E) was amplified (f 5’gca tgt cag atc cac aac gga t 3′; r 5’tgt cag caa tgc atc ctg ca 3′). All PCR products were detected on 1.5 agarose gel (Serva) with Ethidiumbromide (Merck, Germany).StatisticsAnimals were randomized in a blinded manner by drawing lots before the operation. The tibiae (right or left) were also randomized for histological and biomechanical investigation. To determine statistically significant differences in the histomorphometrical and biomechanical results, a Kruskal Wallis followed by Mann-Whitney Test and Bonferroni Holm correction was used (SPSS 14.0, SPSS Inc., Chicago, USA).In the Luciferase group, where a plasmid encoding for Luciferase replaced the BMP-2 encoding plasmid, animals were sacrificed at days 4, 7 and 28. Tissue from the operated tibiae, brain, lungs, liver, spleen, testicles and muscle was taken. Also bone samples from the not operated forelegs were analyzed. The bone was grinded with a cooled grinding device before processing. For analysis, d]furan-1-ylboronic acid 2-Chloro-1-(3-hydroxyazetidin-1-yl)ethan-1-one Fmoc-D-Trp(Boc)-OH 3-Bromo-4′-chloro-1 the tissue of the parenchymatous organs was homogenized and lysed. Total RNA was extracted using ,,RNeasy” Kit?(Quiagen, Germany). Concentration and purity was determined photometrically at 260/280 nm. Approximately 80 ng of total RNA were used for Reverse Transcription PCR. Thereby, single-stranded mRNA was transcribed into complementary DNA (cDNA). In the following non-quantitative PCR the luciferase transcripts were amplified with specific luciferase primers (f 5′ ctg aatResults One animal of the Methyl 5-amino-2,4-difluorobenzoate 28 days Luciferase group died during anaesthesia and was excluded from the study. A connection to the vector administration could not be found. All other animals tolerated the procedure well. Neither the clinical appearance nor the blood specimens suggested an infection at the wound site. In some animals, a transient swelling at the nail insertion site was observed.Radiographic ExaminationRadiographs showed that the implants were correctly positioned and had a similar fitting in all animals. No dislocations, fractures, or other abnormalities were observed postoperatively or after scarifying. Radiographic analysis did not reveal any difference between the groups at any of the time points.Biomechanical testingSimilar to the radiographic examination, gross observation revealed no fractures or abnormalities to any (S)-tert-Butyl 6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carboxylate of the bones. Throughout the groups, the recording of the testing process showed a typical load-displacement curveFigure 3 Histologic preparation stained with Safranin-O/van Kossa. Blue circles are marking a zone of direct bone contact, the yellow circle marks a zone of indirect bone contact.Faensen et al. BMC Musculoskeletal Disorders 2011, 12:163 http://www.biomedcentral.com/1471-2474/12/Page 5 ofwith a steep start and a peak, when the force needed to loosen the implant had been reached (Figure 2). The peak force was set in relation to the length of the tested bone to compensate for differences in length between the specimens. In the blank control group an increase in the strength of fixation was detectable between day 28 and 56. This increase in implant fixation over time was less pronounced in the two other groups. The strength of fixation was significantly lower in the verum group at PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 both time points compared to the blank control PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8833965 group. The difference between the blank control group with no filling of the medullar cavity and the fibrin glue group was not significant at either of the time points (Figure 4).Histomorphometric Analysisdid not differ signifi.