We investigated the effect of hyperbaric oxygen therapy (HBOT) on rat muscles during tibial distraction osteogenesis (DO) at normal and hyperdistraction rates. Animals in groups 1 and 2 were distracted by 0.5 mm/day and those in groups 3 and 4 by 1 mm/day. Groups 2 and 4 received HBOT during distraction. Group 5 served as control. Superoxide dismutase (SOD; U/g protein), malondialdehyde (nmol/g protein), glutathione (mmol/g protein), and protein levels (g/dl) were determined. SOD was significantly higher in group 2 (4.59 ± 0.97) than in controls (2.19 ± 0.7) ( P = 0.0001), and lower in group 4 (3.74 ± 1.70) than in group 2 ( P = 0.011). Malondialdehyde was significantly higher in group 2 (0.72 ± 0.23) than in controls (0.38 ± 0.10) ( P = 0.005). Total protein levels were better preserved with HBOT in distracted muscles: group 2 (3.24 ± 0.37) vs. group 1 (1.88 ± 0.60), and group 4 (3.45 ± 0.70) vs. group 3 (2.03 ± 0.75) (both P = 0.0001). Numbers of fibres were lower in group 1 (4.88 ± 0.59) than in group 2 (6.07 ± 0.86), and in group 3 (5.13 ± 0.36) than in group 4 (6.14 ± 0.74) (both P = 0.001). Numbers of nuclei were higher in group 1 (11.29 ± 2.47) than in group 2 (9.03 ± 1.53) ( P = 0.04), and in group 3 (12.43 ± 3.32) than in group 4 (9.08 ± 1.58) ( P = 0.001). Fibres and nuclei with HBOT were similar to those of controls. HBOT decreased the inflammatory cell infiltrate for group 1 (19.8 ± 8.54) vs. group 2 (4.2 ± 2.53) and group 3 (36.54 ± 11.29) vs. group 4 (21.5 ± 9.23) (both P = 0.001). HBOT improves the adaptation of distracted muscle by increasing fibres and antioxidants while decreasing inflammation.
The oral and maxillofacial surgeon achieves stability in correcting craniofacial deformities by striking a balance between the advancement rate of the bone and soft tissue stress. As we increase our understanding of bone healing in distraction osteogenesis (DO) for craniofacial disorders, we also recognize the importance of the simultaneous adaptation of the surrounding soft tissue envelope, which contributes to the stability of the reconstruction, lessening the risk of relapse. It has been shown in patients that degenerative changes such as necrosis and fibrosis occur at distraction rates above 1 mm/day, with decreased adaptation potential of the muscles adjacent to the distracted bone.
The role of hyperbaric oxygen therapy (HBOT) on the outcome of DO has been studied in the past, showing a beneficial role in increasing both bone mineral density and torsional strength. HBOT is achieved at barometric pressures higher than the ambient pressure (1 atmosphere absolute, ATA). Exposure to HBOT increases the dissolved oxygen content in blood, stimulates fibroblastic activity and collagen production, decreases the inflammatory response, and limits oedema, which improves the microvasculature. Therefore HBOT has been suggested as an adjunctive treatment for various inflammatory and ischaemic diseases. While HBOT reverses local hypoxia in target tissues, it may lead to an increased formation of reactive oxygen species (ROS) with subsequent lipid, protein, and DNA oxidation. The prominent cellular defence mechanisms against ROS are the free radical scavenger enzymes, namely superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx).
We hypothesized that HBOT applied to the distracted rat gastrocnemius muscle at normal and hyperphysiological distraction rates would increase the muscle adaptability during normal and ‘hyperdistracted’ distraction.
Materials and methods
This model of DO in association with HBOT has been used previously in various experimental models of long bones as well as facial bones; however, the use of these models has focused mainly on bone healing aspects of hyperbaric oxygen rather than the soft tissue surrounding the distracted bone. Our work focused on the surrounding soft tissue envelope that contributes to the stability of the reconstruction, with added HBOT as the variable.
Animals and experimental procedures
A total of 46 adult male Wistar albino rats (300 ± 50 g) were used in the current study. This study was performed with the approval of the university ethics committee of the animal care review board. The animals were housed in cages in a regulated environment (23 ± 2 °C and 55 ± 15% relative humidity) under a 12-h light/dark cycle and had access ad libitum to standard laboratory chow and tap water before and after surgery.
Under general anaesthesia (xylazine 5 mg/kg and ketamine 50 mg/kg), an incision was made along the lateral portion of the right hind leg. A cutaneous flap was prepared, the gastrocnemius muscle was split, and the medial part of the tibia was exposed. The distraction device was adapted to the bone. Following osteotomy at the tibial midshaft, the distractor was fixed to the bone with two microscrews above and two microscrews below the osteotomy. The skin was closed with single stitches ( Fig. 1 ). The smallest available distraction apparatus at the study institution was too large for the rat mandible.
Animals were divided randomly into five groups. Groups 1 ( n = 10) and 2 ( n = 10) were distracted by 0.5 mm/day, at a normal rate. Groups 3 ( n = 10) and 4 ( n = 10) were distracted at a rate of 1 mm/day and were considered to be hyperdistracted. Groups 2 and 4 received adjuvant HBOT with daily distraction, whereas groups 1 and 3 were exposed to normoxia. Sham-operated animals served as controls (group 5, n = 6). The animals in groups 1 and 2 were sacrificed on postoperative day 9 and those in groups 3 and 4 were sacrificed on postoperative day 5, with a high dose of ketamine hydrochloride. The amounts bone distraction at the end of the experiments were similar. Tissue specimens were collected in a sterile disposable cup and immediately transported in a cooler on ice to the laboratory. Upon arrival at the laboratory, samples were stored at −70 °C until analyzed. Muscle tissue that was adjacent to the distraction area was harvested.
Hyperbaric oxygen therapy
HBOT was conducted in a small research chamber (0.4 m ). HBOT sessions were 80 min long at 2.5 ATA, including 10 min compression time and 10 min decompression time. The chamber was flushed with oxygen for 10 min to vent the air towards the environment before the compression so that the animals could be pressurized with 100% oxygen. The HBOT was commenced on the day of distraction with one session per day. Group 2 had eight sessions and group 4 had four.
Tissue samples were fixed in 10% buffered formalin. After tissues were fixed, samples were rinsed with distilled water and subsequently dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Sections (5–6 μm) were cut and mounted on glass slides. The sections were deparaffinized with xylene, rehydrated in decreasing concentrations of ethanol, and were stained with haematoxylin and eosin (H&E). The numbers of nuclei and muscle fibres were counted using a light microscope (Olympus CH20; ×100, with immersion oil) and a metric oculometer, and photographs were taken with a light microscope (Leitz Laborlux K). Six different microscopic areas were evaluated for each animal. Assessments and counts were calculated from longitudinal sections of muscle tissue. The numbers of muscle fibres and the numbers of nuclei with respect to their locations (central, intermediate, and periphery) were counted in a magnification area of 100 μm 2 through the oculometer and the average scores were calculated. The inflammatory cell infiltration was similarly assessed in six random microscopic areas for each animal (×100, with immersion oil) and the inflammatory cells were described based on their morphology.
On postoperative day 3, bleeding of the surgical wound was measured by semiquantitative scoring as 0 = no bleeding, 1 = no significant bleeding, 2 = mild bleeding, and 3 = significant bleeding.
Sample preparation and biochemical analysis
Specimens were weighed, washed in 0.9% NaCl, and homogenized in ice-cold 0.15 M KCl 100 g/l. Homogenates of 20% were obtained and sonicated twice at 30-s intervals at 4 °C. Homogenates were centrifuged at 5000 × g for 10 min at 4 °C. All biochemical parameters in homogenates were studied on the same day. All reagents were analytical grade and purchased from Sigma Chemical Co. (St. Louis, MO, USA) and Merck (Darmstadt, Germany).
Copper–zinc SOD assay
Cu–Zn SOD activity was determined with the method of Sun et al. by inhibition of nitroblue tetrazolium (NBT) reduction, with xanthine/xanthine oxidase used as a superoxide generator. One unit of SOD was defined as the amount of protein that inhibits the rate of NBT reduction by 50%.
Glutathione (GSH) assay
Tissue GSH concentrations were determined according to the method of Beutler et al. using metaphosphoric acid for protein precipitation and 5′,5′- dithiobis-2-nitrobenzoic acid for colour development.
Malondialdehyde assay (MDA)
Measurement of thiobarbituric acid reactive substances (TBARS) was used to estimate the MDA content; the levels of TBARS were used as an indicator of lipid peroxidation in this study. The lipid peroxidation end-product was determined as TBARS with a modification of the method of Buege and Aust. One volume of plasma was mixed thoroughly with two volumes of a stock solution of 15% (w/v) trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, and 0.25 mol/l HCl. The mixture was heated for 30 min in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 1000 × g for 10 min. The absorbance of the sample was determined at 535 nm and the TBARS concentration was calculated using the extinction coefficient, 1.56 × 10 5 M −1 cm −1 .
Total protein assay
The total protein concentration was measured by the method of Lowry et al.
All data were expressed as the mean ± standard error of the mean. One-way analysis of variance (ANOVA) was performed with SPSS v. 10.0 (SPSS Inc., Chicago, IL, USA). Correlations between changes in variables were tested using Pearson’s correlation. A P -value of <0.05 was considered statistically significant.
Forty-six animals were treated in this study. There was no intraoperative or any postoperative death during the experimental period. Tissue levels of total protein, SOD, GSH, and MDA in the study groups are summarized in Table 1 . With regard to the histological findings, the average numbers of muscle fibres, nuclei, and inflammatory cells in the muscle fibres are presented in Table 2 . Histology of groups 1–5 showing the inflammatory cell infiltration is shown in Fig. 2 .