Shogren, Kristen L.

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

10531 Background: Osteosarcoma (OS) is a clinically challenging primary malignant bone tumor that has a high rateof local recurrence and metastasis. Disease free survival has been reported to be as low as 19% despite contemporary chemotherapy regimens and surgery. Wnt signaling is known to play a role in osteogenesis and cancer pathogenesis, therefore is a promising target for therapeutic intervention in OS. Secreted Frizzled Related Proteins (sFRPs), Dickkopf-1 (DKK1), and Sclerostin (SOST) are secreted Wnt inhibitor proteins that regulate Wnt signaling in bone. This study investigates the activity of sFRP3, DKK1, and SOST in serum from OS patients with the goal of developing therapeutic Wnt inhibitors and clinically useful biomarkers. Methods: Enzyme linked immunosorbent assayswere used to quantify sFRP3, DKK1 and SOST levels in human serum (n=40 pairs; 20 OS patients, 20 aged matched non-diseased controls). Patients were stratified into 2 age groups: >35 (n=26 pairs) and <30 (n=14 pairs) years of age. Clinical data from the medical records was correlated with experimental results. Using a Wilcoxon signed rank test, a p–value <0.05 was significant. Results: sFRP3 levels were significantly decreased in 55% (22/40) of diseased samples compared to 20% (8/40) of the non-diseased controls (p= 0.007). DKK1 levels were elevated in 40% (16/40) of OS samples compared to 30% (12/40) of the non-diseased controls (p= 0.08). SOST levels were increased in 42.5% (17/40) of diseased samples compared to 32.5% (13/40) of the non-diseased controls (p= 0.38). When stratified by age, the younger patients showed a statistically significant decrease in sFRP3 expression (p<0.001). Serum levels of sFRP3, DKK1, and SOST were not statistically significant between metastatic and non-metastatic OS. Conclusions: Serum sFRP3 levels were significantly decreased in OS patients, a difference that was even more pronounced in the pediatric strata. Since sFRP3 is differentially expressed in patient serum it has potential as a clinical biomarker for initial tumor diagnosis and early detection of recurrence. Decreased sFRP3 expression may be responsible for the up regulation of Wnt signaling in OS. Wnt inhibitors such as sFRP3 have potential as therapeutic agents in OS warranting further investigation.

ABSTRACTOsteosarcoma is a bone tumor that mainly affects children and adolescents. Although its pathogenesis is still not fully understood, activation of Wnt signaling has been implicated in the development and metastasis of osteosarcoma. In this report, we have investigated the effect of the anti‐tumor compound, 2‐methoxyestradiol (2‐ME) on Wnt antagonist frizzled‐related protein b (Frzb), also known as secreted frizzled‐related protein (sFRP)3 in human osteosarcoma (MG63) cells. Our results show that 2‐ME treatment induces Frzb gene promoter activity, and increases Frzb mRNA and protein levels in osteosarcoma cells. In addition, 2‐ME treatment regulates downstream Wnt signaling, increasing the cytoplasmic levels of β‐catenin, and blocking β‐catenin‐mediated Wnt activation in osteosarcoma cells. 2‐ME‐mediated induction of Frzb protein expression is specific to osteosarcoma cells, as it does not affect Frzb expression in normal primary human osteoblasts. Furthermore, 2‐ME‐induced apoptosis and autophagy are blocked in osteosarcoma cells transfected with Frzb siRNAs. Taken together, these studies demonstrate that Frzb protein plays an important role in 2‐ME‐mediated anti‐tumor mechanisms in osteosarcoma cells. J. Cell. Biochem. 118: 1497–1504, 2017. © 2016 Wiley Periodicals, Inc.

Osteosarcoma is a bone tumor that affects children and young adults. 2‐Methoxyestradiol (2‐ME), a naturally occurring estrogen metabolite, kills osteosarcoma cells, but does not affect normal osteoblasts. In order to effectively target osteosarcoma and improve the therapeutic index of the drug 2‐ME, we have encapsulated 2‐ME in a composite of oligo‐(polyethylene glycol) fumarate (OPF) hydrogel and poly (lactic‐co‐glycolic acid) (PLGA) microspheres and investigated the effect of polymer composition on 2‐ME release kinetics and osteosarcoma cell survival. The in vitro study shows that 2‐ME can be released in a controlled manner over 21‐days. The initial burst releases observed on day 1 were 50% and 32% for OPF and OPF/PLGA composites, respectively. The extended release kinetics show that 100% of the encapsulated 2‐ME is released by day 12 from OPF, whereas the OPF/PLGA composites showed a release of 85% on day 21. 2‐ME released from the polymers was biologically active and blocked osteosarcoma cell proliferation in vitro. Also, comparison of 2‐ME delivery in osteosarcoma cells in culture, shows that direct treatment has no effect after 3 days, whereas polymer‐mediated delivery produces anti‐tumor effects that could be sustained for 21 days. These findings show that the OPF and PLGA polymeric system may prove to be useful in controlled and sustained delivery of 2‐ME and could be further explored in the treatment of osteosarcoma. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 2491–2499, 2013.

Abnormal secretion of PTH by the parathyroid glands contributes to a variety of common skeletal disorders. Prior studies implicate platelet-derived growth factor-A (PDGF-A) as an important mediator of selective PTH actions on bone. The present studies used targeted gene profiling and small-molecule antagonists directed against candidate gene products to elucidate the roles of specific PTH-regulated genes and signaling pathways. A group of 29 genes in rats continuously infused with PTH and cotreated with the PDGF receptor antagonist trapidil were differentially expressed compared with PTH treatment alone. Several of the identified genes were functionally clustered as regulators of fibroblast differentiation and extracellular matrix modeling, including the matrix cross-linking enzyme lysyl oxidase (LOX). Treatment with β-aminopropionitrile, an irreversible inhibitor of LOX activity, dramatically reduced diffuse mineralization but had no effect on PTH-induced fibrosis. In contrast, the receptor tyrosine kinase inhibitor Gleevec and the phosphoinositide 3-kinase inhibitor wortmannin each reduced bone marrow fibrosis. In summary, the present studies support the hypotheses that PTH-induced bone marrow fibrosis is mediated by PDGF-A via a phosphoinositide 3-kinase-dependent signaling pathway and that increased LOX gene expression plays a key role in abnormal mineralization, a hallmark of chronic hyperparathyroidism.

Abstract We studied the involvement of interferon-regulated, PKR on 2-ME–mediated actions in human osteosarcoma cells. Our results show that PKR is activated by 2-ME treatment and is necessary for 2-ME–mediated induction of osteosarcoma cell death. Introduction: Osteosarcoma is the most common primary bone tumor and most frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a metabolite of 17β-estradiol, induces interferon gene expression and apoptosis in human osteosarcoma cells. In this report, we studied the role of interferon-regulated double-stranded (ds)RNA-dependent protein kinase (PKR) protein on 2-ME–mediated cell death in human osteosarcoma cells. Materials and Methods: Western blot analyses were used to measure PKR protein and phosphorylation levels. Cell survival and apoptosis assays were measured using trypan blue exclusion and Hoechst dye methods, respectively. A transient transfection protocol was used to express the dominant negative PKR mutants. Results and Conclusions: PKR was increased in 2-ME–treated MG63 cells, whereas 17β-estradiol, 4-hydroxyestradiol, and 16α-hydroxyestradiol, which do not induce cell death, had no effect on PKR protein levels. Also, 2-ME treatment induced PKR kinase activity as indicated by increased autophosphorylation and phosphorylation of the endogenous substrate, eukaryotic initiation factor (eIF)-2α. dsRNA poly (I).poly (C), an activator of PKR protein, increased cell death when osteosarcoma cells were treated with a submaximal concentration of 2-ME. In contrast, a serine-threonine kinase inhibitor SB203580 and a specific PKR inhibitor 2-aminopurine (2-AP) blocked the 2-ME–induced cell death in MG63 cells. A dominant negative PKR mutant protein conferred resistance to 2-ME–induced cell death to MG63 osteosarcoma and 2-ME–mediated PKR regulation did not require interferon gene expression. PKR protein is activated in cell free extracts by 2-ME treatment, resulting in autophosphorylation and in the phosphorylation of the substrate eIF-2α. We conclude from these results that PKR is regulated by 2-ME independently of interferon and is essential for 2-ME–mediated cell death in MG63 osteosarcoma cells.