Evaluation of dimensional behavior of peri-implant tissues in implants immediately exposed or submerged in fresh extraction and healed sites: a histological study in dogs

A total of 40 implants were installed (ICI implant, Galimplant, Sarria, Spain), with 3.5 mm in diameter by 10 mm in length. Eight implants in each dog, half per hemimandible. The surface treatment of this implant model is developed by blasting with three different granulometries of Al₂O₃ and pickling using a hydrofluoric solution (HF) at low temperature and short time, which aims to remove any traces of Al₂O₃. Plus, the conditioning of the surface was performed using hydrochloric acid solution (HCl) and sulfuric acid (H₂SO₄) at high temperature and short time (Fig. 1). Twenty titanium healing abutments with 3.5 mm in diameter and 6 mm in length were used.

Five American foxhound dogs of approximately 1 year of age were used in this study. The Ethics Committee for Animal Research at The University of Murcia (Spain) approved the study protocol, which followed the guidelines established by the European Union Council Directive of February 2013 (R.D.53/2013). Clinical examination determined that all animals were in good general health; moreover, all animals presented intact maxillae, without occlusal trauma or mucosal lesions.

The animals were pre-anesthetized with acepromazine 0.12–0.25 mg/kg, buprenorphine 0.01 mg/kg, and medetomidine 35 mg/kg. This mixture was injected intramuscularly in the femoral quadriceps. Animals were then taken to the operating theater, where an intravenous catheter was inserted into the cephalic vein, and propofol (0.4 mg/kg/ min) was continuously infused to maintain the general anesthesia. Conventional dental infiltration anesthesia (articaine 40 mg, 1% epinephrine) was injected at the surgical intraoral sites. All procedures were carried out under the supervision of a veterinary surgeon.

Initially, an impression of each hemimandible was performed to make a surgical guide indicate the implant position, which was predetermined to correspond with the distal root and the center of the crown teeth. Sixty days previous to the surgery, the left mandibular premolars (P2, P3, P4) and molar (M1) were extracted to heal the alveolus sites [21]. In the surgery to place the implants, equally to previous surgery, the teeth of the right hemimandibles were sectioned in a bucco-lingual direction using a tungsten carbide bur so that the roots could be extracted individually without damaging the remaining bony walls. After that, full-thickness mucoperiosteal flaps were increased. The socket of the distal root of each premolar was used as experimental site. For the left sides, a full-thickness mucoperiostal flap was used. All implants were positioned in the crestal bone level. After implant placement, a randomization (randomization.​com) was performed to determine which implants received healing abutment and the submerged implants, forming four groups: implant installed in fresh extraction and submerged (group 1), implants in fresh extraction and immediately exposed (group 2), implants installed in healed site and submerged (group 3), and implants in healed site and immediately exposed (group 4). The height of the healing abutments was determined to stay 0.5 mm less of the contact with the corresponding antagonist teeth. No grafting materials were used between the implants and the bony plates. The flaps were closed using single nonabsorbable sutures (Silk® 4-0, Sweden & Martina, Due Carrare). After the surgical procedures, animals received antibiotic treatment (amoxicillin 500 mg, twice a day) and analgesics (ibuprofen 600 mg, three times a day) via the systemic route. Moreover, dogs were fed a soft diet for 7 days, and plaque control was maintained by the application of Sea 4 (Sea 4 teeth, Blue Sea Laboratories, Alicante, Spain). Wounds were inspected daily for clinical postsurgical complications. Two weeks after surgery, sutures were removed. All animals were sacrificed at 12 weeks after the implant insertion by means of an overdose of Pentothal Natrium® (Abbott Laboratories, Madrid, Spain).

The hemimandibles were removed with care to preserve the integrity of both peri-implant hard and soft tissues, washed in saline solution and fixed in 10% buffered formalin, and sent for processing at the Laboratory of Ucam-Biotecnos (Murcia, Spain). Specimens were dehydrated in ascending series of alcohol rinses and embedded in a glycol methacrylate resin (Technovit 7200 VLC; Kulzer, Wehrheim, Germany). After polymerization, the specimens were sectioned along its longitudinal axis with a high-precision diamond disk in the IsoMet® 1000 (Buehler, Lake Bluff, IL, USA), at about 150 μm down to 30 μm. A total of two slides were obtained for each implant. The slides were stained with Picrosirius Red Stain (Polysciences, Inc., Warrington, USA) and observed in a normal transmitted light microscope and a polarized light microscope (Nikon, Tokyo, Japan). Buccal bone wall level in comparison with lingual bone wall height after remodeling was expressed as a linear measurement from the implant shoulder to the first bone-implant contact, showed in the Fig. 2 corresponding with the A-B distance. The buccal and lingual tissue thickness was measured in the level corresponding with the implant shoulder (A line) from the implant to the external epithelium portion of the mucosa, showed in the Fig. 2 corresponding with the C-D distance. The measurements were performed by an expert examiner in histology (SG).

Means, medians, and standard deviations of crestal bone height and tissue thickness were calculated for all groups. All data sets (n = 10) were tested for normality using the Shapiro–Wilk test, and the data did not show normal distribution. The Friedman test was performed for intergroup comparisons in buccal or lingual recorded measures for A-B and C-D parameters followed by the Dunn’s multiple comparison test for further comparison of different groups. Furthermore, Wilcoxon matched-pairs signed-rank test was used for the comparison of two groups. The significance level was set at P < 0.05.

A power analysis was conducted to determine appropriate sample size; although it was determined that 6 samples from each group would generate a 95% confidence limit (G3Power), 10 samples were proposed for each situation to increase the level of significance.

Direct contact was observed between living bone, and all implants without the presence of soft tissues were observed in all groups. However, during the healing, the crestal areas were accompanied by decreases in the dimensions of the buccal as well as the lingual bone walls in different proportions for each group (Figs. 3 and 4). For all implants, keratinized oral epithelium was continuous with junctional epithelium facing the implants and the healing abutments. Subjacent connective tissue with a dense network of collagen fibers was observed.

After evaluating all measurements, the distance from the top of the implant collar (line A) to the first contact of the implant with the bone (line B) was measured for buccal and lingual aspect. Mean, median, standard deviation, and standard error for each group evaluated for lingual as well as buccal sites are summarized in Table 1 and showed in the Fig. 5. The buccal and lingual dimensions showed statistically significant differences at 12 weeks among the groups, which are showed in the Table 3 and the distribution data represented in the Fig. 7a. A-B distance evaluated in the group 3 was significantly lower in both buccal and lingual measured groups, whereas for group 2, A-B distance resulted higher than the other groups.

The overall mean of the tissue thickness from the top of the implant collar (line C) to the more external portion of the tissues (line D) was also calculated. Parameters such as mean, median, standard deviation, and standard error for each group evaluated for lingual as well as buccal sites are summarized in Table 2 and showed in the Fig. 6. Crestal bone height was higher for group 2 both at buccal and lingual sites. The statistical analysis also revealed differences among groups of the measured parameters, which are presented in the Table 3 and the distribution data represented in the Fig. 7b.