Effects of radiatherapy on new bone formation, osseointegration, normal surrounding tissues and mesenchymal cells

Abstract

Radiotherapy is the procedure in which occurs release of irradiation in specific areas, aiming at the treatment of tumors. However acute or chronic damage may develop in the irradiated area and in surrounding normal tissues. In bone tissue, injuries vary from osteopenia to osteroadionecrose and due to these changes patients irradiated patients with implants have shown higher failure rate. The objective of this study will be to evaluate the effects of radiation, simulating a full dose of a conventional radiation treatment for cancer patients, in the bone regeneration and osseointegration of implants in the femurs of rats, surrounding tissues normal to the irradiated area, as well as in the differentiation and gene expression of mesenchymal cells present in these femurs, and to evaluate the present nuclear damage in these mesenchymal cells. For that, 160 rats will receive cylindrical implants of commercially pure titanium (cpTi) the right and left femurs. The animals will be divided into 4 groups: a) implant group (Ip): implant placement surgery (control group - healthy tissue without irradiation); b) Group immediate irradiation (IRI): placement of implants surgery followed by immediate irradiation; c) Group late irradiation (IRT): placement of the implant surgery and irradiation after 4 weeks; d) Group irradiation (Ir): irradiation and four weeks after surgery to implant placement. The animals will be exposed to single dose of irradiation with 70 Gy. The rats will be randomly euthanized at periods of 3 days, 2 and 6 weeks after treatment. During the experiment, the animals will be carefully monitored for clinical changes such as diet, hair loss, infection of surgical wounds and weight loss. The level of IL-6 and TNF-± in the blood will be determined by ELISA imunoenzimatic test after 12 and 48 hours of radiation and the in time of euthanasia. In each period 08 animals will undergo computed microtomography (¼CT) and subsequent histological and histomorphometric, to evaluate new bone formation and bone-implant interface. The expression of PARP p-85, TGF-beta and VEGF in the bone-implant interface will be determined by immunohistochemistry. The other 08 animals in each period, excluding the period of 3 days, will be submitted to the push-out test to evaluate the strength of bone-implant fixation, obtained in osseointegration. At the time of euthanasia, in all animals, the surrounding skin will be removed, fixed and processed by conventional histological technique HE and picrosirus red for evaluation respectively of this degree of inflammation and the diameter of collagen fibers. In addition immunohistochemical staining will be performed for anti PARP p-85, TGF-beta and VEGF in the skin tissue. In muscle tissue, prior to euthanasia, will be measured contractility activity of this muscle in the surgical area using electromyography and after euthanasia will be evaluate the presence of muscular fibrosis in slides stained with Masson trichrome. The differentiation and activities of the mesenchymal cells present in the femurs, will be evaluated by in vitro cell viability, total protein content, alkaline phosphatase activity, cell cytoskeleton and formation of mineralized nodules. Damage to the cell nucleus will be checked using the micronucleus test. The gene expression of markers such as alkaline phosphatase, integrin beta1, collagen 1, osteocalcin, osteopontin, osteonectin, bone sialoprotein, TGF-beta, osterix, Runx2 stimulating factor and macrophage colony - MCS-F will be assessed on mesenchymal cells by real time-PCR (reverse transcriptase polymerase chain reaction). All data will be statistically analyzed initially using the normality test aimed at applying appropriate statistical test. The significance level will be the standard value of 5%. (AU)