Establishment of a preclinical ovine screening model for the investigation of bone tissue engineering strategies in cancellous and cortical bone defects.

BACKGROUND: New tissue engineering strategies for bone regeneration need to be investigated in a relevant preclinical large animal model before making the translation into human patients. Therefore, our interdisciplinary group established a simplified large animal screening model for intramembranous bone defect regeneration in cancellous and cortical bone. METHODS: Related to a well-established model of cancellous drill hole defect regeneration in sheep, both the proximal and distal epimetaphyseal regions of the femur and the humerus were used bilaterally for eight drill hole cancellous defects (Ø 6 mm, 15 mm depth). Several improvements of the surgical procedure and equipment for an easier harvest of samples were invented. For the inclusion of cortical defect regeneration, a total of eight unicortical diaphyseal drill holes (6 mm Ø) were placed in the proximal-lateral and distal-medial parts of the metacarpal (MC) and metatarsal (MT) diaphyseal bone bilaterally. Acting moments within a normal gait cycle in the musculoskeletal lower limb model were compared with the results of the biomechanical in vitro torsion test until failure to ensure a low accidental fracture risk of utilized bones (ANOVA, p < 0.05). The model was tested in vivo, using thirteen adult, female, black-face sheep (Ø 66 kg; ± 5 kg; age ≥ 2.5 years). In a two-step surgical procedure 16 drill holes were performed for the investigation of two different time points within one animal. Defects were left empty, augmented with autologous cancellous bone or soft bone graft substitutes. RESULTS: The in vitro tests confirmed this model a high comparability between drilled MC and MT bones and a high safety margin until fracture. The exclusion of one animal from the in vivo study, due to a spiral fracture of the left MC bone led to a tolerable failure rate of 8 %. CONCLUSIONS: As a screening tool, promising biomaterials can be tested in this cancellous and cortical bone defect model prior to the application in a more complex treatment site.

Pull-out strength of monocortical screws placed in the maxillae and mandibles of dogs.

BACKGROUND: Mini-implants can facilitate orthodontic tooth movement by serving as anchors. The purpose of this investigation was to determine whether the pull-out strength of screws in bone varies depending on the site of insertion in the maxilla or the mandible. MATERIALS: Fifty-six titanium screws (2 mm diameter, 6 mm length, Synthes USA, Monument, Colo) were placed in 4 beagle dogs (14 screws per dog) within 30 minutes after they were killed. The screws were inserted to obtain monocortical anchorage, at predetermined sites in the anterior, middle, and posterior regions of the jaws bilaterally. Two screws were placed in the posterior palate in each dog. The jaws were harvested, and bone blocks, each containing a screw, were prepared for mechanical testing. The bone/screw block was aligned on a custom-made fixture, and the maximum force (F max ) at pullout was recorded. Cortical bone thickness was measured after extraction of the screw. Statistical analyses to test for differences were conducted with ANOVA and Tukey-Kramer tests. RESULTS: Screws placed in the anterior mandibular region had significantly ( P < .05) lower F max (134.5 +/- 24N, mean +/- SE) than those placed in the posterior mandibular region (388.3 +/- 23.1N). Regression analyses suggested a weak (r = 0.39, P = .02) but significant correlation between F max and cortical bone thickness. CONCLUSIONS: The bone supporting monocortical screws would most likely withstand immediate loading and support tooth-moving forces.

Novel mechanism of inhibition of HIV-1 reverse transcriptase by a new non-nucleoside analog, KM-1.

2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1)) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) that was designed to bind at an unconventional site on human immunodeficiency virus type 1 reverse transcriptase (RT) (Skillman, A. G., Maurer, K. W., Roe, D. C., Stauber, M. J., Eargle, D., Ewing, T. J., Muscate, A., Davioud-Charvet, E., Medaglia, M. V., Fisher, R. J., Arnold, E., Gao, H. Q., Buckheit, R., Boyer, P. L., Hughes, S. H., Kuntz, I. D., and Kenyon, G. L. (2002) Bioorg. Chem. 30, 443-458). We have investigated the mechanism by which KM-1 inhibits wild-type human immunodeficiency virus type 1 RT by using pre-steady state kinetic methods to examine the effect of KM-1 on the parameters governing the single nucleotide incorporation catalyzed by RT. Analysis of the pre-steady-state burst phase of dATP incorporation showed that KM-1 decreased the amplitude of the reaction as previously shown for other NNRTIs, because of the slow equilibration of the inhibitor with RT. In the ternary enzyme-DNA-KM-1 complex (E-DNA-I), incorporation of the next nucleotide onto the primer is blocked. However, unlike conventional NNRTIs, the inhibitory effect was caused primarily by weakening the DNA binding affinity and displacing DNA from the enzyme. Wild-type RT binds a 25/45-mer DNA duplex with an apparent K(d) of 3 nm, which was increased to 400 nm upon saturation with KM-1. Likewise, the apparent K(d) for KM-1 binding to RT increased at higher DNA concentrations. We therefore conclude that KM-1 represents a new class of inhibitor distinct from nevirapine and related NNRTIs. KM-1 can bind to RT in both the absence and presence of DNA but weakens the affinity for DNA 140-fold so that it favors DNA dissociation. The data suggest that KM-1 distorts RT conformation and misaligns DNA at the active site.