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Vascular graft infections (VGI) are severe complications in prosthetic vascular surgery with an incidence ranging from 1 to 6%. In these cases, synthetic grafts are commonly used in combination with antimicrobial agents. Expanded polytetrafluoroethylene (ePTFE) is in clinical use as a synthetic graft material and shows promising results by influencing bacterial adhesion. However, the literature on antibiotic-bound ePTFE grafts is scarce. Gentamicin is a frequently used antibiotic for local treatment of surgical site infections, but has not been evaluated as antimicrobial agent on ePTFE grafts. In this study, we examine the antimicrobial efficacy and biocompatibility of novel types of gentamicin-coated ePTFE grafts in vitro. ePTFE grafts coated with gentamicin salt formulations with covalently-bound palmitate were evaluated in two drug concentrations (GP1.75% and GP3.5%). To investigate effects from types of formulations, also suspensions of gentamicin in palmitate as well as polylactide were used at comparable levels (GS + PA and GS + R203). Antibacterial efficacies were estimated by employing a zone of inhibition, growth inhibition and bacterial adhesion assay againstStaphylococcus aureus(SA). Cytotoxicity was determined with murine fibroblasts according to the ISO standard 10993-5. Gentamicin-coated ePTFE grafts show low bacterial adherence and strong antibacterial properties in vitro against SA. Bactericidal inhibition lasted until day 11. Highest biocompatibility was achieved using gentamicin palmitate GP1.75% coated ePTFE grafts. ePTFE grafts with gentamicin-coating are effective in vitro against SA growth and adherence. Most promising results regarding antimicrobial properties and biocompatibility were shown with chemically bounded gentamicin palmitate GP1.75% coatings.

Background Clinical cartilage repair strategies can be tested using the sheep model as suggest by the European Medicines Agency. To characterize variation within the joint a full biomechanical mapping is necessary. The aim of this study is to establish a loading model, to map regional differences within the knee and determine reference areas for area specific replacement techniques. Methods A porous indenter was selected to evaluate 22 defined test locations (femoral condyles, tibia plateau, patella, femoral groove) on ovine knees (n = 7). A high-dynamic force-controlled micro creep and creep-recovery indentation test system applied five loading (0.11 MPa) and unloading (5.6 kPa) cycles for 60 s each and recorded creep-recovery. Needle indentation was used to measure cartilage thickness and calculate total strain. Findings Steady state behaviour was observed from the third cycle and further evaluated. Little variation of stiffness in N/mm was found within the patella (4.3SD0.5) and femoral groove (8.1SD0.7) compared to larger variations in the femur (7.9SD2.0) and tibia (7.5SD3.2). Creep indentation showed values of 14.5%(SD2.7%) for the patella and 17.4%(SD3%) for the femoral grove opposed to 13.4%(SD4.3%) for the femoral condyles and 21.8%(SD6.6%) for the tibia plateau. Similar trends were observed analysing creep-recovery. Values were normalized to cartilage thickness which ranged between 0.36 mm and 1.14 mm. Interpretation Our setup allows a reliable evaluation of zonal differences. Homogenous biomechanical behaviour is found within the patella and femoral groove whereas significant biomechanical variation within the femoral condyles and tibia plateau indicates the need for site-specific cartilage repair products.

The aggressive fast-growing osteosarcoma is the most common primary malignant bone tumor. The relevance of estrogen as a key player in bone metabolism and bone tumor is well-known. At the molecular level, estrogen activates the estrogen receptor α (ERα) as a natural ligand of this receptor. ERα acts as a transcription factor by binding to the "estrogen response element" (ERE) and regulates the expression of a various number of genes. Epigenetic processes, e.g. the methylation of the "cytosine-phosphatidyl-guanine (CpG) islands" can change the transcription of target genes and subsequently the protein expression. As DNA methylation is generally associated with gene transcription repression, up until now little is known about the ERα methylation in osteosarcoma cells. The aim of the present pilot study was to evaluate the methylation status of ERα in osteosarcoma cells SAOS-2 and MG 63 after stimulation with estrogen.SAOS-2 and MG 63 cells were cultured in DMEM. After treatment with 10 nmol estrogen (E2) for 24 h, the expression of ERα was detected by immunocytochemistry (ICC). As controls we used untreated cells. Staining was evaluated semi-quantitatively by the immunoreactive score of Remmele and Stegner (IRS). To determine mRNA gene expression, extracted RNA was transcribed into c-DNA and a quantitative real-time-PCR (qRT-PCR) was carried out. The semi quantitative evaluation of the ERα mRNA was based on the 2(-ΔΔct) method using untreated cells as reference control. One microgram of each extracted genomic DNA sample was converted with bisulfite and a real-time methylation-specific PCR (rt-MSP) was performed.The estrogen-stimulated SAOS-2 cells showed a significant increase of ERα expression. A 7-fold up-regulation of ERα mRNA confirmed the results of immunocytochemistry. Methylation of the ERα promoter was not detected in treated cells. In contrast, we identified methylation of the ERα promoters in untreated cells. The staining of MG 63 cells showed a weak gain of ERα expression in the stimulated cells, as well as a weak increase of the ER-α mRNA (2-fold). Methylation of the ERα promoters was not detectable in either treated or untreated cells.The methylation status of ERα in osteosarcoma cells is affected by estrogen. These findings indicate that epigenetic changes of genomic DNA regulate ERα synthesis. Taken together, our results suggest that SAOS-2 cells can be an interesting model for further investigating ERα synthesis. In addition, the evaluation of ERα methylation in osteosarcoma specimens is in progress.

Background: Adapted from results in the field of cervical cancer, a direct connection between HPV infection and oropharyngeal carcinoma development could be established. Aim of this study was to evaluate p16 and TA-MUC1 in laryngeal cancer and their correlation to diagnostic, since TA-MUC1 is primarily restricted to malignancies. Methods: Paraffin-embedded laryngeal cancer specimens (n=129) and normal tissue (n=5) were analyzed for TA-MUC1 expression using hPankoMab-GEXTM antibody and evaluated according the immunoreactive score. Survival was assessed via log-rank test and Kaplan-Meier-survival analysis. Results: Significant correlation with tumor grading and staging was exhibited by TA-MUC1staining, while being negative in normal tissues. Expression of p16 significantly increased in T4 compared to T1 tumors. Significant differences in overall survival were found in correlation to TNM-classification, grading and relapse. TA-MUC1 showed a positive trend correlating to p16. Conclusion: Because of this positive trend, we suggest a HPV association in head and neck tumors. Most likely due to an insufficient quantity of HPV-positive patients, no statistical significance could be established. However, targeting TA-MUC1 would improve tumor therapy by linking hPankoMab-GEXTM to the overexpressed galectin. Systematic analysis of HPV-association should be performed generally in laryngeal cancer to gain further information about the interaction of HPV and malignancies.

During the last decade, several tissues and biomaterials for medical applications in replacing bony tissues have been developed. Three-dimensional cell clusters and mandibular bone grafts are two distinct examples of these developments. The characterization of the complex three-dimensional structures, however, is still mainly restricted on the twodimensional analysis of histological slices. The present paper examines the quantitative analysis of mandibular bone grafts and three-dimensional cell clusters on the basis of synchrotron radiation-based micro computed tomography measurements. An automated search of pre-defined microstructures through component labeling is applied to the real datasets in order to identify features that reside independently from other components. The examples demonstrate three levels of complexity: rather large pieces of bone augmentation material that touch each other, individual adipocytes difficult to automatically segment in a wet cluster and osmium-stained adipocyte exhibiting higher X-ray absorption than the surrounding tissue. Although the structures of interest such as the cells can be labeled, de-clustering of the components requires the incorporation of erosion and dilation algorithms.

Two new tumor-like cell lines were established which developed spontaneously in vitro from normal human primary osteoblast-like cells originating from non-oncogenic bone surgery.The tumor cell properties studied included morphology, proliferation characteristics in normal and low-serum media, and anchorage-independent growth in soft agarose.Karyotyping of the cells showed numerous rearrangements and abnormalities. These results pointed to the tumorigenic potential of the cells and demonstrate the importance of biosafety in tissue engineering and therapeutic cell applications when prolonged culture conditions are required.