Materials and methods

The protocol and guidelines for this study were approved by the Institutional Animal Care and Investigation Committee of Cumhuriyet University Medical Faculty, Sivas, Turkey.

Thirty adult male New Zealand white rabbits, weighing 2.0–3.2 kg (mean weight, 2.7 kg) were used as experimental animals. The animals were allocated to 3 groups: 10 rabbits were used for systemic alendronate administration; 9 for local alendronate administration; and 11 were selected for the control group.

All animals were operated on under general anaesthesia. For the anaesthesia, they were given intramuscular 5 mg/kg xylazine (Rompun 2%; Bayer, Istanbul, Turkey) and 50 mg/kg ketamine HCl (Ketalar; Eczacıbası, Istanbul, Turkey).

In this study, extra oral bone-borne distraction devices consisted of an 8 mm expansion screw (Leone, Firenze, Italy). Three holes soldered with 0.9 mm stainless steel wire for bone attachment were used.

All surgical equipment was sterilized in an autoclave. Sterile gowns, gloves, surgical masks, and theatre caps were used. The mandible was shaved and disinfected with iodine. After surgical preparation of the experimental side of the mandible, a 2.5 cm long submandibular skin incision was made. The subcutaneous tissues were exposed with careful dissection down to the periosteum and the bone was exposed with a periosteal incision. With a reciprocal saw, a vertical corticotomy line was outlined between the premolars and extended to the inferior mandibular border under saline irrigation. The distractor device was fixed with two posterior (7 mm) and one anterior (9 mm) titanium screws. The bone cut was completed using a thin osteotome through the vertical corticotomy line, and mobilization of bone fragments was achieved. The distraction device’s activation was tested. The gap between the bone fragments was narrowed with reversely directed activation of the distractor. The periosteal flaps were repositioned and closed with 3.0 catgut sutures.

In the locally applied alendronate group, the DO gap was filled with a gelatin sponge soaked in 2 mg of alendronate sodium (alendronate sodium trihydrate; Sigma Chemical Co., St. Louis, MO, USA) in 0.1 ml distilled water. In the systemic application group, a 10 mg alendronate tablet (Merck Sharp and Dohme, Germany) dissolved in 10 ml water was used. A concentration of 0.5 mg/kg was given by a gastric tube beginning on the operation day until the end of the distraction period.

All animals were administered intramuscular ceftriaxon 25 mg/kg (Rocephin; Roche, Basel, Switzerland) and intracutaneous carprofen 4 mg/kg (Rimadyl; Pfizer, New York, IL, USA) postoperatively twice a day for 5 days. The animals were given a soft diet and kept single in cages during the study.

After a latency period of 5 days, distraction was started at a rate of 0.4 mm twice a day for 9 days. After a consolidation period of 28 days, all animals were killed with 200 mg/kg of sodium penthotal (Pentothal; Abbott, USA). The operated mandibles were dissected subperiosteally and fixed in a 10% formalin solution. Subsequently, a plain radiograph was taken of the regions of newly formed bone for assessment.

Radiologic and histomorphometric analysis

To assess the volumetric density and morphology of the regenerate, the excised bone was scanned using peripheral quantitative computerized tomography (QCT) (Brilliance 16 slice; Philips Electronics, Amsterdam, Netherlands). The slices were centred at the middle of the regenerate, as identified by the scout view of the QCT system. The 0.8 mm thick slices were transferred to an Extended Brilliance Workspace and evaluated by multiplane reconstruction and a volume-rendering method using MX View software. Drawn slices were evaluated to yield an estimate of the area of the regenerate and density of these samples. Distraction zones were evaluated using dual energy X-ray absorptiometry (DEXA) (Hologic QDR 4500, USA) in the lengthened mandibles. Bone mineral density (BMD) and bone mineral content (BMC) were also measured.

For histologic and histomorphic evaluation each hemi mandible was cut in the axial plane after being decalcified in 10% formic acid. A 3 mm × 5 mm × 4 mm sample of bone was harvested from the centre of the distraction gap using a diamond tipped circular saw and processed for histomorphometric analysis. The inferior part was used for histologic analysis ( Fig. 1 ). The bone specimens were embedded in paraffin, cut in 5 mm thick sections with a microtome (AS 325; Shandon, Waltham, UK) and stained with haematoxylin-eosin for histologic and histomorphometric evaluation. The slices were determined using systematic random sampling for histomorphometric evaluation. From the superior part of the hemi mandible, every tenth section was stained with haematoxylin-eosin, and 20 sections were acquired from this sampling. Then, every second slice was taken; eventually 10 slices were acquired. Volume fraction method 15 was used for histomorphometric analysis to determine new bone formation. The bone tissue/total tissue ratio was used for comparison. A 100 dot square grid was used for counting the new bone ratio; the dots were 1 cm apart. A special microscope (Jenamed; BW Optic, Aschendorf, Germany) was used for projecting the grid onto the histologic slice area of the distraction gap new bone formation with 20× magnification. In this case, the distance separating each dot from the other was 50 μm at the tissue level. The dots on new bone were counted and rationed to the total tissue. From each slice, 5 areas were counted using systematic random sampling. Data were analysed statistically using the Mann–Whitney U -test.

Representative radiographs of the distraction area. Control, local and systemic alendronate groups, respectively.

All materials were fixed in 10% buffered paraformaldehyde for 48 h, and then decalcified in ethylenediamine tetra-acetic acid (EDTA) solutions. Tissue specimens were prepared in an autotechnicon, embedded in paraffin, and sectioned with a microtome. The sections were stained with haematoxylin-eosin. Stained specimens were investigated using a Nikon Eclipse E400 (Nikon, Tokyo, Japan) light microscope. After each specimen was stained, the same area was photographed using a Nikon camera.

A Coolpix 5000 photograph attachment was made. A photograph of the Nikon micrometre microscope slide also was taken during the procedure. All photographs were then transferred into a PC environment and analysed (Clemex Vision Lite 3.5 Image Analysis; Clemex Technologies, Longueuil, Quebec, Canada). The length was calibrated by comparing the photograph of the specimen with the photograph of the Nikon micrometre microscope slide, which was taken under the same magnification.

A 0.5 mm ² area was designated using the Clemex Vision Lite 3.5 Image Analysis program, and osteoblasts, osteoclasts, collagen fibres, and fibroblasts were marked with the same Image Analysis program in a 0.1 mm ² area. Damaged cells were not evaluated. The marked cells were counted automatically with the same image analysis program. New bone forming areas were measured with the same image analysis program in a 0.5 mm ² area. New bone formation regions per unit area (491959.30 μm ² ) were calculated at the same time. All data were analysed using the Mann–Whitney U -test. The reader was blind to the origin of the specimen.

Evaluation of the newly formed tissues in distraction areas was done by estimating their respective volume fractions. Eleven specimens were fixed in 10% neutral-buffered formaldehyde, decalcified in 10% formic acid, and embedded in Paraplast (McCormick Scientific, St Louis, MO, USA). The specimens were cut in the sagittal plane with a microtome setting of 8 μm. Every twentieth section was sampled in a systematic random fashion and stained with haematoxylin-eosin. This sampling fraction yielded on average 20–23 sections per specimen. After defining the distraction areas on these stained sections, resampling was carried out by using only every second of them to obtain an approximate total of 10 sections, a sufficient number for such estimations. An estimation of volume fractions was performed on computer images of the distraction areas, which were obtained by directly scanning the prepared slides on a high-resolution scanner. The volume fractions of the fibrous tissue and marrow within the ossified distraction gap were used for the comparisons.

All data were analysed statistically using the nonparametric Mann–Whitney U and Kruskal–Wallis tests to assess statistical significance.

Results

Thirty distracted hemi mandibles were evaluated. During the study, due to a faulty distractor device and infection, 1 rabbit in the control group and 1 rabbit in the systemic alendronate group were excluded from the study and substituted with new ones. The animals that completed the study had good general health. In a range between 5 and 7 mm, bone lengthening was achieved in the mandible of all animals. Thus, laterognathia and malocclusion were observed.

After stabilization, radiolucencies were observed in the distraction region of all groups according to radiographic examination. Callus formation was shown in the distraction gap and distraction areas of all samples ( Fig. 1 ).

No statistically significant differences were seen in terms of callus area values between the three groups. The QCT images revealed that differences in the density of the regenerate were statistically significant between groups as demonstrated by the mean radiographic scores given in Table 1 . Although there were no statistical differences between systemic alendronate (753.20 ± 112.85 Hounsfield unit) and local alendronate groups (703.95 ± 162.99 Hounsfield unit), the density of regenerate was significantly higher in systemic and local alendronate groups than in the control group (463.95 ± 191.19 Hounsfield unit), ( p < 0.05), ( Table 1 ), ( Fig. 2 ).

Demonstration of quantitative CT images of the regenerates. Control, local and systemic groups demonstrated, respectively.

Histologic findings indicated that bone formation was sparse in all groups and distraction gaps were filled with intramembranous new bone formation and fibrovascular tissue. When histologic analysis of new bone areas of the study and control groups was performed, there were no statistically significant differences between the systemic alendronate group and the local alendronate group. In a comparison of the study and control groups, the results showed that systemic and local alendronate groups were more effective in accelerating new bone formation than the control group with a statistical difference of ( p < 0.05) ( Table 2 ; Fig. 3 ).

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