2010 - Volume 37 - Issue 1

5-9
Alveolar Bone Loss: A Shorter-Time Study Model in Mice
by Cristiane Alencar, Dalva Maria Pereira Padilha, Juliana Balbinot Hilgert & Elken Gomes Rivaldo

Mucoperiosteal flap surgery (MPS) of the mouth is conducted in several clinical situations. However, MPS triggers a resorption process that leads to alveolar bone ridge loss. Rodents are routinely used in studies evaluating alveolar bone loss (ABL), and the experimental time involved in these experiments is usually 21 days. This study aims at establishing whether the alveolar bone resorption area differs between 10 and 21 days after MPS in mice mandibles. MPS was performed in the vestibular aspect of the left mandible (LM) of 20 male CF1 mice Mus domesticus. The right mandible (RM) was used as control. Animals formed identical groups for each experimental time. Animals were euthanized 10 days (ten days group, TG) or 21 days (twenty one days group, TWG) after the surgical procedure. All mandibles were hemisectioned, cleaned and stained for stereomicroscopic inspection. Digital images were obtained and the alveolar bone loss area measured (mm2) using image analysis software. The results demonstrate that a significant loss is observed in the left mandible (LM) (Student’s t test, p<0.01), as compared to the RM, in both groups. No statistically significant difference was observed in the ABL area (p>0.05) between TWG and TG. This investigation leads to the conclusion that it is possible to reduce experimental times when using the MPS model in mice.

13-18
Technical Report: Scintigraphic Evaluation of Bone Formation in Göttingen Minipigs
by Schwarz ML, Seidling R, Mauermann E, Werner A, Steil V, Forsch E, Obertacke U, Becker K, Lehmann L

In experiments and processes requiring the application of nuclear tracers in large animals, statutory provisions and safety standards as well as a variety of techniques have to be regarded and employed. In order to sufficiently analyze questions pertaining to osseointegration as well as the possibility of ectopic bone formation in Göttingen minipigs, we decided to use scintigraphic examinations using 99mTc-HDP (Technetium - hydroxymethane diphosphonate). In this study, metallic implants coated in different forms with rhBMP-2 (recombinant human bone morphogenetic protein-2) were surgically introduced into the pigs’ femora. A total of 26 adult female minipigs (Ellegard, Dalmose, Denmark) averaging 40 months in age were post-surgically evaluated through the application of a radionuclide and its subsequent distribution using a scintillation camera. Each animal received approximately 10 MBq/kg BW (mega Becquerel per kilogram bodyweight). This paper describes the procedures of anaesthesia, the quite challenging transvaginal- urethral catheterization, the application of a catheter in the jugular vein, the radionuclide injection and the disposal of the sacrificed animals under statutory provisions and safety standards. The technical report reveals that the scintigraphic evaluation in large animal experiments is a practicable– yet sophisticated – method of examination and also strives to encourage further research groups to implement this elegant procedure.

23-26
Kinetics and Penetration into Inflammatory Tissue Cage Fluid of Cefepime Administered to Rabbits
by R. Rule, M. Vita & P. Martino

The kinetics and penetration of cefepime into inflammatory tissue cage fluid were determined in rabbits. Ten adult healthy rabbits were used. Concentrations of cefepime were measured in serum and induced inflammatory exudate by biological methods. The kinetic analysis was performed by mean of a non-compartmental model. Pharmacokinetic results in serum (S) and inflammatory tissue cage fluid (ITCF) (means± standard error) were: half life of elimination [t1\2 (S) ]= 1.6 ± 0.2 and (ITCF) 3.7 ± 0.3 h; area under the curve [AUC (S)]= 225.3 ± 21.4 and (ITCF) 208.0 ± 13.6 (μg/ml/h]; maximum concentration [Cmax (ITCF)]= 37.7 ± 3.6 μg/ml and time to reach Cmax [tmax (ITCF)]= 1.8 ± 0.4 h and the penetration into inflammatory tissue cage fluid [P (ITCF)] = 92.3 ± 8.7 %. In conclusion, the cefepime administered to rabbits penetrate rapidly and nearly completely into inflammatory tissue cage fluid.

31-40
Involvement of BMP-2, TGF-ß2 and TGF-ß3 Signaling in Initial and Early Stages of Heterotopic Ossification in a rat Experimental Model
by S Suutre, A Toom, A Arend, G Selstam

This study focused on the localization and expression of bone morphogenetic protein 2 (BMP-2) and different isoforms of transforming growth factor ß (TGF-ß1, TGF-ß2 and TGF-ß3) in the initial and early stages of heterotopic ossification (HO) employing an animal model mimicking the situation after total hip arthroplasty (THA). Bone growth was induced in rats using ß-tricalcium phosphate implants immersed either in osteoinductive rhBMP-2 solution or in saline and implanted at the site where the HO is usually expected to develop after THA. Implants were removed at 3 or 21 days after the operation and handled according to stereology principles. mRNA expression and protein staining of growth factors in different types of tissues was determined by in situ hybridization and immunohistochemistry, respectively. After three days, TGF-ß3 content in the undifferentiated mesenchymal-like cells in the rhBMP-2 treated implants was, as assessed by immunohistochemistry, 49.6% higher compared to the saline treated group (p=0.024). This was also supported by in situ hybridization of mRNA of TGF-ß3, which showed stronger expression in rhBMP-2 treated group. Immunohistochemical investigation showed that after 21 days the connective tissue in the rhBMP-2 treated implants contained more TGF-ß1, TGF-ß2 and TGF-ß3, compared to BMP-2 and osteoblasts contained significantly (27.2%) more TGF-ß3 compared to TGF-ß1 (p=0.045). In the formed HO the proportion of the TGF-ß2 and TGF-ß3 producing bone tissue was increased by 32.1% and 47.8% respectively, compared to the TGF-ß1 producing bone tissue (p=0.007 and p=0.006) and although this difference was not so clear at mRNA level, this suggests that TGF-ß2 and TGF-ß3 signaling seem to play an important role during initial and early stages of HO formation.

43-54
The Impact of a Germ Free Perinatal Period on the Variation in Animal Models of Human Inflammatory Diseases – A Review
by Louise F. S. Lauritsen, Majbritt Ravn Hufeldt, Bent Aasted, Camilla Hartmann Friis Hansen, Tore Midtvedt, Karsten Buschard & Axel Kornerup Hansen

Bacteria prime the immune system in early life, which in the first place is relevant for the development of oral tolerance. For some disease models, such as those for inflammatory bowel disease, germ free status for an entire life span, leads to the absence of prominent disease symptoms, while for other models, such as the Type 1 diabetes-prone NOD mouse, germ free status in early life would increase the incidence to a maximum. Basically both reactions are dependent on how the immune system has been primed in early life, i.e. with which bacteria and at which age. After early life priming, the gut regulatory immunity seems to be stable and less prone to be influenced by the gut flora. However, disease development later in life will still be dependent on contact with microorganisms to induce the inflammatory response. The aim of this review is to analyze whether it is reasonable to assume that variation in animal models, and thereby reduced groups size in experiments, may be achieved if animals are reared germ free with subsequent inoculation of a standardized gut flora at a standard age.