Tag Archives: bone morphogenetic proteins
4-Thiazolidinone-based derivatives rescue TNAα-inhibited osteoblast differentiation in mouse mesenchymal precursor cells
Kh. V. Malysheva1,2,3, N. S. Finiuk1, O. K. Pavlenko4, D. Ya. Havrylyuk5,
R. B. Lesyk5, R. S. Stoika1, O. G. Korchynskyi1,3
1Institute of Cell Biology, NAS of Ukraine, Lviv;
2Insitute of Animal Biology, NAAS of Ukraine, Lviv;
3Centre for Innovative Research in Medical and Natural Sciences,
Rzeszow University and Medical Faculty, Poland;
4Ivan Franko National University of Lviv, Ukraine;
5Danylo Halytsky Lviv National Medical University, Ukraine;
e-mail: olexkor@hotmail.com
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease of yet unknown etiology. Tumor necrosis factor α (TNFα) is recognized as a regulatory substance that plays a central role in RA development and progression. On the other side, the bone morphogenetic protein (BMP) and Wnt signaling pathways are key mechanisms that induce and support cartilage and bone formation and maintenance. Previous studies showed that the pro-inflammatory cytokines TNFα and interleukin 1β (IL-1β) are central players in the inhibition of activity of skeletogenesis. The aim of this study was to evaluate the anti-inflammatory activity of novel 4-thiazolidinone-based derivatives towards TNFα–induced pro-inflammatory effects during bone formation. We performed in vitro evaluation of functional effects of 4-thiazolidinones denoted as Les-4368, Les-4370, Les-3882 and Les-3288 that were used in different doses (0.02, 0.1, 0.3 and 1.0 μM) on the TNFα-mediated inhibition of the BMP-induced osteoblast differentiation in mouse mesenchymal precursor (stem) cells of C2C12 line. Treatment of these cells with TNFα completely inhibited their myogenic differentiation, as well as strongly inhibited the BMP-induced osteogenesis. Strikingly, the treatment of C2C12 cells with Les-4368 and Les-3882 rescued the osteoblast differentiation from negative control of TNFα, and, moreover, converted this cytokine from the inhibitor of osteogenesis into its stimulator. Western-blot analysis of Inhibitory κBα (I-κBα) degradation was used to elucidate a mechanism of the anti-inflammatory effects. Les-3882 was more active, and it stimulated osteoblast differentiation at low dose (0.1 μM), presumably, via modulation of the NF-κB signaling pathway.
Generation of optimized preparations of bone morphogenetic proteins for bone regeneration
Kh. V. Malysheva1,2, I. M. Spasyuk3, O. K. Pavlenko3,
R. S. Stoika1, O. G. Korchynskyi1,4
1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv;
2Insitute of Animal Biology, National Academy of Agrarian Sciences of Ukraine, Lviv;
3Ivan Franko National University of Lviv, Ukraine;
4Centre for Innovative Research in Medical and Natural Sciences,
Medical Faculty of Rzeszow University, Poland;
e-mail: olexkor@hotmail.com
Correction of inherited skeletal abnormalities, traumas affecting wide bone areas and non-healing fractures require efficient bone formation and regeneration. Bone morphogenetic proteins (BMPs) are signaling molecules that play a crucial role in bone and cartilage formation and regeneration. Osteoinductive properties of existing hydroxyapatite-based osteoplastic materials are frequently insufficient for efficient bone regeneration, thus raising a requirement for novel matrices involving BMPs for highly efficient local induction of bone formation at the area of the bone defect. The aim of this study was conducting in vitro optimization of osteoinductive properties of recombinant BMPs preparations to be used in bone regenerative practice. Recombinant BMPs were produced in human embryonic kidney 293 cells upon their transfection or co-transfection with plasmids expressing BMP2 and BMP7 at different ratios. A quality of BMP preps was validated based on their ability to induce in vitro osteoblast differentiation of C2C12 cells. Alkaline phosphatase that is widely used as a marker of osteoblast differentiation was measured spectrophotometrically. We found that the most effective inducer of osteoblast differentiation was recombinant BMP preparation produced upon cotransfection of 85% of BMP2 and 15% of BMP7 plasmids, that is most likely due to generation of conditions most favorable for formation of BMP2/7 heterodimers. Frozen BMP2/7 preparations stored for 3 h in experimental setup and for several weeks in routine work do not lose their osteoinductive properties compared with freshly prepared BMP2/7 preparations and can be successfully used for generation of highly efficient bone regenerative matrices.