A total of 75 male rats (Rattus Norvegicus Albinus, Wistar), aged between 70–90 days and weighing approximately 250–300 mg, were randomly divided into three groups (n = 25) according to the type of implant surface: polished group (PG), animals received implants with a polished surface; machined group (MG), animals received implants with a machined surface; and treated group (TG), animals received implants with a treated surface.
The randomization of the rats into the three groups, was conducted by drawing lots in an envelope containing one of the three group names.
This study follows ARRIVE guidelines and all methods were carried out in accordance with relevant guidelines and regulations, approved by the Ethics Committee on Use of Animals of the Faculty of Dentistry of Araçatuba (UNESP) (process n° 00253–2012).
This study used 75 mini-implants with internal insertion manufactured by Implalife Biotechnology (Jales, São Paulo, Brazil), for the tibia region of the rat, with dimensions of 2 mm in diameter and, 7 mm in length having machined, polished, and treated types of surfaces. The subtraction method was used on the surfaces of the treated implants, where acid solutions baths of nitric acid, hydrochloric and phosphoric acid were used. Surface treatment was carried out entirely by the implant manufacturing company.
Study samples were anesthetized with 0,2 ml Ketamine Hydrochloride (Ketamina Agener®, União Química Farmacêutica Nacional SA, São Paulo, SP, Brazil) and 0.1 ml Xylazine (Dopaser®, Laboratório Calier do Brasil, Osasco, SP, Brazil), for 250 mg of body weight. After trichotomy on the anterior surface of the right posterior limb and antisepsis with polyvinylpyrrolidone iodine degermante (10%, Riodeine Degermante®, Rioquímica, São José do Rio Preto, SP, Brazil), the superior third of the tibia was infiltraded using 0.1 ml of Lidocaine Hydrochloride with 1:100,000 epinephrine (Alphacaíne lidocaine 2% epinephrine 1:100,000 DFL®, Rio de Janeiro, RJ, Brazil), to ensure local hemostasis. The incisions were made by planes and the flap was detached, and the bone tissue was exposed to perform the bicortical osteotomy.
The milling implants installation was performed with a 2.0 mm diameter drill, electric motor (BLM 600®; Driller, São Paulo, SP, Brazil) at a speed of 1500 rpm and a speed reducer handpiece (1:16; (KaVo®, Kaltenbach & VoigtGmbH & Co., Biberach, Germany), under constant irrigation of sterile 0.9% sodium chloride solution. The implants were installed according to the group to which the animal belonged. The skin and muscular flaps were repositioned and closed with a tight suture in layers.
All the animals were injected intramuscularly with 1 mg/kg/day of sodium dipyrone (Ariston Indústrias Químicas e Farmacêuticas Ltda, São Paulo, Brazil) and a 20,000 I.U. of penicillin G benzathine (Fontoura Wyeth S.A. Industrias Farmacêuticas, São Bernardo do Campo, SP, Brazil) per animal for immediate postoperative period. The animals were euthanized by anesthetic overdose (about three times the anesthetic dose) with Ketamine Hydrochloride (Ketamina Agener®, União Química Farmacêutica Nacional SA, São Paulo, SP, Brazil) and Xylazine (Dopaser®, Laboratório Calier do Brasil, Osasco, SP, Brazil) on days 3, 7, 15, 21 and 40 postoperatively.
Analysis by scanning electron imcroscopy and X-Ray spectroscopy by energy dispersion
The implants in each group were analyzed for surface topography using Scanning Electron Microscopy (SEM) with an energy dispersion X-Ray Spectroscopy (EDS) system (model EVO LS15, Zeiss, Germany), to analyze the chemical composition and elementary mapping of the surfaces17.
Histological and histometric analysis
The tissue samples from the bone/implant interface were obtained and fixed in 10% formaldehyde solution for 48 hours, washed in running water for 24 hours, decalcified in 20% ethylenediamine tetraacetic acid for 5 weeks, and then dehydrated using alcohols and diaphanized. The pieces obtained were placed in separete paraffin blocks and 6 µm thick, cuts were obtained, which were later stained using hematoxylin and eosin and mounted on histology slides.
The images obtained were analyzed using an optical microscope (Jenamed 2 Histology, Carl Zeiss, Germany), and captured using a photographic camera (AxioCamICc1, Carl Zeiss Microimaging system) connected to the microscope and the computer. The images were captured using the application Axio Vision (Release 4.8.2 SP1 12-2011, Carl Zeiss, Germany).
Histometric analyses were performed using the ImageJ image analysis software, version 1.47. The linear extent of contact between the newly formed tissues and the implant surface (LECOI) was calculated as a percentage, from the initial organization of the clot (3 days) to the bone deposition over the entire implant (40 days).
The data obtained in each type of comparison were subjected to statistical analysis using the statistical analysis program in experimental work BioEstat 5.3.