Abstract
This study aimed to investigate soft tissue changes in distraction osteogenesis depending on various distraction and consolidation periods. Fifteen male rabbits were divided into three groups and evaluated for five parameters. An external custom-made device was placed on the right side of the mandible. The postoperative latent period was 4 days. The groups were distracted for different periods (6 days, 3 mm; 11 days, 5.5 mm, 16 days, 8 mm). For histomorphometric analyses, specimens were retrieved from both study and control sides of the mandible and were examined with light microscopy. The number of muscle fibres and nuclei on the study side increase proportionally with the distraction period. Regeneration in the first group continued in its natural pattern; in the second group, there seemed to be degeneration rather than regeneration; and in the third group there seemed to be a balance between regeneration and degeneration processes. In conclusion, when a massive amount of bone is to be obtained, instead of distracting the bone at once, it is thought to give better results if the total amount of distraction is divided into several time periods .
Deformities of the maxillofacial region are conventionally reconstructed using various orthognathic surgery procedures, which may be associated with complications and unwanted results . One of the most important problems in orthognathic surgery is adaptation of the soft tissues. In some cases, the soft tissues surrounding the bone do not adapt to the new position during the massive bone movements resulting in functional and aesthetic problems. Techniques have been developed to avoid such problems .
New techniques have been developed for the reconstruction of anterior–posterior and transversal-vertical deformities; one of these methods is distraction osteogenesis . During the distraction procedure adaptive changes that occur in the soft tissues are known as distraction histogenesis .
Materials and methods
The aim of this study is to determine the best adaptation process by investigating the histomorphometric changes in muscle tissues after distraction procedures using different amounts and times.
Fifteen albino, New Zealand type, male rabbits were used. They were 7–8 months old and weighed 2.5–3 kg. They were sheltered and fed in 15 separate cages according to the ECPVAEOSP (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purpose) Standards of the European Union.
The distraction device used in the study was designed specifically for the study method and the size of the animals. The part of the distraction device left outside the tissues is produced from a titanium–aluminium alloy, which is light, resistant to rusting and can be sterilized. The pins that are inserted in the tissue are made of the same material.
The masseter muscles of all the rabbits were measured to show the relationship between soft tissue and distraction amount and time. The mean value was calculated as 27.5 mm. The distraction amounts were chosen according to the mean length of the masseter muscles. The rabbits were randomly divided into three groups each including five rabbits. Bone lengthening of 11% (3 mm), 20% (5.5 mm), and 29% (8 mm) was achieved in groups 1, 2 and 3, respectively. The aim in choosing these study groups was to create models of cases that need high, average or low distraction amounts. The surgical procedures were performed on the right side of the mandible.
After induction of general anaesthesia (100 mg/kg ketamine HCl and 10 mg/kg xylazin HCl) the right sides of the mandibles were shaved and cleaned using an antiseptic solution (povidone iodine). 1 ml local anaesthetic (Ultracaine D-S forte, an ampoule of 2 ml that includes epinephrine, 0.012 mg/ml, and articaine HCl, 40 mg/ml) was injected into the masseter region subcutaneously.
The cutaneous and subcutaneous tissues were sequentially dissected. Masseter muscle was scraped upwards with dissection in the axial direction while muscle fibres that will be facing the distraction line were left intact. After exposure of the bone a corticotomy was performed. The transcortical pins of the distraction device were fixed 1.5 cm over the incision line before the bone segments were split by maintaining a 1 cm distance between the pins ( Fig. 1 ). The distraction device was fixed with screws to the bone fragments following bone splitting. Muscle and subcutaneous tissues were closed sequentially with 3.0 catgut and left to heal.
For 4 days postoperatively, 5 mg/kg gentamycin sulphate (50 cc flacon, genta-vet, VETAŞ) was injected intramuscularly two times a day. The rabbits were killed by ether inhalation at different times. The postoperative latent period was 4 days. An extraoral device was used to carry out 0.5 mm distraction daily (two times a day, each 0.25 mm) on the rabbit mandibles. The groups were distracted for different times (6 days, 3 mm; 11 days, 5.5 mm and 16 days, 8 mm). The consolidation time is twice as long as the distraction time.
Table 1 shows the study protocol. Specimens of 2.5 cm 3 were retrieved from the distracted side of the mandible for the study group and from the left side of the mandible for the control group. Tissues were shaped as two cubes of 1 cm 3 . After chemical procedures, they were fixed and stained with haematoxylin-eosin for histomorphometric analysis. Ten specimens were obtained from each tissue sample.
| 1. Group | 2. Group | 3. Group | |
|---|---|---|---|
| Number of rabbits | 5 | 5 | 5 |
| Distraction region | Right side of mandible | Right side of mandible | Right side of mandible |
| Latent period | 4 days | 4 days | 4 days |
| Rhythm of distraction | 2 × 0.25 mm/days | 2 × 0.25 mm/days | 2 × 0.25 mm/days |
| Speed of distraction | 0.5 mm/days | 0.5 mm/days | 0.5 mm/days |
| Duration of distraction | 6 days | 11 days | 16 days |
| Amount of distraction | 3 mm | 5.5 mm | 8 mm |
| Duration of consolidation | 12 days | 22 days | 32 days |
| Derivation of tissue specimens | After 22 days | After 37 days | After 52 days |
Each specimen was assessed with a light microscope (Olympus CH20) by different researchers twice to prevent the possible errors that can occur with manual assessments. The numbers of nuclei and muscle fibres in the muscle tissues were counted in a magnification area of 100 μm through an oculometer and the average scores of two assessments were calculated. The locations of the nuclei were evaluated. Each muscle fibre was analyzed and the location of nuclei in the centre and periphery of the myofibres was determined.
The groups were compared in the following way. In group 1 (amount of distraction 3 mm) the study sides of the mandibles and the control sides of the mandibles were compared. In group 2 (distraction 5.5 mm) the study side of the mandibles and the control sides of the mandibles were compared. In group 3 (distraction 8 mm) the study side of the mandibles and the control sides of the mandible were compared. The control sides of the mandibles in every group were assessed comparatively. The study sides of mandible in every group were assessed comparatively.
The statistical tests used were the nonparametric Kruskal–Wallis test and the Wilcoxon rank sum test (SPSS 13.00 for Windows). Results with p < 0.05 were considered significant and those with p > 0.05 not significant.
Results
Location and number of nuclei and muscle fibres in muscle tissues were examined on a magnification area of 100 μm through an oculometer ( Table 2 ). Photomicrographs of specimens taken from groups with different distraction amounts are shown in Figs 2–5 .
| Group 1 | Group 2 | Group 3 | ||||
|---|---|---|---|---|---|---|
| Left side control | Right side study | Left side control | Right side study | Left side control | Right side study | |
| Numbers of fibres | 4.05 | 4.7 | 4.5 | 5.3 | 4.2 | 4.4 |
| Numbers of nuclei | ||||||
| Periphery | 7.9 | 9.1 | 6.6 | 7.9 | 7.6 | 8.7 |
| Centre | 2 | 2.8 | 1.15 | 3.2 | 2.6 | 3 |
| Intermediate | 3.3 | 3.8 | 1.9 | 3.8 | 1.3 | 3.6 |
| Sum | 13.3 | 15.7 | 9.6 | 14.9 | 11.5 | 15.3 |
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