The research protocol was submitted to and approved by the Ethical Committee of the University of Medical Sciences, School of Dentistry, Havana, Cuba (prot. 013/2013).
Sample and location for animal treatment and maintenance
Eight female Pelibuey sheep, with a mean body weight of approximately 35 kg and a mean age of approximately 3 years, were provided by the Centro Nacional para la Producción de Animales de Laboratorio (CENPALAB) in Havana, Cuba. The surgical sessions were performed at the Centro de Cirugía Experimental (CENCEX) Facultad de Medicina “Victoria de Girón”, Universidad de Ciencias Médicas in Havana, Cuba. The animals were kept at the facilities of CENPALAB during the experimental period.
Randomisation and allocation concealment
The ARRIVE checklist was followed as a guide. According to the Three R requirements [20], aiming to reduce the number of animals, the interferences among animals was eliminated, adopting a split-mouth design. No previous experiments were available that studied healing at the repositioned bony plate, so to describe the event of healing with sufficient approximation, a relevant difference in bone formation within the sinus was set to 10%, assuming a standard deviation of approximately 8%. Considering these values clinically relevant, seven pairs of subjects were calculated to be able to reject the null hypothesis that this response difference is zero with a power of 0.8 and α = 0.05.
Two different procedures for antrostomy closure were adopted, one using a resorbable membrane (control site) and the other repositioning the bone plate (experimental site). The randomisation was performed electronically (www.randomization.com) by an author not involved in any surgical procedures (DB). The surgeon (AP) was informed of the treatment randomly selected only at the end of the grafting procedure.
The examiner of the histological slides (AP) was carefully trained before the evaluation. The measurements were performed twice, and mean values used.
Anaesthetic procedures
The animals fasted for 24 h preoperatively, but were allowed to drink water ad libitum. Anaesthesia was induced by 0.4 mg/kg of midazolam (Dormicum; Roche, Basel, Switzerland) and 10 mg/kg of ketamine (Ketamina-50; Liorad, Havana, Cuba), and orotracheal intubation was performed. The anaesthesia was maintained with a mixture of oxygen and 2–3% isoflurane (Isoflurane-vet; Merial, Toulouse, France) at a rate of 5 L/min. The surgical sites were rinsed with 0.12% chlorhexidine digluconate (Periogard™; Colgate-Palmolive Ltd, New York, NY, USA) and trichotomy was performed. After general anaesthesia, 1.5–2 cc of 2% mepivacaine HCI with 1:100,000 epinephrine was injected at the surgery site. All surgeries were performed under sterile conditions, using good clinical and laboratory practices.
Clinical procedures
Through an extra-oral approach, an oblique incision was made bilaterally along the sagittal axis between the facial tuberosity and the inferior orbital rim. The skin and periosteum were elevated separately, and the bony facial sinus wall was exposed on both sides of the maxilla (Fig. 1a).
A 12-mm large and 8-mm high antrostomy was prepared using a burr (H254E Komet Dental, Trophagener Weg 25, Lemgo, Germany) and the bone plate was removed bilaterally (Fig. 1b). The sinus mucosa was subsequently carefully elevated with sinus floor elevators, exposing the medial bony plate of the maxillary sinus (Fig. 1c). A landmark was positioned, twisting a steel wire through a hole above the access window (Fig. 1d).
After sinus mucosa elevation, the obtained space was filled with a biphasic calcium phosphate (60% HA, 40% β-TCP) (Easy-graftTM CRYSTAL; Sunstar GUIDOR, Etoy, Switzerland) on both sides (Fig. 1d).
Experimental and control sites were randomly chosen. The control site was covered with a polylactic acid blended with a citric acid ester membrane (GUIDOR® matrix barrier; Sunstar Americas Inc., Chicago, IL, USA) and secured with a minimum amount of cyanoacrylate glue (Indermil® x fine; Henkel-Loctite Corp. Dublin, Republic of Ireland) accurately positioned in four points (one at each corner; Fig. 1e, f). Cyanoacrylate glue was stored at 4 °C and used immediately after leaving the refrigerator, to keep the viscosity low. At the experimental site, similar procedures were applied (Fig. 2a–c), and the bony window was repositioned in place and secured with the four points of cyanoacrylate (one at each corner; Fig. 2d). Suturing in layers was then performed.
Maintenance after surgery
Gentamicin (Gentamicin-5® Bela-Pharm GmbH, Vechta, Germany) 8 mL/100 kg was administered every 12 h during the first postoperative day, and every 24 h during the following 3 days. The sheep were kept in an animal house individually, in a roofed shed to reduce distress after surgery, with a concrete floor. The boxes were cleaned daily and the animals had free access to water. The diet was based on a balanced specific food made of cereals, protein, and concentrates of vitamins and minerals, with added green forages. The wounds were cleaned by the veterinarians of the centre every day during the first week, and then inspected three times per week for clinical signs of complications for the duration of the experiment.
Euthanasia
After 4 months, the animals were anaesthetised and then euthanised with an overdose of pentobarbital sodium and subsequently perfused with 10% formalin. The maxilla was retrieved en bloc, trimmed, and immersed in formalin solution.
Histological preparation
All histological procedures were performed in the Laboratorio de Histologıa de la Facultad de Odontologıa de la Universidad de Ciencia Medica in Havana, Cuba. Bilateral maxillary block sections, each containing one sinus, were obtained and maintained in a 4% formaldehyde solution. The blocks were then cut in a buccolingual plane at the level of the landmark reference, using a diamond band saw fitted in a precision slicing machine (Exakt; Apparatebau, Norderstedt, Germany). One hemiblock was dehydrated in a series of graded ethanol and subsequently embedded in resin (Technovit® 7200 VLC; Kulzer, Friedrichsdorf, Germany). One section was obtained and reduced to a thickness of approximately 60 μm using a cutting-grinding device (Exakt®; Apparatebau, Norderstedt, Germany). Subsequently, the histological slides were stained with Stevenel’s blue and alizarin red and examined under a light microscope for histometric analysis.
Histological evaluations
The histological measurements were performed using an Eclipse Ci microscope (Nikon Corporation, Tokyo, Japan), equipped with a digital video camera (Digital Sight DS-2Mv, Nikon Corporation, Tokyo, Japan), connected to a computer and using the software NIS-Elements D 4.10 (Laboratory Imaging, Nikon Corporation, Tokyo, Japan).
Histological measurements within the augmented sinus were performed in four different regions: (i) subjacent to the sinus elevated mucosa (submucosa), (ii) centre of the grafted area (middle), (iii) the original base of the sinus (base), and (iv) immediately under the replaced bone window or the membrane (sub-window) (Fig. 3a).
In the antrostomy area, histology was performed in two regions: (i) the bony window or membrane area (centre) and (ii) the osteotomy regions on the maxilla (edges) (Fig. 3b).
A point counting procedure [21] was performed to identify the tissue composition within the described regions. A lattice, with squares of 75 μm in dimensions, was superposed over the tissues at a magnification of × 100.
The percentages of the new mineralised bone, soft or connective tissue, pure graft, graft interpenetrated by bone, and remnants of cyanoacrylate were evaluated. The total tissue percentages in the elevated space that included submucosa, middle, base, and sub-window regions were also calculated.
Data analysis
Mean values and standard deviations (SDs) as well as the 25th, 50th (median), and 75th percentiles were calculated for each outcome variable. The main outcome variable was the percentage of the new mineralised bone within the sinus.
The IBM SPSS statistics software (IBM Inc., Chicago, IL, USA) was adopted for analyses. The Wilcoxon test was used to assess the differences between the repositioned bone plate and collagen membrane sites. The level of significance was set at α = 0.05.