- Original Article
- Open Access
Effect of alpha-tocopherol on bone formation during distraction osteogenesis: a rabbit model
© The Author(s) 2011
Received: 21 March 2011
Accepted: 17 June 2011
Published: 15 July 2011
The purpose of this study was to evaluate the effects of alpha-tocopherol on distraction osteogenesis.
Materials and methods
Right tibias of 30 New Zealand white rabbits were distracted at a rate of 0.5 mm/day for 20 days with a circular external fixator. Experimental group rabbits (n = 15) were administered i.m. 20 mg/kg/day alpha-tocopherol for 30 days. Radiographic examinations were performed at the 20th, 30th and 40th days. Bone scintigraphy was performed at the 5th and 20th days. Serum total antioxidant capacity (TAC) was measured at the 5th and 30th days. All animals were sacrificed and the right tibias of all animals were harvested for histopathologic examination at the 40th day.
Radiologic scores were statistically similar at the 20th day. However, the experimental group demonstrated higher radiologic scores at the 30th and 40th days. A scintigraphic baseline study at the 5th day of the study showed statistically similar osteoblastic activities in both groups. However, at the 20th day, osteoblastic activity was significantly higher in the experimental group. Serum TAC values were also significantly higher in the experimental group at the 30th day. At necropsy, histopathologic examination revealed statistically significantly higher scores in the experimental group.
The results of this study show that alpha-tocopherol has beneficial effects on new bone formation during distraction osteogenesis.
The technique of “distraction osteogenesis” is frequently used in the treatment of bony loss, pseudoarthrosis, chronic osteomyelitis, limb length discrepancy, biologic reconstruction after wide tumoral resection, and deformity [1–6]. One major problem with this method, however, is the prolonged time required for the newly formed bone in the distraction gap to consolidate and become strong enough for weight-bearing . Various clinical and experimental investigations have been focused on the acceleration of bone formation and consolidation, and have thereby aimed to shorten the framing time [8–11].
Distraction osteogenesis is recognized as being “intramembranous ossification,” which can be assumed to be a special form of fracture healing . Fracture healing after injury involves inflammation, repair and remodeling . At the inflammatory stage, polymorphonuclear leukocytes (PMNLs), macrophages and mast cells migrate into the fracture site, and osteoclasts begin to remove necrotic bone [14, 15]. Activation of PMNLs produces oxygen free radicals, which cause lipid peroxidation and are known to impair fracture healing [16, 17]. Antioxidant administration has been shown to be beneficial in suppressing the damaging effects of oxygen free radicals in cells during fracture healing [18–21].
Consequently, we hypothesized that alpha-tocopherol, which is a potent antioxidant, may also have favorable effects on the quality of new bone formation during distraction osteogenesis and shorten the time required for consolidation. Thus, in this study, the effect of alpha-tocopherol on bone formation during distraction osteogenesis was investigated.
Materials and methods
In this study, 30 adult New Zealand white rabbits (mean weight 1,800 g; range 1,500–2,000 g) were used. The animals were fed a standard laboratory diet and water and had a 12 h day/night cycle. The rabbits were housed separately in standard cages in a temperature-controlled room (20–22°C). Before initiating the study, approval from the local ethics committee was obtained. The study was carried out in the Center for Experimental Animals at the same institution. The rabbits were randomized into experimental and control groups each consisting of 15 animals.
Surgical procedures (day 1)
Experimental intervention (day 1–30)
The experimental group of rabbits received 20 mg/kg alpha-tocopherol intramuscularly starting on the first day of study, and a daily injection of alpha-tocopherol was given for 30 days thereafter. The control group did not receive any corresponding treatment.
Radiological follow-up and evaluation (days 20, 30 and 40)
Radiologic evaluation system 
Lack of bone formation
Bone formation filling 25% of the defect
Bone formation filling 50% of the defect
Bone formation filling 75% of the defect
Bone formation filling 100% of the defect
Onset of union
Complete radiologic union
Onset of union
Complete radiologic union
Formation of intramedullary channel
Formation of cortex
Total score (maximum)
Scintigraphic method (days 5 and 20)
Before the scintigraphic assesment, the rabbits were sedated with 10 mg/kg ketamine hydrochloride. In the scintigraphic study, 3 ± 0.5 mCi/0.5 cc technetium–99 m methylene diphosphonate was injected into the ear vein of each rabbit. Three hours after the injection of the radiopharmaceutical, the subject was positioned laterally under the gamma camera (Millenium, General Electric, Milwaukee, WI, USA) equipped with a low energy, high-resolution collimator, and planar acquisition was initiated for 10 min using a 15% window centered over the 140 keV photopeak. Rectangular regions of interest (ROIs) were drawn on both tibias (the region of distraction osteogenesis and the contralateral healthy leg) at approximately similar locations. Counts were derived from both ROIs in order to calculate the osteoblastic activity ratio (count for the lesion/count for the contralateral side). Scintigraphic assessments were performed on the 5th and 20th days of the study, before and after the distraction period.
Total antioxidant capacity (TAC) measurement (days 5 and 30)
Plasma TAC was measured using a Randox total antioxidant status kit (Total Antioxidant Status, Randox, Crumlin, UK) in which ABTS (2,2-azino-di-[3-ethylbenzthiazolin-6-sulfanate]) is incubated with a peroxidase and H2O2 to produce the radical cation ABTS+. This has a stable blue-green color, which is measured at 600 nm. Antioxidants present in the sample suppress this color production to an extent that is proportional to their concentration. The suppression of the absorbance of the ABTS+ radical cation by serum antioxidants was compared with the suppression caused by Trolox (6-hydroxy-2,5,7-tetramethylchroman-2-carboxylic acid), which is included as part of the TAC kit. The results are expressed as mmol/l of the Trolox equivalent .
Histopathologic evaluation (day 40)
Histopathologic evaluation system 
Predominantly fibrous tissue with some cartilage
Equal amounts of fibrous and cartilage tissue
Predominantly cartilage tissue with some woven bone
Equal amounts of cartilage and woven bone
Predominantly woven bone with some cartilage
Entirely woven bone
Woven bone and some mature bone
Lamellar (mature) bone
The Wilcoxon signed-rank test was used for repeated measurements of the same group and the Mann–Whitney U test was employed to compare groups used. Statistical significance (P < 0.05) was determined based on the 95% confidence interval.
Summary of results (mean radiologic and histopathologic scores, TAC values, and scintigraphic counts for the groups, as well as statistical significance)
Experimental group (SD)
Control group (SD)
5th day TAC value (mmol/L of Trolox equivalents)
2.58 ± 0.28
2.59 ± 0.14
30th day TAC value (mmol/L of Trolox equivalents)
2.75 ± 0.28
2.50 ± 0.08
5th day scintigraphic ROI (distracted/normal, ratio)
1.06 ± 0.20
0.97 ± 0.18
20th day scintigraphic ROI (distracted/normal, ratio)
3.63 ± 1.06
1.65 ± 0.54
20th day X-ray score
1.73 ± 0.45
1.46 ± 0.51
30th day X-ray score
4.60 ± 0.91
3.46 ± 0.83
40th day X-ray score
7.73 ± 1.09
5.46 ± 0.83
40th day histopathological grade
9.86 ± 0.35
8.00 ± 0.92
Scintigraphic baseline study on the 5th day of study showed statistically similar osteoblastic activities in both groups (P = 0.233). However, on the 20th day, the osteoblastic activity was significantly higher in the experimental group (0.000).
TAC measurement results
Serum TAC values were statistically similar in both groups on the 5th day of the study (P = 0.389). However, on the 30th day, TAC values were significantly higher in the experimental group (P = 0.001).
In this study, possible favorable effects of alpha-tocopherol on the quality of new bone formation during distraction osteogenesis were investigated. We have shown that the administration of alpha-tocopherol provided better results as far as the radiologic, scintigraphic and histopathologic evaluations were concerned.
Oxygen-derived free radicals are highly toxic molecules that produce cellular damage by causing both structural and functional impairment in almost all components of the cell, but mainly the cell membrane. They initiate a chain reaction leading to cell membrane damage via lipid peroxidation, thereby causing cell lysis . Alpha-tocopherol is a natural macromolecule that acts as a biological antioxidant in the cell membranes, inhibiting lipid peroxidation by scavenging peroxy and alkoxy radicals and thus breaking chain reactions [26, 27].
Distraction osteogenesis is widely used for the treatment of various challenging musculoskeletal disorders. Prolonged time spent with external fixation is one of the disadvantages that can cause complications such as pin tract infection, loosening, muscle weakness and contractures . Furthermore, prolonged framing time decreases the compliance of patients and causes psychological and behavioral problems .
Distraction osteogenesis is considered intramembranous ossification, which can be assumed to be a special form of fracture healing . During the initial ischemic stage, considerable amounts of oxygen-derived free radicals are produced due to the activation of inflammatory cells [30, 31]. Likewise, Prasad et al. measured the predictors of oxidative stress in fracture patients and found that oxidative stress was directly proportional to the number of fractures, and that it peaked at the 3th week after the fracture and continued until the 4th week .
On the other hand, various experimental studies have been carried out to accelerate and shorten fracture healing with the administration of antioxidants. Göktürk et al. demonstrated that the administration of zymosan—which induces oxygen-free radicals through the stimulation of NADPH oxidase in polymorphonuclear leukocytes—impaired fracture healing in a rat model . Yilmaz et al. have demonstrated the positive effects of ascorbic acid, a well-known antioxidant, on fracture healing . Moreover, there are also studies that have shown the beneficial effects of alpha-tocopherol on fracture healing, whereas its effects on distraction osteogenesis have not been investigated [18–20]. Therefore, to our best knowledge, our study is the first report in this regard.
The fact that serum TAC values were not significantly different from those of the control group on the 5th day of our study, but they were significantly greater on the 30th day, suggests that alpha-tocopherol exerts a favorable effect during the ischemic stage but not during the inflammatory period. Radiologic and histologic evidence of callus formation and maturation were also found to be directly proportional to serum TAC values in the experimental group. It is also worth noting that decreasing the ischemic stage using antioxidants would not only induce osteoblastic activity but it would also impede the osteoclastic resorption of newly formed bone due to oxygen-derived free radicals.
Overall, based on our study, we may conclude that the administration of supplemental alpha-tocopherol in patients treated with distraction osteogenesis may shorthen the framing time and increase the quality of the regenerated bone. Further clinical studies are necessary to check its effects on humans and also to ascertain whether it should be used prophylactically or continuously until the end of the consolidation period. However, when compared with normal fracture healing, alpha-tocopherol may be much more effective at decreasing the repetitive ischemic cycles that are produced during distraction osteogenesis.
This study was funded by the Animal Research Center at our institution. We, the authors and our affiliations, declare that we have no relevant financial involvement with any commercial organisation with direct financial interest in the subject or materials discussed in this manuscript.
Conflict of interest
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited.
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