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

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.

Isolation and Characterization of CD271⁺ Stem Cells Derived from Sheep Dermal Skin.

BACKGROUND: Mesenchymal stem cells (MSCs) have great promise in the field of regenerative medicine due to their differentiation potential into several lineages. Besides the bone marrow, MSCs can be obtained from the dermis, which represents a large stem cell reservoir in the skin. Sheep provide an appropriate large animal model for preclinical studies. In this study, we focused on the isolation and characterization of MSCs from sheep dermis as an alternative to bone marrow MSCs (bmMSCs). METHODS: Primary ovine cells were obtained from the dermis for comparison with bone marrow. CD271(+)/45(-) dermal MSCs (CD271-dMSCs), which were sorted by flow cytometry, and plastic-adherent bmMSCs were examined for morphology, proliferation and senescence-associated ß-galactosidase activity in both low and high oxygen conditions. CD271 expression on cultured cells was assessed by flow cytometry. Adipogenic and osteogenic potentials of CD271-dMSCs were evaluated by oil red O and von Kossa staining. Chondrogenic capacity of CD271-dMSCs and CD271(+)/CD45(-) bone marrow cells (CD271-bmMSCs) was detected using immunohistochemistry and measurement of sulfated glycosaminoglycans. RESULTS: The cell proliferation assay demonstrated no significant difference between CD271-dMSCs and bmMSCs under low oxygen conditions. Cultured CD271-dMSCs revealed much more CD271 expression compared to CD271-bmMSCs. CD271-dMSCs and CD271-bmMSCs showed basically similar expression of the cartilage-specific proteins aggrecan and collagen type II, although with a stronger staining in CD271-bmMSC-derived cultures. Remarkably, there was co-expression of CD271 and aggrecan during chondrogenic differentiation, suggesting an involvement of CD271 in chondrogenesis. CONCLUSION: Based on these findings, CD271-dMSCs might serve as an appropriate alternative cell source in preclinical research.

Gene silencing of chordin improves BMP-2 effects on osteogenic differentiation of human adipose tissue-derived stromal cells.

Although bone morphogenic protein (BMP)-2 is known to potently induce osteogenic differentiation of human mesenchymal stem cells, strong individual differences have been reported. In part, this is due to internal antagonists of BMP-2 for example, noggin and chordin, secreted by differentiating cells. This enabling study was performed to prove the hypothesis that osteogenic effects of BMP-2 can be improved by transient nonviral gene silencing of chordin. We investigated the effect of siRNA against chordin on osteogenic differentiation in human adipose tissue-derived stromal cells (hASC). Cells of two different donors were isolated after liposuction and proliferated for passage 4 or 5. On seeding, hASCs were transfected with siRNA using a commercial liposomal transfection reagent. Subsequently, cells were differentiated in the presence or absence of BMP-2 (100 ng/mL). Noncoding siRNA as well as siRNA against noggin served as a control. Osteogenic differentiation of hASC was determined by alkaline phosphase (ALP) activity and matrix mineralization. ALP activity of hASC treated with siRNA against chordin was increased for cells of both donors. In contrast, silencing of noggin had no effect in any of the donors. In combination with BMP-2, silencing of either chordin or noggin showed strongly improved ALP activity compared with the control group that was also supplemented with BMP-2. Mineralization was observed to start earlier in groups that received siRNA against chordin or noggin and showed increased amounts of incorporated calcium on day 15 compared with the control groups. Silencing chordin in hASCs was successful to increase BMP-2 effects on osteogenic differentiation in both donors, while effects of noggin silencing were reliably observed only in one of the two investigated donors. In contrast to noggin silencing, chordin silencing also increased osteogenic differentiation without supplemented BMP-2.

Change in nicotine-induced VEGF, PGE2 AND COX-2 expression following COX inhibition in human oral squamous cancer.

Cigarette smoke has been documented to be related to the development of cancer. However, the exact mechanism for the carcinogenic action of cigarette smoke is still unknown. Nicotine is recognized to be the major compound in cigarette smoke and has been suggested to play a role in oral cancer via a cyclooxygenase (COX)/ prostaglandin-dependent pathway. This study was designed to evaluate the action of nicotine in the oral cancer cell and to further examine whether COX-2 is responsible for expression of tumor-associated angiogenic vascular endothelial growth factor (VEGF) in vitro. Viability of human oral squamous cancer cells (BHY) was measured using MTT assay. Protein expression was determined by Western blot and immunoassay kits. We found that exposure of BHY cells to nicotine (200 µg/mL for 6 hours) resulted in 2.9-fold induction of COX-2 expression as well as a 4-fold increase in VEGF levels compared with a control group. Pretreatment with celecoxib inhibited nicotine-induced change in the expression of VEGF and COX-2. The results suggest that stimulation of COX-2 and VEGF expression can contribute as important factors in the tumorigenic action of nicotine in oral cancer progression. This effect can be blocked by celecoxib, suggesting an interaction of nicotine and COX-2 pathways.