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1.  Non-invasive monitoring of BMP-2 retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes 
Non-invasive imaging can provide essential information for the optimization of new drug delivery-based bone regeneration strategies to repair damaged or impaired bone tissue. This study investigates the applicability of nuclear medicine and radiological techniques to monitor growth factor retention profiles and subsequent effects on bone formation. Recombinant human bone morphogenetic protein-2 (BMP-2, 6.5 μg/scaffold) was incorporated into a sustained release vehicle consisting of poly(lactic-co-glycolic acid) microspheres embedded in a poly(propylene fumarate) scaffold surrounded by a gelatin hydrogel and implanted subcutaneously and in 5-mm segmental femoral defects in 9 rats for a period of 56 days. To determine the pharmacokinetic profile, BMP-2 was radiolabeled with 125I and the local retention of 125I-BMP-2 was measured by single photon emission computed tomography (SPECT), scintillation probes and ex vivo scintillation analysis. Bone formation was monitored by micro-computed tomography (μCT). The scaffolds released BMP-2 in a sustained fashion over the 56-day implantation period. A good correlation between the SPECT and scintillation probe measurements was found and there were no significant differences between the non-invasive and ex-vivo counting method after 8 weeks of follow up. SPECT analysis of the total body and thyroid counts showed a limited accumulation of 125I within the body. Ectopic bone formation was induced in the scaffolds and the femur defects healed completely. In vivo μCT imaging detected the first signs of bone formation at days 14 and 28 for the orthotopic and ectopic implants, respectively, and provided a detailed profile of the bone formation rate. Overall, this study clearly demonstrates the benefit of applying non-invasive techniques in drug delivery-based bone regeneration strategies by providing detailed and reliable profiles of the growth factor retention and bone formation at different implantation sites in a limited number of animals.
doi:10.1016/j.jconrel.2008.11.023
PMCID: PMC3974410  PMID: 19105972
Drug delivery; Controlled release; Bone morphogenetic protein-2; Single photon emission computed; tomography; Scintillation probes; Micro-computed tomography
2.  RNA-Dependent Protein Kinase Is Essential for 2-Methoxyestradiol-Induced Autophagy in Osteosarcoma Cells 
PLoS ONE  2013;8(3):e59406.
Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Surgical resection and adjunctive chemotherapy are the only widely available options of treatment for this disease. Anti-tumor compound 2-Methoxyestradiol (2-ME) triggers cell death through the induction of apoptosis in osteosarcoma cells, but not in normal osteoblasts. In this report, we have investigated whether autophagy plays a role in 2-ME actions on osteosarcoma cells. Transmission electron microscopy imaging shows that 2-ME treatment leads to the accumulation of autophagosomes in human osteosarcoma cells. 2-ME induces the conversion of the microtubule-associated protein LC3-I to LC3-II, a biochemical marker of autophagy that is correlated with the formation of autophagosomes. Conversion to LC3-II is accompanied by protein degradation in 2-ME-treated cells. 2-ME does not induce autophagosome formation in normal primary human osteoblasts. In addition, 2-ME-dependent autophagosome formation in osteosarcoma cells requires ATG7 expression. Furthermore, 2-ME does not induce accumulation of autophagosomes in osteosarcoma cells that express dominant negative mutant RNA-dependent protein kinase (PKR) and are resistant to anti-proliferative and anti-tumor effects of 2-ME. Taken together, our study shows that 2-ME treatment induces PKR-dependent autophagy in osteosarcoma cells, and that autophagy could play an important role in 2-ME-mediated anti-tumor actions and in the control of osteosarcoma.
doi:10.1371/journal.pone.0059406
PMCID: PMC3602192  PMID: 23527187
3.  Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells 
BMC Cancer  2012;12:93.
Background
Osteosarcoma is a bone tumor that often affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rate in the past decades, local recurrence and metastases still develop in 40% of patients. A definite therapy is yet to be determined for osteosarcoma. Anti- tumor compound and a metabolite of estrogen, 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. In this report, we have investigated whether interferon (IFN) pathway is involved in 2-ME-induced anti-tumor effects in osteosarcoma cells.
Methods
2-ME effects on IFN mRNA levels were determined by Real time PCR analysis. Transient transfections followed by reporter assays were used for investigating 2-ME effects on IFN-pathway. Western blot analyses were used to measure protein and phosphorylation levels of IFN-regulated eukaryotic initiation factor-2 alpha (eIF-2α).
Results
2-ME regulates IFN and IFN-mediated effects in osteosarcoma cells. 2 -ME induces IFN gene activity and expression in osteosarcoma cells. 2-ME treatment induced IFN-stimulated response element (ISRE) sequence-dependent transcription and gamma-activated sequence (GAS)-dependent transcription in several osteosarcoma cells. Whereas, 2-ME did not affect IFN gene and IFN pathways in normal primary human osteoblasts (HOB). 2-ME treatment increased the phosphorylation of eIF-2α in osteosarcoma cells. Furthermore, analysis of osteosarcoma tissues shows that the levels of phosphorylated form of eIF-2α are decreased in tumor compared to normal controls.
Conclusions
2-ME treatment triggers the induction and activity of IFN and IFN pathway genes in 2-ME-sensitive osteosarcoma tumor cells but not in 2-ME-resistant normal osteoblasts. In addition, IFN-signaling is inhibited in osteosarcoma patients. Thus, IFN pathways play a role in osteosarcoma and in 2-ME-mediated anti-proliferative effects, and therefore targeted induction of IFN signaling could lead to effective treatment strategies in the control of osteosarcoma.
doi:10.1186/1471-2407-12-93
PMCID: PMC3414746  PMID: 22429849
2-Methoxyestradiol; osteosarcoma; Interferon; ISRE; GAS
4.  Enhanced Bone Morphogenetic Protein-2-Induced Ectopic and Orthotopic Bone Formation by Intermittent Parathyroid Hormone (1–34) Administration 
Tissue Engineering. Part A  2010;16(12):3769-3777.
Bone morphogenetic proteins (BMPs) play a central role in local bone regeneration strategies, whereas the anabolic features of parathyroid hormone (PTH) are particularly appealing for the systemic treatment of generalized bone loss. The aim of the current study was to investigate whether local BMP-2-induced bone regeneration could be enhanced by systemic administration of PTH (1–34). Empty or BMP-2-loaded poly(lactic-co glycolic acid)/poly(propylene fumarate)/gelatin composites were implanted subcutaneously and in femoral defects in rats (n = 9). For the orthotopic site, empty defects were also tested. Each of the conditions was investigated in combination with daily administered subcutaneous PTH (1–34) injections in the neck. After 8 weeks of implantation, bone mineral density (BMD) and bone volume were analyzed using microcomputed tomography and histology. Ectopic bone formation and almost complete healing of the femoral defect were only seen in rats that received BMP-2-loaded composites. Additional treatment of the rats with PTH (1–34) resulted in significantly (p < 0.05) enhanced BMD and bone volume in the BMP-2 composites at both implantation sites. Despite its effect on BMD in the humerus and vertebra, PTH (1–34) treatment had no significant effect on BMD and bone volume in the empty femoral defects and the ectopically or orthotopically implanted empty composites. Histological analysis showed that the newly formed bone had a normal woven and trabecular appearance. Overall, this study suggests that intermittent administration of a low PTH dose alone has limited potential to enhance local bone regeneration in a critical-sized defect in rats. However, when combined with local BMP-2-releasing scaffolds, PTH administration significantly enhanced osteogenesis in both ectopic and orthotopic sites.
doi:10.1089/ten.tea.2010.0173
PMCID: PMC2991197  PMID: 20666615
5.  Preferential Expression of the Secreted and Membrane forms of Tumor Endothelial Marker 7 transcripts in Osteosarcoma 
Anticancer research  2009;29(11):4317-4322.
Background
High expression of tumor endothelial marker 7 (TEM7) is correlated with osteogenic sarcoma (OS) metastasis and poor survival of patients. The TEM7 gene produces four alternatively spliced transcripts with distinct functional domains; the expression pattern of these transcripts in OS is unknown.
Materials and Methods
mRNA expression was assessed in 5 OS cell lines, 7 normal bone, and 9 OS tumor specimens by reverse transcriptase polymerase chain reaction.
Results
All OS cell lines, 6/9 tumors but none of the bone specimens expressed mRNA of TEM7 secreted forms 1 and 2. A total of 3/5 OS cell lines, 8/9 of tumors and 4/7 of bone specimens expressed mRNA of the TEM7 intracellular form. One out of 5 cell lines, 2/7 tumors and none of the bone specimens expressed mRNA of the TEM7 membrane form. The secreted forms had 20-fold higher expression in metastatic (LM7) compared to non-metatstatic (SAOS-2) cells.
Conclusion
The mRNA of secreted and the membrane forms of TEM7 are preferentially expressed in OS.
PMCID: PMC2800050  PMID: 20032373
TEM7; alternative splicing; osteosarcoma; PCR; metastasis
6.  Osteoblastic and Osteolytic Human Osteosarcomas can be Studied with a new Xenograft Mouse Model Producing Spontaneous Metastases 
Cancer investigation  2009;27(4):435-442.
There is no animal model that reflects the histological and radiographical heterogeneity of osteosarcoma. We assessed seven osteosarcoma cell lines for their potential to develop orthotopic tumors and lung metastasis in SCID mice. Whereas radiologically, 143B developed osteolytic tumors, SaOS-LM7 developed osteoblastic primary tumors. The mineralization status was confirmed by assessing the alkaline phosphatase activity and the microarray expression profile. We herein report a xenograft orthotopic osteosarcoma mouse model to assess osteoblastic and osteolytic lesions, which may contribute in the search for new diagnostic and therapeutic approaches.
doi:10.1080/07357900802491477
PMCID: PMC2723944  PMID: 19212826
Osteosarcoma; Animal Model; Xenograft; Orthotopic; Lung metastasis
7.  2-Methoxyestradiol-induced Cell Death in Osteosarcoma Cells is Preceded by Cell Cycle Arrest 
2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17β-Estradiol (E2), induces cell death in osteosarcoma cells. To further understand the molecular mechanisms of action, we have investigated cell cycle progression in 2-ME-treated human osteosarcoma (MG63, SaOS-2 and LM8) cells. At 5 μM, 2-ME induced growth arrest by inducing a block in cell cycle; 2-ME-treatment resulted in 2-fold increases in G1 phase cells and a decrease in S phase cells in MG63 and SaOS-2 osteosarcoma cell lines, compared to the appropriate vehicle controls. 2-ME-treatment induced a 3-fold increase in the G2 phase in LM8 osteosarcoma cells. The results demonstrated steroid specificity, as the tumorigenic metabolite, 16α-hydroxyestradiol (16-OHE), did not have any effect on cell cycle progression in osteosarcoma cells. The cell cycle arrest coincided with an increase in expression of the cell cycle markers p21, p27 and p53 proteins in 2-ME-treated osteosarcoma cells. Also, MG63 cells, transiently transfected with cDNA for a ‘loss of function mutant” RNA-dependent protein kinase (PKR) protein, were resistant to 2-ME-induced cell cycle arrest. These results suggest that 2-ME works in concert with factors regulating cell cycle progression, and cell cycle arrest precedes cell death in 2-ME-treated osteosarcoma cells.
doi:10.1002/jcb.21758
PMCID: PMC2821714  PMID: 18384113
estrogen metabolite; MG63 cells; cell cycle arrest; PKR
8.  Non-invasive screening method for simultaneous evaluation of in vivo growth factor release profiles from multiple ectopic bone tissue engineering implants 
The purpose of this study was to develop and validate a screening method based on scintillation probes for the simultaneous evaluation of in vivo growth factor release profiles of multiple implants in the same animal. First, we characterized the scintillation probes in a series of in vitro experiments to optimize the accuracy of the measurement setup. The scintillation probes were found to have a strong geometric dependence and experience saturation effects at high activities. In vitro simulation of 4 subcutaneous limb implants in a rat showed minimal interference of surrounding implants on local measurements at close to parallel positioning of the probes. These characteristics were taken into consideration for the design of the probe setup and in vivo experiment. The measurement setup was then validated in a rat subcutaneous implantation model using 4 different sustained release carriers loaded with 125I-BMP-2 per animal. The implants were removed after 42 or 84 days of implantation, for comparison of the non-invasive method to ex-vivo radioisotope counting. The non-invasive method demonstrated a good correlation with the ex-vivo counting method at both time-points of all 4 carriers. Overall, this study showed that scintillation probes could be successfully used for paired measurement of 4 release profiles with minimal interference of the surrounding implants, and may find use as non-invasive screening tools for various drug delivery applications.
doi:10.1016/j.jconrel.2008.05.004
PMCID: PMC2601638  PMID: 18554743
Controlled drug delivery; non-invasive screening; scintillation detector; radiolabelled growth factor; method validation
9.  Double-Stranded RNA-Dependent Protein Kinase Is Involved in 2-Methoxyestradiol–Mediated Cell Death of Osteosarcoma Cells 
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.
doi:10.1359/JBMR.060914
PMCID: PMC1955766  PMID: 17014383
estrogen metabolite; MG63 cells; interferon; protein kinase; double-stranded RNA-dependent protein kinase

Results 1-9 (9)