Influence of low level laser therapy on osseointegration of implants installed in the tibia of systemically nicotine-modified rats or not: biomechanical, histological, histomorfometric, histoquimical and immunohistochemical evaluation
The purpose of this study is to evaluate the influence of low level laser therapy (LLLT) on osseointegration of implants placed in the tibias of nicotine systemically modified (or not) rats, from the biomechanical, histomorphometric, histochemical and immunohistochemical through of markers stand point.120 rats (Wistar) will be assigned into two major experimental groups (n=60) will undergo two-daily-applications of saline (SS) or nicotine (NIC). Thirty days after the beginning of administrations, all the animals will be submitted to titanium implant (2.2 mm x 4 mm) placement in the right and left tibia. The surgical alveoli will be prepared with a cutter (2 mm) mounted in a surgical motor (35 N of torque and 1200 rpm), under constant irrigation. Then, the groups will be assigned into the following experimental groups (n=30): SS Group - no local treatment of surgical alveoli prior to implant placement; SS/LLLT Group - irradiation of surgical alveoli with low level laser prior to implant placement; NIC Group - no local treatment of surgical alveoli prior to implant placement; NIC/LLLT Group - irradiation of surgical alveoli with low level laser prior to implant placement. Thera Lase® (InGaAIP 660 nm) will be used in continuous mode and in contact with the area, with a power of 35 mW, energy of 0.14 J, energy density of 4.9 J/cm 2, for 4 seconds. Ten animals from each group will be euthanized at 15, 30 and 60 days after implant placement. The right tibia will be submitted to reverse-torque analysis and, then undergone decalcification laboratory processing in order to prepare histological sections of 4 µm thick. These histological sections will be subsequently stained either by hematoxylin and eosin technique or picrosirius red or subjected to immunohistochemical reactions by immune-peroxidase technique with the following primary polyclonal antibodies: VEGF, HIF-alpha, RUNX2, BMP-2, OCN, ALP, RANKL, OPG and TRAP. The left tibias will be submitted to the laboratory processing without decalcification and submitted to histometric analysis. The histochemical analysis will be performed by polarized light microscopy and the histological, histometric and immunohistochemical analysis by bright field optical microscopy. All immunohistochemical reactions will be semi-quantitatively analyzed. The data obtained from the histochemical, histometric and immunohistochemical analysis will be subjected to statistical analysis (p<0.05).
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