This study was performed in accordance with the Swedish Animal Protection Law (Animal Welfare Act 1988:534) and EU Directive 2010/63/EU on the protection of animals used for scientific purposes. The study was approved by the Ethical Committee of the University of Lund, Sweden (ethical approval no. M-192-14) and reported according to the ARRIVE (Animal Research Reporting of In Vivo Experiments) guidelines [26].
Ten female Göttingen minipigs (Ellegaard Göttingen minipigs A/S, Dalmose, Denmark), with a mean weight of 34.62 ± 2.61 kg and a mean age of 20.92 ± 0.79 months, were used in the study. The animals were acclimatised for a minimum of 1 week in standard pens (three or four animals per pen) under controlled environmental conditions. A standard soft food diet (# 801586; Special Diet Services (SDS), Witham, UK) was given. Nine animals were used for data collection with one for calibration.
Surgical procedure and terminal procedure
Tooth extraction and implant placement was performed three months apart in an animal surgery operating suite under full narcosis and aseptic conditions. The animals were fasted overnight prior to surgery to prevent vomiting. The authors placed all implants and undertook all measurements.
Dexmedetomidine (Dexdomitor, Orion Pharma Animal Health, FI-02101 Espoo, Finland; 25–35 µg/kg) and tiletamine -zolazepam (Zoletil 100 Vet, 06511 Carros cedex, France, 50–70 mg/kg) were injected intramuscularly as a pre-medication anaesthetic. Thereafter, each animal was individually dosed with Propofol (PropoVet multidose, Orion Pharma Animal Health, FI-02101 Espoo, Finland) in the range of 40–100 mg/hr to maintain anaesthesia. Pain relief was given pre-emptively and for up to 4 days post-surgically by means of Carprofen (Rimadyl Vet, Orion Pharma Animal Health, FI-02101 Espoo, Finland; 4 mg/kg, s.i.d.) in combination with buprenorphine (Vetergesic Vet, Orion Pharma Animal Health, FI-02101 Espoo, Finland; 0.03 mg/kg). During anaesthesia the animals were intubated for ventilator-assisted breathing and vital parameters (pulse oximetry, rectal temperature, blood pressure, CO2 levels) were continuously monitored.
Intra-operatively, a local anaesthetic, Xylocaine in combination with adrenaline (20 mg/ml and 12.5 µg/ml respectively, Astra AB, Södertälje, Sweden) was administered (1.8 mL infiltrate injection Astra AB, Södertälje, Sweden; 20 mg/ml and 12.5 mg/ml) per hemi-mandible.
Two surgical interventions were performed in each of the 10 animals. First, the mandibular premolars (P2-P4) and first molar (M1) were extracted via a flapless approach. After a 12-week healing period [27], the alveolar ridge was exposed on both sides of the mandible following incision and reflection of the muco-periosteal flap. Gentle bone grinding was performed to flatten the crest of the ridge under cooled sterile saline irrigation (Fig. 1a-c).
Implant dimensions
The implants selected for the study were BLX® Roxolid® SLActive® 4.5 mm x 8 mm implants (The Straumann Group, Basel, Switzerland) that had a body diameter of 3.5 mm at the shoulder tapering to 1.9 mm at the apex. The cutting threads commenced 1 mm apical to the shoulder and terminated at the apex. The outer diameter of the cutting threads was 4.5 mm in the coronal portion of the implant gradually reducing to 3.6 mm at the apex. Thus, the threads of the implants were 0.5 mm wider than the implant body at 1 mm from the implant shoulder. The neck of the implant consisted of 0.5 mm region of microthreads and a 0.5 mm coronal unthreaded region adjacent to the shoulder.
Inter-implant distance
On one side of the mandible, osteotomies were prepared in the centre of the ridge using a drill template, with the positions of the implants marked with a needle drill (Ø 1.6 mm; The Straumann Group, Basel, Switzerland). The implant centres were fixed at 7.5 mm, 6.5 mm and 5.5 mm apart, so that four implants could be placed with inter-implant thread distances of 3 mm, 2 mm or 1 mm (Fig. 1d-f). The needle drill was left in place after the first drill hole so that the drill template remained in place for the positioning of the other three implants. Osteotomy creation followed the manufacturer’s guidelines for hard bone type (BLX drills Ø 2.8 mm, 3.2 mm, 3.7 mm and 4.2 mm, The Straumann Group, Basel, Switzerland). Four BLX Roxolid® SLActive® 4.5 mm x 8 mm implants (The Straumann Group, Basel, Switzerland) were placed at crestal bone level according to the manufacturer’s instructions. Closure screws were placed and the soft tissue repositioned and closed with resorbable sutures.
Buccal wall thickness
On the left side of the mandible, three implants were placed with the distance from the buccal wall to the centre of each implant was measured with a dental probe with 0.5 mm increments; the proposed implant centre was marked with a needle drill (as for the inter-implant distance above) and distances of 2 mm, 3 mm and 4 mm were measured to give implant thread to buccal wall distances of > 1 mm, 0.5–1 mm, or < 0.5 mm (Fig. 1g-i). The implants were placed with a minimum of 4 mm distance between implants. Osteotomy creation and implant placement followed the same procedure as for the inter-implant distance protocol above. Due to the risk of microfractures of the buccal bone plate in the < 0.5 mm group, a slow resorbing collagen membrane (Straumann® Jason® membrane, Straumann AG, Basel, Switzerland) was placed over the buccal aspect of all implants on the left side to serve as a barrier membrane to protect the bone. No randomization was performed for either procedure.
The relative dimensions of the implant, maximum outer thread diameter, and osteotomy diameter at the implant neck, as well as the inter-implant distances and buccal wall thickness are depicted in Fig. 2. All observations, including measured maximum insertion torque, as well as the inter-implant distance and buccal wall thickness measured by means of a dental probe, were recorded and photographs taken from all sites at implantation and termination.
Post-surgery
Antibiotic prophylaxis was administered by means of a combination of benzylpenicillin procaine + dihydrostreptomycin (Streptocillin Vet, Boehringer Ingelheim Vetmedica, Ingelheim am Rhein, Germany) 25 mg/mg and 20 mg/kg s.i.d. i.m. If required further anaesthesia was provided as described above.
Twelve weeks after surgery all animals were sacrificed by intra-cardiac injection of a 20% solution of pentobarbital (Pentobarbitalnatrium, Apoteket AB; Stockholm, Sweden), 60 mg/ml.
Statistical analysis
Descriptive statistics (means, SDs, medians, and ranges) were calculated, and paired comparisons were performed using the Wilcoxon signed rank test. Associations with each of the outcomes, adjusted for the effects of the animals, were examined using multivariable mixed linear regression models with adjustment for different group effects and multiple comparisons by the Dunnett-Hsu method.