H. Jahr

Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study.

BackgroundAlthough pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation.MethodsBMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05.ResultsBiochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control.Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation.ConclusionsPEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo.

253 INHIBITING CALCINEURIN ACTIVITY UNDER PHYSIOLOGICAL CONDITIONS SELECTIVELY PREVENTS ARTICULAR CHONDROCYTE HYPERTROPHY

Results: Loss of a5 integrin expression was related to chondrocyte dedifferentiation in the osteoarthritic articular cartilage, while a1, a2 and a3 integrin expression increased in fibrous areas in osteoarthritic cartilage. The aV integrin was expressed in hyperthrophic cartilage and increased in the articular cartilage of osteoarthritic rats, where bone was developed and neovascularization was observed. The effect of GDF-5 (growth differentiation factor 5) and BMP-7 (bone morphogenetic protein 7) on integrins expression during chondrocytes differentiation was also analyzed. A micromasses culture system of mouse embryo mesenchymal cells, were treated with GDF-5 or BMP-7 for 4 and 6 days. The expression of integrins was analyzed by immunohistochemistry. GDF-5 induced the expression of a5 sub-unit while BMP-7 induced the expression of aV sub-unit. This suggests a switch in the signal for chondral differentiation towards hypertrophy, where GDF-5 could keep the state of the articular chondrocyte and BMP-7 would induce hypertrophy. The typical chondrocyte phenotype in the articular cartilage is preserved due to the presence of Indian hedgehog (Ihh), which is dependent on a5 integrin and GDF-5 to keep the articular cartilage and prevent hypertrophy. This was supported by decrease of Ihh expression in articular cartilage during OA. Treating mouse knees organ cultures with Cyclopamine an Ihh signaling inhibitor, articular cartilage ossification was observed similarly with OA rat knees, probably by a process apparently similar to endochondral ossification. Conclusions: The typical chondrocyte phenotype in the articular cartilage is preserved due to the presence of Indian hedgehog (Ihh), which is dependent on a5 integrin and GDF-5 to keep the articular cartilage phenotype and prevent hypertrophy and OA.

245 INHIBITING CALCINEURIN ACTIVITY UNDER PHYSIOLOGICAL TONICITY: A WIN-WIN SITUATION FOR CELL-BASED CHONDRAL LESION

OGT and O-GlcNAcase expression were assessed by western blot analysis employing specific antibodies. Furthermore OGT expression was localized in the cartilage by immunohistochemistry. Results: In human OA cartilage, the level of O-GlcNAcylated proteins was significantly increased in comparison to that of healthy cartilage. We also observed an increased expression of the different isoforms of OGT in OA cartilage and OA chondrocytes when compared to healthy controls. The level of O-GlcNAcylated proteins was also increased in OA rabbit cartilage at all the weeks studied. We also observed an increased expression of the different isoforms of OGT in the OA cartilage in comparison to control animals. The peak of expression of these proteins was observed 12 weeks after surgery. Furthermore, in OA cartilage we observed a decreased expression of O-GlcNAcase at all the times studied. By immunohistochemical studies, OGT was mainly localized inside the chondrocytes in all the samples analyzed, including both superficial and deeper zone chondrocytes. Conclusions: These results demonstrate that OA could be associated with a dysregulation in the hexosamine biosynthesis pathway that could lead to an accumulation of O-GlcNAcylated proteins. Our data support the hypothesis that O-GlcNAc formation may play an important role in the development of chronic and age-related diseases.

4.6 Tracking bone marrow stromal cells with iron labelling

Tracking bone marrow stromal cells with iron labelling E. Farrell, P. Wielopolsky, H. Jahr, J. Verhaar, H. Weinans, G. Krestin, F. O‘Brien, M. Bernsen, G. van Osch; Center For Bioengineering, Trinity College Dublin & Royal College of Surgeons in Ireland, Dublin, Ireland, Radiology, Erasmus MC, Rotterdam, Netherlands, Orthopaedics, Erasmus MC, Rotterdam, Netherlands, X, Royal College of Surgeons, Dublin, Ireland, Orthopaedics & Otorhinolaryngology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands

23 CALCINEURIN – NUCLEAR FACTOR OF ACTIVATED T CELLS (NFAT) PATHWAY REGULATES ADULT HUMAN ARTICULAR CHONDROCYTE DIFFERENTIATION

Calcineurin (Cn) – Nuclear Factor of Activated T cells (NFAT) pathway regulates adult human articular chondrocyte differentiation Anna E. van der Windt1, Holger Jahr1, Paul van der Laan1, Predrag Pavljasevic1, Eric Farrell1, Jan A. Verhaar1, Harrie Weinans1, Gerjo J. van Osch1,2 1Orthopaedics, Erasmus MC, University Medical Center, Rotterdam, Netherlands; 2Othorhinolaryngology, Erasmus MC, University Medical Center, Rotterdam, Netherlands g.vanosch@erasmusmc.nl