Rat Osteosarcoma Cells as a Therapeutic Target Model for Osteoregeneration via Sclerostin Knockdown

Abstract

There are various conceptually different strategies to improve bone regeneration and to treat osteoporosis, each with distinct inherent advantages and disadvantages. The use of RNA interference strategies to suppress the biological action of catabolic factors or antagonists of osteogenic proteins is promising, and such strategies can be applied locally. They are comparably inexpensive and do not suffer from stability problems as protein-based approaches. In this study, we focus on sclerostin, encoded by the SOST gene, a key regulator of bone formation and remodeling. Sclerostin is expressed by mature osteocytes but also by late osteogenically differentiated cells. Thus, it is difficult and requires long-term cultures to investigate the effects of SOST silencing on the expression of osteogenic markers using primary cells. We, therefore, selected a rat osteosarcoma cell line, UMR-106, that has been shown to express SOST and secrete sclerostin in a comparable fashion as late osteoblasts and osteocytes. We investigated the effects of differentiating supplements on SOST expression and sclerostin secretion in UMR-106 cells and found that addition of 100 ng/ml of bone morphogenetic protein (BMP)-2 strongly induced sclerostin secretion, whereas dexamethasone inhibited secretion. Effects of silencing SOST in UMR-106 cells cultured in various differentiation media including BMP-2 and/or dexamethasone were determined next with the aim to find promising test conditions for a readout system for the evaluation of future small interfering RNA release formulations for local induction of bone formation. We found a direct correlation between attenuated SOST expression and an increase in the osteogenic potential of UMR-106 cells. The combination of SOST silencing and BMP-2 could synergistically improve osteogenic factors. A lowered proliferation rate in silenced groups may indicate a faster switch to differentiation.

Microtissues from mesenchymal stem cells and siRNA-loaded cross-linked gelatin microparticles for bone regeneration

“…Medium was removed and samples were washed with warm PBS (37 ​°C). The DNA quantity was measured in a Plate Reader Synergy H1 (BioTek, Bad Friedrichshall, Germany) using Quant-iT™ PicoGreen™ dsDNA (Invitrogen™, Darmstadt, Germany) following a previously described protocol [ 14 , 21 ]. A standard curve of increasing DNA dilutions (Invitrogen™, Darmstadt, Germany) in a range of 0.001–10 ​μg/ml was used for quantification of unknown samples.…”

“…Medium was removed and samples were washed with warm PBS (37 ​°C). Cell lysis and measurement of ALP activity were performed following a previously described protocol [ 14 , 21 ] in a Plate Reader Synergy H1 (BioTek, Bad Friedrichshall, Germany). ALP activity was normalized to the DNA content per microtissue determined at the same time points.…”

Mineralizing Gelatin Microparticles as Cell Carrier and Drug Delivery System for siRNA for Bone Tissue Engineering

“…DNA quantity was determined for normalization of ALP activity and calcium content. DNA quantity in microtissues was measured in a Plate Reader Synergy H1 (BioTek, Bad Friedrichshall, Germany) using Quant-iT™ PicoGreen™ dsDNA (Invitrogen™, Darmstadt, Germany) following a previously described protocol [ 10 , 11 ]. A standard curve of increasing DNA dilutions (Invitrogen™, Darmstadt, Germany) in a range of 0.001–10 µg/mL was used for the quantification of unknown samples.…”

“…Both can bind and induce the degradation of complementary messenger RNA (mRNA) by activating the RNA-induced silencing complex (RISC) [ 9 ]. Previous studies have shown that RNAi can improve bone regeneration in vitro [ 10 , 11 , 12 , 13 ] and in vivo [ 14 , 15 ]. While the use of siRNA allows a very specific downregulation of proteins that control cell differentiation, miRNAs usually have several targets, with a multitude of consequences to cellular processes.…”