated in vivo conditions, by maintaining quiescence and facilitating niche exit via osteogenic differentiation following appropriate cues. Our findings also underline the importance of 3D heterotypic cell-cell interactions in the regulation of MSC behaviour, suggesting that multicellular cocktails and/or 3D-based delivery strategies may be beneficial for bone repair. Source


Frith J.E.,University of York | Thomson B.,Smith and Nephew Research Center | Genever P.G.,University of York
Tissue Engineering - Part C: Methods | Year: 2010

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along the osteogenic, chondrogenic, and adipogenic lineages and have potential applications in a range of therapies. MSCs can be cultured as monolayers on tissue culture plastic, but there are indications that they lose cell-specific properties with time in vitro and so poorly reflect in vivo MSC behavior. We developed dynamic three-dimensional (3D) techniques for in vitro MSC culture using spinner flasks and a rotating wall vessel bioreactor. We characterized the two methods for dynamic 3D MSC culture and compared the properties of these cultures with monolayer MSCs. Our results showed that under optimal conditions, MSCs form compact cellular spheroids and remain viable in dynamic 3D culture. We demonstrated altered cell size and surface antigen expression together with enhanced osteogenic and adipogenic differentiation potential in MSCs from dynamic 3D conditions. By microarray analysis of monolayer and spinner flask MSCs, we identified many differences in gene expression, including those confirming widespread changes to the cellular architecture and extracellular matrix. The upregulation of interleukin 24 in dynamic 3D cultures was shown to selectively impair the viability of prostate cancer cells cultured in medium conditioned by dynamic 3D MSCs. Overall, this work suggests a novel therapeutic application for dynamic 3D MSCs and demonstrates that these methods are a viable alternative to monolayer techniques and may prove beneficial for retaining MSC properties in vitro. © 2010 Mary Ann Liebert, Inc. Source


Saleh F.A.,University of York | Whyte M.,Smith and Nephew Research Center | Ashton P.,University of York | Genever P.G.,University of York
Stem Cells and Development | Year: 2011

Emerging data suggest that mesenchymal stem cells (MSCs) are part of a periendothelial niche, suggesting the existence of heterotypic cell-cell crosstalk between endothelial cells and MSCs that regulate MSCs in their local microenvironment. We determined the effects of paracrine factors secreted by human umbilical vein endothelial cells (HUVECs) on MSC survival, proliferation, and differentiation by using an optimized, serum-free HUVEC-conditioned medium (CM). HUVEC-CM induced a significant increase in the size and number of colony-forming units-fibroblast (CFU-F) and CFU-osteoblast (CFU-O) and stimulated the proliferation of MSCs as determined by 5-bromo-2′- deoxyuridine incorporation, compared with non-CM. We also demonstrated that CM significantly enhanced the osteogenic differentiation of MSCs as shown by alkaline phosphatase enzyme histochemistry and von Kossa staining of mineralized nodules as well as by quantitative reverse transcriptase-polymerase chain reaction analysis of osteogenic markers. In contrast, there was no effect on the adipogenic differentiation of MSCs. Bioinformatic integration of HUVEC and MSC gene expression datasets identified several candidate signaling pathways responsible for mediating these effects, including fibroblast growth factor, Wnt, bone morphogenetic protein, and Notch. These data suggest strongly that endothelial cells secrete a soluble factor (or factors) that stimulates progenitor cell activity and, selectively, the osteogenic differentiation of MSCs that could contribute to niche exit. © 2011, Mary Ann Liebert, Inc. Source


Ehrenfried L.M.,University of Cambridge | Farrar D.,Smith and Nephew Research Center | Cameron R.E.,University of Cambridge
Journal of the Royal Society Interface | Year: 2010

This study investigates the in vitro degradation properties of composites consisting of a porous tricalcium phosphate (TCP) foam filled with degradable poly(DL-lactic acid) (PDLLA) via either in situ polymerization or infiltration. The motivation was to develop a material for bone repair that would be initially mechanically strong and would develop porosity during degradation of one of the components. A thorough analysis of the physical in vitro degradation properties has been conducted and reported by the same authors elsewhere. Synchrotron microcomputer tomography analysis (conducted at ID19, ESRF, Grenoble, France) allowed detailed insights to be gained into the process of the composites' degradation, which was discovered to be strongly influenced by the manufacturing method. The polymer phase of in situ-polymerized TCP-PDLLA degraded as a bulk sample, with faster degradation in the centre of the sample as a whole. In contrast, the polymer phase of infiltrated TCP-PDLLA degraded as individual polymer spheres with faster degradation in the centre of each sphere. 2010 The Royal Society. Source


Plaas A.,Rush University Medical Center | Li J.,Rush University Medical Center | Riesco J.,Rush University Medical Center | Das R.,Rush University Medical Center | And 2 more authors.
Arthritis Research and Therapy | Year: 2010

Introduction: Intraarticular hyaluronan (HA) is used clinically for symptomatic relief in patients with knee osteoarthritis (OA); however, the mechanism of action is unclear. In this study, we examined the effects of a single injection of HA on joint tissue pathology, mechanical allodynia and gait changes (measured by stride times) in a murine model of OA.Methods: OA was induced in the right knee joint (stifle) of 12-week-old male C57BL/6 mice by transforming growth factor β1 (TGFβ1) injection and treadmill running for 14 days. Gait parameters were quantified by using TreadScan, mechanical allodynia was evaluated with von Frey filaments, and joint pathology was evaluated by scoring of macroscopic images for both cartilage erosion and periarticular fibrosis. HA or saline control was injected 1 day after TGFβ1 injection but before the start of treadmill running.Results: OA development in this model was accompanied by significant (P < 0.01) enhancement of the stance and propulsion times of affected legs. HA injection (but not saline injection) blocked all gait changes and also protected joints from femoral cartilage erosion as well as tibial and femoral tissue fibrosis. Both HA injection and saline injection attenuated acute allodynia, but the HA effect was more pronounced and prolonged than the saline injection.Conclusions: We conclude that videographic gait analysis is an objective, sensitive and reproducible means of monitoring joint pathology in experimental murine OA, since stance time appears to correlate directly with OA severity. A single injection of HA prevents acute and prolonged gait changes and ameliorates the cartilage erosion and periarticular fibrosis normally seen in this model. We speculate that the capacity of HA to prevent cartilage erosion results from its normalization of joint biomechanics and its inhibitory effects on periarticular cells, which are involved in tissue hyperplasia and fibrosis. This effect of exogenous HA appears to mimic the protective effects of ablation of Adamts5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) on experimental murine OA, and we speculate that a common mechanism is involved. © 2011 Plaas et al.; licensee BioMed Central Ltd. Source

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