Effects of caffeic acid phenethyl ester and melatonin on distraction osteogenesis: an experimental study
© Erdem et al.; licensee Springer. 2014
Received: 15 August 2013
Accepted: 12 December 2013
Published: 3 January 2014
The aim of this experimental animal model study is to investigate the effects of caffeic acid phenethyl ester (CAPE) and melatonin on the maturation of newly-formed regenerated bone in distraction osteogenesis.
Unilateral femoral lengthening(extension) was applied to 39 adult male Wistar albino rats, which were randomly allocated to 3 groups of 13; control, melatonin and CAPE groups. Through a 7-day latent waiting period and 15 days of distraction, melatonin of 25 mg/kg and CAPE of 10 μmol/kg were administered to the respective groups. The animals were sacrificed on Day 82. Radiographic, histological and biomechanical evaluations were made and measurements were taken.
At the end of 82 days, the distraction osteogenesis area was seen to be completely filled with new bone formation in all 3 groups both radiologically and histologically. Biomechanically, the maximum torsional fracture strength (Maximum Torque (N-m)) of the melatonin group was higher compared to that of the control group, although it was not statistically significant (p > 0.05). The maximum torsional momentum of the CAPE group was statistically significantly high (p < 0.05). The degree of rigidity (N-m/deg) of both the melatonin and CAPE groups was higher than that of the control group and the CAPE group was found to be statistically significantly higher than the melatonin group (p < 0.05).
Melatonin and CAPE increase the maturation of new bone in distraction osteogenesis. These effects are probably due to the reducing effect on bone resorption by inhibiting NF-κB and free oxygen radicals.
Distraction osteogenesis, which was popularised by Ilizarov (Ilizarov and Ledyaev1992), is nowadays successfully used in many orthopaedic reconstructive surgical procedures. It is used in the treatment of chronic orthopaedic problems such as congenital and developmental limb length discrepancy, deformities, post-traumatic segmental bone loss, pseudoarthrosis and osteomyelitis successfully (Ilizarov and Ledyaev1992; Alam et al.2009; Sen et al.2007). Distraction osteogenesis has three stages, the longest and most problematic of which is the consolidation stage. In this stage, the lengthy duration of the fixator restricts movement of the joint and increases the risk of pin site infection. Various methods have been recommended to reduce the external fixator time (EFT) and the external fixator index (EFI) and these have been reported with success in literature (Alam et al.2009; Catagni et al.2011; Emara et al.2011; Lovisetti and Sala2013). These may be listed as the use of autogenous bone graft, the application of ultrasonography (USG), electromagnetic waves, bone morphogenic protein, compression-distraction of the regenerative area, biphosphanate, calcitonin and platelet-rich plasma (PRP).
Melatonin is a hormone secreted by the pineal gland which regulates the biorhythm. It is known as antioxidant, and has the property of removing free oxygen radicals. In-vitro studies have shown melatonin to have a positive effect on osteoblastic differentiation and bone healing (Roth et al.1999). In a study using rat tibia, Halici et al. (2010) showed melatonin to have an accelerating effect on bone healing. In an in vitro study, Nakade et al. (1999) showed melatonin stimulates proliferation and type 1 collagen synthesis in human bone cells, and suggesting that melatonin may act to stimulate bone formation. In an another study, Koyama et al. showed melatonin causes an inhibition of bone resorption and increase in bone mass by decreasing the RANKL- mediated osteoclast formation (Koyama et al.2002).
Cafffeic acid phenethyl ester (CAPE) is one of the constituents of propolis, which is produced by honeybees. Various in vitro studies have shown this to have antiseptic, antibacterial, anti-inflammatory, immunomodulator, antioxidant and antimutagenic effects (Elmali et al.2002). Our experimental investigation aimed to compare the effects of melatonin and CAPE with a control group in an animal model of distraction osteogenesis.
Materials and method
Approval for the study was granted by Gaziosmanpaşa University Medical Faculty Animal Experiments Ethics Committee and the study was supported by Gaziosmanpaşa University Scientific Research Projects Commission. The study comprised 39 (it is not consistent with the number given in the first page (33))adult male Wistar albino rats with a mean weight of 384 gr (350–400). The rats were randomly allocated to 3 groups of 13, as Group 1, Melatonin group, Group 2, CAPE group and Group 3, control group.
Radiological evaluation system (Lane and Sandhu 1987 )
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 radiological union
Formation of intramedullary channel
Formation of cortex
Maximal total score
Histological Evaluation System (Huddleston et al. 2000 )
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
One way variance analysis (ANOVA) was used in the comparison between the 3 groups when normal distribution was shown according to the Kolmogorov Smirnov normality test on the continuous variables used in the study. In paired comparisons between the groups, the Sheffe and Tamhane tests were used according to the variance homogeneity status. Continuous variables were stated as arithmetical mean and standard deviation (SD). A p value of <0.05 was accepted as statistically significant. Calculations were made using a statistical software package (PASW v18, SPSS Inc, Chicago, IL).
Mean radiological and histological scores of the groups at the end of 82 days
Mean ± SS (median)
9,18 ± 0,60 (9)
9,09 ± 0,54 (9)
8,64 ± 0,50 (9)
9,09 ± 0,30 (9)
9,00 ± 0,44 (9)
8,82 ± 0,40 (9)
The concept of distraction osteogenesis, as described by Codivalla (1994) and developed by Ilizarov (Ilizarov and Ledyaev1992) opened a new era in orthopaedic reconstructive surgery. Problematic orthopaedic cases such as congenital or developmental extremity shortness, deformities, pseudoarthrosis, extremity reconstruction following bone tumour resection and osteomyelitis started to be treated with this method (Ilizarov and Ledyaev1992; Alam et al.2009; Sen et al.2007).
There are three phases to distraction osteogenesis (Emara et al.2011). The first stage as the latent phase starts with an osteotomy and lasts for mean 7–10 days. In the second stage as the distraction stage, lengthening is made with a unilateral or circular external fixator applied to the proximal and distal bone fragments of the osteotomy. In the third and final stage as the consolidation stage, the regeneration, formed during the lengthening matures. The longest of these stages is the consolidation stage. It is the stage of maturation of the regeneration formed between the two ends of the osteotomy line during distraction. Extension of this stage creates patient dissatisfaction and intolerance. In addition, that it can cause various complications such as pin site infection, fixator loosening and stiffness in neighbouring joints has been reported in literature. Several authors have proposed various methods to reduce external fixator duration at this stage of the procedure and have reported successful results. Compression-distraction on the fixator, grafting with autogenous bone graft, the use of bone morphogenic protein, PRP, biphosphanate and calcitonin are the most prominent methods (Alam et al.2009; Emara et al.2011; Lovisetti and Sala2013).
The histopathology of bone formation in the distraction stage was elucidated by Aronson (1997). The bone formation between the ends of the osteotomy, which is named regeneration, occurs by intramembranous bone formation, as after the osteotomy, firstly type 1 collagen fibres are formed then these collagen fibres are aligned paralel to the lengthening vector and start to cluster on the transverse axis. At this stage, chondroblasts start to appear and cartilage production and bone ossification on this starts. At the end of the distraction phase, osteon canals and osteoblastic activity are seen between the bone ends and mineralisation starts. In the consolidation stage, according to the Wolf principle, this regeneration area remodels to bone tissue as the intramedullary canal and the sharp cortex surrounding it (Emara et al.2011). The duration of this regeneration formation and the duration of its maturation are defined by several factors. The age of the patient, diabetes, the use of immunosuppressive medication, cigarette smoking, blood circulation of the osteotomy side and external or internal stability of the bone are important factors (Catagni et al.2011; Emara et al.2011). Therefore, young, healthy, albino rats were selected for this study. The osteotomies were made with a low-speed motor without excessive periosteal dissection as Kojimoto et al. (1988) reported that protecting the periosteum rather then the endosteum of the bone had a more positive effective on callus formation. Following the recommended 7-day latent phase (Ilizarov and Ledyaev1992), lengthening of 2 × 0.175 mm was started. From the radiographs taken in the 82ndday, the lengthening rhythm was seen to be sufficient.
Various treatment protocols have been recommended by several authors for maturation in the consolidation stage of the regeneration which is formed in the distraction stage. Yamane et al. (1999) showed that ED-71, which is a Vitamin D product, enhanced thickening in the callus tissue after the distraction stage. Mizumoto et al. (2003) demonstrated an increase in bone mineral density at the distraction osteogenesis stage with the use of rh-BMP. In a study by Li et al. (Yamane et al.1999), thrombin peptide 508 (TP508) was seen to contribute to the bone maturation and consolidation phases of distraction-osteogenesis. Sen et al. (2006) reported positive effects of calcitonin on distracted segment consolidation. Shimazaki et al. (2000) demonstrated that low-dose USG had a positive effect on maturation. In an experimental study by Sen et al. (2007), NSAIDs were seen to have a negative effect on maturation.
In experimental studies, the formation of free oxygen radicals (FOR) have been seen in the reperfusion stage following temporary ischaemia in the inflammation stage of bone healing (Erdem et al.2010). FOR, especially superoxide anion are a mediator in the formation of osteoclasts and have a significant effect in bone resorption (Garett et al.1990) Göktürk et al. (1995) showed the negative effects of FOR on bone healing. We did not encounter any studies on the effects of CAPE on the outcome of distraction osteogenesis. Caffeic acid phenethyl ester (CAPE) is one of the molecular agents of propolis, which is produced by honeybees, and is known to have anti-inflammatory, anticarcinogenic, immunomodulatory and antioxidant properties (Lovisetti and Sala2013; Nakade et al.1999). In recent years, it has started to be used frequently in medical treatments because of these properties. Elmali et al. (2002) reported that the effect of CAPE on osteoarthritis in an experimental rabbit model was shown by inhibiting nuclear factor kapa B (NF-κB) which leads to cartilage damage. In another experimental animal model, Ang et al. (2009) showed that CAPE inhibited osteoclast differentiation and activation by inhibiting NF-κB associated with RANKL. In the current study, positive effect of CAPE were seen in the distraction-osteogenesis consolidation stage. This is thought to be due to making the FOR ineffective and inhibiting NF-κB which plays a key role in the differentiation and activation of osteoclasts. This effect was demonstrated in the statistically high values of the biomechanical breaking strength and rigidity of the newly-formed bone tissue compared to those of the control group (p < 0.005).
Melatonin is secreted by the pineal gland. It has a regulatory effect on body heat and circadian and seasonal rhythms. Melatonin is a significant free oxygen radical scavenger (Erdem et al.2010; Cuzzocrea and Reiter2001; Satomura et al.2007). There is a positive effect of melatonin on bone formation. This effect was shown as an increase in osteoblastic activity by Roth et al. (1999). In the current study, the rates of biomechanical breaking and level of bone rigidity were found to be high in the melatonin group compared to the control group. When the biomechanical breaking point and rigidity value of the CAPE group were compared with the melatonin group, although the values were high, no statistical significance was determined.
In an experimental study, Clafshenkel et al. (2012) recommended that, bone defects implanted with CA-Melatonin scaffolds show greatest degree of bone remodelling both quantitatively and qualitatively. The histological analysis of the samples clarified the osteoid tissue mineralization and bone formation. The addition of PRP with CA did not show the same improvement as in the case of Melatonin. Shimazaki et al. (2000) concluded that ultrasound can accelerate bone maturation in distraction osteogenesis in rabbits, even in states of poor callotasis. Histological analysis showed no tissue damage attributed to ultrasound exposure. Lesalchot et al. (2011) investigated the influence of rh-BMP-2 on the consolidation phase in a distraction osteogenesis model. Qualitative radiographic evaluation revealed hypertrophic calluses. Densitometric analysis revealed the bone mineral content (BMC) was significantly higher in the rh-BMP-2 treated animals.
Li et al. (2005) stated that recombinant human bone morphogenetic protein- 2 (rhBMP-2) reinforced the consolidation phase of distraction osteogenesis in rabbit models. To compare the effects of melatonin and CAPE with USG, rhBMP-2 and PRP, further experimental and human studies should be studied.
The use of CAPE and melatonin can be beneficial in the maturation of the newly-formed regeneration and reducing the external fixator time and index in distraction osteogenesis and due to the biomechanical torsional breaking strength and high rigidity rate, will reduce the possibility of refracture and angulation which may be seen following fixator removal.
Gaziosmanpasa University Scientific Research Projects Funds has provided financial support for this experimental study, grant number [Project No:2006/29].
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