Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells

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

Evaluation of Osteoconductive Scaffolds in the Canine Femoral Multi-Defect Model

Treatment of large segmental bone defects remains an unsolved clinical challenge, despite a wide array of existing bone graft materials. This project was designed to rapidly assess and compare promising biodegradable osteoconductive scaffolds for use in the systematic development of new bone regeneration methodologies that combine scaffolds, sources of osteogenic cells, and bioactive scaffold modifications. Promising biomaterials and scaffold fabrication methods were identified in laboratories at Rutgers, MIT, Integra Life Sciences, and Mayo Clinic. Scaffolds were fabricated from various materials, including poly(L-lactide-co-glycolide) (PLGA), poly(L-lactide-co-ɛ-caprolactone) (PLCL), tyrosine-derived polycarbonate (TyrPC), and poly(propylene fumarate) (PPF). Highly porous three-dimensional (3D) scaffolds were fabricated by 3D printing, laser stereolithography, or solvent casting followed by porogen leaching. The canine femoral multi-defect model was used to systematically compare scaffold performance and enable selection of the most promising substrate(s) on which to add cell sourcing options and bioactive surface modifications. Mineralized cancellous allograft (MCA) was used to provide a comparative reference to the current clinical standard for osteoconductive scaffolds. Percent bone volume within the defect was assessed 4 weeks after implantation using both MicroCT and limited histomorphometry. Bone formed at the periphery of all scaffolds with varying levels of radial ingrowth. MCA produced a rapid and advanced stage of bone formation and remodeling throughout the defect in 4 weeks, greatly exceeding the performance of all polymer scaffolds. Two scaffold constructs, TyrPC[subscript PL]/TCP and PPF4[subscript SLA]/HA[subscript PLGA Dip], proved to be significantly better than alternative PLGA and PLCL scaffolds, justifying further development. MCA remains the current standard for osteoconductive scaffolds.United States. Army Medical Research and Materiel Command (Armed Forces Institute of Regenerative Medicine)United States. Office of Naval ResearchUnited States. Air Force. Office of the Surgeon GeneralUnited States. NavyNational Institutes of Health (U.S.)United States. Veterans AdministrationCleveland Clinic Foundatio

C-Reactive Protein, Erythrocyte Sedimentation Rate and Orthopedic Implant Infection

BACKGROUND: C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) have been shown to be useful for diagnosis of prosthetic hip and knee infection. Little information is available on CRP and ESR in patients undergoing revision or resection of shoulder arthroplasties or spine implants. METHODS/RESULTS: We analyzed preoperative CRP and ESR in 636 subjects who underwent knee (n=297), hip (n=221) or shoulder (n=64) arthroplasty, or spine implant (n=54) removal. A standardized definition of orthopedic implant-associated infection was applied. Receiver operating curve analysis was used to determine ideal cutoff values for differentiating infected from non-infected cases. ESR was significantly different in subjects with aseptic failure infection of knee (median 11 and 53.5 mm/h, respectively, p=<0.0001) and hip (median 11 and 30 mm/h, respectively, p=<0.0001) arthroplasties and spine implants (median 10 and 48.5 mm/h, respectively, p=0.0033), but not shoulder arthroplasties (median 10 and 9 mm/h, respectively, p=0.9883). Optimized ESR cutoffs for knee, hip and shoulder arthroplasties and spine implants were 19, 13, 26, and 45 mm/h, respectively. Using these cutoffs, sensitivity and specificity to detect infection were 89 and 74% for knee, 82 and 60% for hip, and 32 and 93% for shoulder arthroplasties, and 57 and 90% for spine implants. CRP was significantly different in subjects with aseptic failure and infection of knee (median 4 and 51 mg/l, respectively, p<0.0001), hip (median 3 and 18 mg/l, respectively, p<0.0001), and shoulder (median 3 and 10 mg/l, respectively, p=0.01) arthroplasties, and spine implants (median 3 and 20 mg/l, respectively, p=0.0011). Optimized CRP cutoffs for knee, hip, and shoulder arthroplasties, and spine implants were 14.5, 10.3, 7, and 4.6 mg/l, respectively. Using these cutoffs, sensitivity and specificity to detect infection were 79 and 88% for knee, 74 and 79% for hip, and 63 and 73% for shoulder arthroplasties, and 79 and 68% for spine implants. CONCLUSION: CRP and ESR have poor sensitivity for the diagnosis of shoulder implant infection. A CRP of 4.6 mg/l had a sensitivity of 79 and a specificity of 68% to detect infection of spine implants

Carrageenan-based hydrogels for the controlled delivery of PDGF-BB in bone tissue engineering applications

One of the major drawbacks found in most bone tissue engineering approaches developed so far consists in the lack of strategies to promote vascularisation. Some studies have addressed different issues that may enhance vascularisation in tissue engineered constructs, most of them involving the use of growth factors (GFs) that are involved in the restitution of the vascularity in a damaged zone. The use of sustained delivery systems might also play an important role in the re-establishment of angiogenesis. In this study, !-carrageenan, a naturally occurring polymer, was used to develop hydrogel beads with the ability to incorporate GFs with the purpose of establishing an effective angiogenesis mechanism. Some processing parameters were studied and their influence on the final bead properties was evaluated. Platelet derived growth factor (PDGF-BB) was selected as the angiogenic factor to incorporate in the developed beads, and the results demonstrate the achievement of an efficient encapsulation and controlled release profile matching those usually required for the development of a fully functional vascular network. In general, the obtained results demonstrate the potential of these systems for bone tissue engineering applications.This work was supported by the European NoE EXPERTISSUES (NMP3-CT-2004-500283), the European STREP HIPPOCRATES (NMP3-CT-2003-505758), and by the Portuguese Foundation for Science and Technology (FCT) through the project PTDC/FIS/68517/2006 and through the V. Espirito Santo's Ph.D. grant (SFRH/BD/39486/2007)

Severe Osteogenesis Imperfecta in Cyclophilin B–Deficient Mice

Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB–deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB–deficient cells and tissues from CypB–knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone

Signální proteinově-funkciolizované zlaté nanočástice pro jaderné cílení v buňkách osteosarkomu pro použití v radiosenzibilizačních experimentech

The standard therapy for malignant primary bone tumors such as osteosarcoma involves major surgeries. For tumors located in difficult regions such as the pelvis, surgical intervention could lead to serious side effects for example loss of a limb and/or function, loss of bowel, bladder and sexual function as well as problems with wound healing and surgical complications. Therefore, exploring other approaches that can improve or complement current surgical techniques is important. Hence, sensitizing primary bone tumors to radiation could offer an additional strategy that could complement surgery and significantly improve survival and quality of life. Gold nanoparticles (AuNPs) have been shown to enhance radiosensitivity by increasing the local dose of radiation inside tumors. Therefore, the referred procedure of preparation and functionalization of gold nanoparticles may be used for investigation whether DNA repair inhibition in the presence of AuNPs leads to an effective radiosensitizing strategy for primary bone tumor cells and explore the mechanism of how this may be happening. In our work, we prepared gold nanoparticles and verified the relation between the size of the AuNPs and their uptake in tumor 143B cells and also investigated whether the optimal size of the AuNPs should not be smaller than the size of nuclear envelope pores (20-50 nm). Hence, two different AuNPs systems were prepared: the first one with AuNPs core size of about 5 nm (BS) and the second one with AuNPs core size of about 50 nm (ZA). For cellular AuNPs uptake enhancement, we functionalized the AuNPs with signaling peptides. For this purpose we prepared PEG-coated AuNPs functionalized with signal peptides for targeted transport into the cytoplasm (CPP) and into the cell nucleus (CPP + NLS). The toxicity of the AuNPs systems was assessed by MTS assay. We prepared stable functionalized AuNPs systems of both sizes. With the functionalizing of the AuNPs using signal peptides (CPP, NLS), the AuNPs penetrated into the cell nucleus. The referred procedure of preparation and functionalization of gold nanoparticles may be used for investigating inhibition of DNA repair in the presence of AuNPs and it could lead to new understanding in overcoming radioresistance in primary bone tumor cells.Standardní terapie pro zhoubné primární kostní nádory jako např. osteosarkom zahrnují závažné operace. Pro nádory nacházející se v obtížných oblastech, jako je pánev, operační intervence můžou vést k závažným vedlejším efektům, jako je například ztráta končetiny nebo její funkce, ztráta střeva, žaludku a sexuálních funkcí, jakožto i problémy s hojením ran a další operační komplikace. Proto výzkum dalších přístupů je důležitý pro vylepšení nebo doplnění současných operačních technik. Z tohoto důvodu může senzibilizace primárních kostních nádorů vůči radiaci nabídnout další strategický přístup, který může sloužit jako doplněk operaci a výrazně vylepšit přežití a kvalitu života pacientů. Zlaté nanočástice (Au NPs) vykazují zvýšenou radiosenzitivitu pomocí zvýšení lokální dávky radiace v nádoru. V naší práci jsme připravili zlaté nanočástice a ověřili spojitost mezi velikostí AuNPs a jejich absorpcí do buněk 143B a také jsme zkoumali, zda optimální velikost AuNPs nebude menší než velikost pórů obalu (20-50 nm). Proto byly připraveny dva systému AuNPs: první s velikostí jader AuNPs okolo 5 nm (BS) a druhý s velikostí jádra kolem 50 nm (ZA). Pro vylepšení buněčného příjmu AuNPs jsme funkcionalizovali částice signálními peptidy. Pro tento účel jsme připravili AuNPs obalené PEG, které jsme následně funcionalizovali pro cílený transport do cytoplasmy (CPP) a do jádra buňky (CPP + NLS). Toxicita onou systémů AuNPs byla posouzena MTS testem. Připravili jsme stabilní funkcionalizované AuNPs obou velikostí. Pomocí funcionalizace AuNPs pomocí signálních peptidů (CPP, NLS), částice úspěšně penetrovali do jádra buňky. Popsaná procedura přípravy a funkcionalizace zlatých nanočástic může bát použita pro výzkum inhibitorů opravy DNA za přítomnosti AuNPs a může vést k novému porozumění překonání radioodolnosti buněk v primárních kostních nádorech

Preferential expression of the secreted and membrane forms of tumor endothelial marker 7 transcripts in osteosarcoma

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-metastatic (SAOS-2) cells. CONCLUSION: The mRNA of secreted and the membrane forms of TEM7 are preferentially expressed in OS