Transverse Maxillary Deficiency

Transverse maxillary deficiency is a frequent problem among patients seeking orthodontics care. It is distinguishd by a narrow maxilla in relation to the rest of the craniofacial structures, a narrow palatal vault, and often a posterior crossbite. According to an epidemiological study by da Silva et al. (2007), the prevalence of some forms of posterior crossbite in the primary dentition is 20.81%. An extended history of palatal expansion as a way to treat dental crowding ,crossbites, anteroposterior discrepancies, and airway issues has made it a common treatment modality in most orthodontic practices today. The etiologies of transverse maxillary deficiency are various. It can be due to genetic factor, environmental factor, soft tissue influences, cleft palate, low tongue position, CIII anteroposterior skeletal discrepancies, and habits (Malandris and Mahoney, 2004). Rapid maxillary expansion (RME) is one of the most common methods used by orthodontics to treat this problem.
RME is based on the concept of widening the dental arch by means of opening the midpalatal suture. The concept dates back to 1860 when Angel clarified rapid expansion in an article to the dental community. From the time of its origin, the concept of rapid maxillary expansion has been used to correct posterior crossbites, increase nasal permeability, and increase arch perimeter to relieve crowding and tooth size-arch length discrepancies. RME utilizes orthopedic forces to separate the two halves of the maxilla at the mid-palatal suture ( Isaacson and Ingram, 1964).
Bell (1982) explained the mechanics of rapid maxillary expansion simply when he reported, 'If the applied transverse forces are of adequate magnitude to overcome the bioelastic strength of the sutural elements, orthopedic separation of the maxillary segments can occur.' At the same time, expansion appliances condense the periodontal ligament, bend the alveolar processes, tilt the anchor teeth, and gradually open the midpalatal suture when the forces exceed the limits required for orthodontic tooth movement (Bishara and Staley,1987).
Usually, rapid maxillary expansion is done with a jackscrew at the rate of 0.5-1 mm per day, whereas slow maxillary expansion is done at a rate of 1 mm per week. According to Isaacson and Ingram (1964), a single activation of the expansion screw creates 3-10 pounds of force, with a smaller load being produced per activation in younger patients as compared with more mature patients. The midpalatal suture becomes more interdigitated with age and; therefore, heavier forces are needed to overcome the partially interlocked suture in older adolescents and adults. These forces decay rapidly following activation, but the rate of decay decreases within several minutes. Active expansion usually takes place for 2-3 weeks followed by 3-6 months of the appliance being left in place as the suture reorganizes (Isaacson and Ingram ,1964).
The relapse after RME is a complex phenomenon because the regulation of bone metabolism and stresses created on midpalatal and circumaxillary sutures depend on many factors. Although the reason for early relapse is not fully understood, rate and quality of bone formation in the midpalatal suture during and after expansion may influence the post-treatment relapse (Saito and Shimizu, 1997).
Insufficient bone formation in the suture could lead to early relapse since the suture cannot withstand the tension and pressure of facial bone structures . It would be potentially beneficial; therefore, to improve bone generation and mineralization in the expanded mid-palatal suture to prevent relapse and to shorten the retention period (Saito and Shimizu, 1997). A variety of agents have been evaluated, either locally or systemically, to stimulate bone formation in the sagittal suture and midpalatal suture of experimental animals after rapid expansion. Example of tested materials are bone morphogenic protein (Liu et al., 2013), GSK-3?? inhibitor (Jiang et al., 2013), vitamin C (Uysal et al., 2011a), vitamin E (Uysal et al., 2009a), vitamin D (Uysal et al., 2009b), zoledronic acid (??zt??rk et al., 2012), periostin -like factor (Zhong et al., 2011), electrical stimulation (Uysal et al., 2010a), laser (da Silva et al., 2012), systemic propolis (Altan et al., 2013), Ginkgo biloba (Kara et al., 2012b), lithium (Tang et al., 2011), boron (Uysal et al., 2009c), resveratrol (Uysal et al., 2011b), and thymoquinone (Kara et al., 2012a).

The arrangement of craniofacial sutures in human is very comparable to the arrangement seen in other species, such as rabbits, rats and mice which have been used as study tools to examine suture biology and pathology (Opperman, 2000). In this study, the rabbit sagittal suture was used as a model for human midpalatal suture. Small rodents have primitive bone structures and don't have haversian systems (Nunamaker,1998) and although little is known about the importance of this anatomical difference between rodents and humans, this makes bone repair in these animals varied from that seen in human beings. Rabbits have haversian systems that are similar to that of human being, which is avital advantage in terms of extrapolation of results obtained with such animals for human bone repair. However, the fast healing process in these animals compared with humans, make them an important bioassay for screening of comparable technologies, but doubtful for a direct transfer of information to the human clinical situation (Nunamaker, 1998). In addition, rabbits as an experimental animal are cheap, easier to care and keep, having primitive bone and soft tissue response as humans, and ethically better accepted for experiments than sheep or dogs . Moreover, because the bone used for the histological section is very small, it was very useful to analyse a histological picture of the entire expanded area on a single histological section. The exclusive use of males avoided the possible hormonal changes present in females since this could compromise the results .Animal was age selected as normally young human with less interdigitation between both palatine bones.
Accessibility is one of the most important aspects that is considered before conducting a study and because most of the examined craniofacial sutures respond in the same way to the force applied, most researchers used the sutures that are easy to access such as the sagittal suture of rabbits and rats (Lee et al., 2001; Sasaki et al., 2003; Byron et al., 2004; Liu et al., 2013). In addition, the soft tissues covering the sagittal suture of rabbits is very thin and devoid of muscle attachments, which simplify the procedure.
The intermaxillary suture of rabbit was not chosen in this study because of the difficulty of fitting the appliance to the anterior teeth of rabbits, the possible interference of the appliance with feeding of the animals and the fact that the incisors of rabbits are continuously erupting which would lead to failure of the expansion appliance.
5.1 Suture Expansion
In the present study, the force of expansion was 100 g using 0.8mm round wire spring with double coil. The majority of experimental studies used helical springs because the forces of these springs decay as the suture expands. Many experimental studies on rats and rabbits used an expansion force of 50-100g to expand the sagittal or interpremaxillary suture (Zahrowski and Turley, 1992; Lee et al., 2001; Liu et al., 2011).
The amount of suture expansion gained in the present study was about 3 mm after 7 days of expansion in all animal groups, with no any significant difference among the control and various experimental groups. Therefore, the amount of suture expansion will not be considered as a factor affecting the bone formation ratio between the different groups, as it is nearly identical in all groups. The same expansion appliance was used in many other studies and gained nearly the same amount of suture separation (Lee et al., 2001; Lai et al., 2013).
5.2 Platelet Rich Plasma Study
Platelet rich plasma is a form blood coagulum that have a highly concentrated number of platelets. The employ of PRP offers an easy and cost-effective way to obtain high concentrations of growth factors for tissue healing and regeneration. It is an amount of plasma fraction of autologous blood having platelet concentration above base line gained by two different steps of centrifugation.
The platelets are the major regulators of the inflammatory phase of healing and play an important role in the proliferation and differentiation phase (Intini, 2009). Violation of the vascular structure as a result of injury leads to the formation of fibrin and platelet aggregation. A stable blood clot is then formed by coagulation of the blood. Afterward, several growth factors are released into the injured tissue from the platelet and other cells that promote and support healing and tissue formation. To improve these effects surgeons developed higher concentration of platelets compared to base line.
The employ of growth factors such as human platelet rich plasma has broken new ground in tissue engineering. Since the time of its inception, PRP has been used in various regenerative procedures such as defect around implants, periodontal bone defects (Becen et al., 2007), in guided tissue regeneration, as a soft tissue regenerator in chronic ulcers of foot (Knox et al., 2006), knee joint replacement (Everts et al., 2006b), as a conductive agent in sinus lift procedures (Kassolis and Reynolds, 2005). Furthermore, PRP was applied in mandibular reconstructive procedures (Cieslik-Bielecka et al., 2008; Marx et al., 1998), and recently PRP has been used in dermatology for promoting hair growth (Miao et al., 2013a, Miao et al., 2013b) and for facelift procedures and dermal abrasion (Don et al., 2007).
All the previous studies tested platelet rich plasma on the healing of iatrogenic or surgically created defects and up to our knowledge, the present study was the first to test the efficacy of human platelet rich plasma on the regeneration of orthopaedically expanded suture in an animal model. The remodeling of orthopaedically expanded suture is a completely different phenomenon from the healing of bony defects that heal by secondary intention. In our study, human platelet rich plasma was used instead of rabbit platelet rich plasma because several studies have indicated presence of several growth factors in the human platelet rich plasma which are not present in animal PRP (Weibrich et al., 2002; Fiedler et al., 2002), but to our knowledge, there is no publication as yet, about the growth factors of rabbit platelet rich plasma in the literature. In addition, preparation of platelet rich plasma from the animal blood is not a standardized procedure such as platelet rich plasma preparation from human blood. The critical effective amount of platelet in platelet rich plasma from different animal species, levels of growth factors in different animal species and similarities or differences in their mechanisms of action with platelet rich plasma of humans have still to be defined. Until then, the animal platelet rich plasma preparations/studies should be interpreted carefully ( Parizi et al., 2012).
In the present study, the human platelet rich plasma was used as a xenograft on rabbits, completely tolerated with no histological signs of acute or chronic inflammatory response being noted. The same observation was noted by other authors. Ranly et al. (2007) evaluated human platelet rich plasma with demineralized bone matrix on bone formation in nude mice. Shafei-Sarvastani et al. (2012) evaluated the effect of combined use of human platelet rich plasma with hydroxyapatite and coral on bone healing in rabbits. Parizi et al. (2012) used human platelet rich plasma combined with Persian gulf coral on experimental bone healing in the rabbit model.
5.2.1 Relapse Ratio of the Expanded Sagittal Suture
In this study, the relapse ratio of the expanded sagittal suture is measured one week after removal of retainer which was maintained for two weeks after expansion on digital radiograph using an image analysis software. Up to our knowledge this is the first study that estimated the relapse ratio with application of human platelet rich plasma in animal model of sagittal suture. The relapse ratio of the expanded suture in both single and double human platelet rich plasma treated groups was significantly less than the control animals. The single platelet rich plasma application reduced the relapse of the expanded suture by 57% whereas the double application of platelet rich plasma reduced the relapse of the expanded suture by 70%, as compared to the relapse of the control animals. This finding indicates that the degree of relapse after rapid expansion of the sagittal suture is reduced significantly by the application of hPRP and the effects are dependent on the frequency of application. The less relapse observed in platelet rich plasma treated groups could be attributed to more dense fibrous tissue in the expanded sagittal suture of single hPRP in addition to early bone formation of double hPRP treated groups as shown in radiographic and histological-histomorphometric observations of the expanded suture. The less relapse of the expanded suture in platelet rich plasma treated groups makes a strong indicator for their clinical application on patients with midpalatal suture to hasten bone formation and to reduce the retention period recommended after expansion. The clinical application of platelet rich plasma is a simple procedure that require few minutes of preparation from the patient own blood. Although an additional laboratory procedure is required for the preparation of platelet gel, it can always be prepared by an assistant thereby to reduce the procedure time.
5.2.2 Radiographic Density of the Expanded Sagittal Suture

The density of the expanded sagittal suture in the control and hPRP treated groups was measured on digital radiograph of the cranial specimen of the rabbits after two, four, and six weeks after commencement of retention. The density of the suture was measured by an image analysis software program. This software has been used in many studies to measure bone density. C??lio-Marino et al. (2012) used this software to measure the radiographic density of the impacted lower third molar socket after the application of autologous platelet-rich plasma. Antonello et al. (2013) also used the same software to measure the density of alveolar bone repaired following extraction of impacted third molars by the platelet-rich plasma.
The radiographic density of the expanded sagittal suture at two, four and six weeks was significantly higher in double hPRP than single hPRP and control groups. The density of the single hPRP group was higher than the control group with no significant difference at two and four weeks, but reaches a significant level at six weeks. The results indicated that the application of hPRP has a positive effect on the density of regenerated bone of the expanded sagittal suture with the weekly apart second application of hPRP resulted in more dense suture. Our observations come in accordance with many other studies. Cieslik-Bielecka et al. (2008) showed that the bone density of odontogenic cysts-cavities filled with platelet rich plasma reached about 69.5% after six months of treatment. Molina-Minano et al. (2009) used digital radiographic images to investigate the density of surgically created defects in rabbit tibia filled with platelet rich plasma and found a significant increase in bone density one month after surgery; however, the density after two months is no longer significantly different from control defects. C??lio-Marino et al. (2012) evaluated the performance of autologous PRP applied in tooth socket. Thirty extractions of bilateral impacted mandibular third molars were performed in 15 volunteers. They observed a significant faster bone formation on periapical radiograph in sockets treated with PRP in the first, second, and third month post-extraction in comparison with control non-treated sockets. Shafei-Sarveastani et al. (2012) investigated the effect of hPRP with or without hydroxyapatite or coral on healing of critical- sized defect of the forelimb of rabbits and found that the radiological evidence of bone healing was enhanced when hPRP was used. Our results are not in agreement with those of Mooren et al.(2007) because they showed that the goat PRP was not able to enhance early bone healing in a goat skull bone healing model. In addition, our radiological results are not in accordance with the observations of Aghaloo et al.(2005) who failed to show a radiographic increase in bone density with the addition of combined PRP to freeze-dried bone in non-critical-sized defects in the rabbits' cranium.
5.2.3 Histology and Histomorphometry
The histological observations of the expanded sagittal suture showed regeneration in all study groups. At two weeks, the hPRP treated groups showed denser collagen fibers than the control group. The double hPRP group showed early appearance of bone islands which was not noted in the single hPRP and control groups. The osteoid islands start to appear in the control and single hPRP groups at four weeks and at six weeks. There was nearly complete suture closure by mature bone in double hPRP group, while the single hPRP group showed more maturation than the control group.
In this study, possible favorable effects of hPRP on the induction of new bone formation after rapid suture expansion were investigated by using a histomorphometric method. Histomorphometric parameters in this work were osteoblast and blood vessel numbers counted by light microscopic lens, in addition, the total area of expanded suture with associated new formed osteoid area within fibrous tissue were calculated by Image-J software. Bone histomorphometry is a dependable method that is always used in estimation of bone growth in experimental, in vivo and in vitro researches. Uysal et al., (2009a) used computer-assisted image'analysis software,Image-J, to evaluate the effects of vitamin E administration on bone formation in response to expantion of the inter-premaxillary suture in rats, histomorphometrically. Uysal, et al.,(2009b) used computer-assisted image'analysis software(AnalySIS 2.1) to evaluate the effects of ED-71 on bone regeneration in response to expantion of the inter-premaxillary suture in rats, histomorphometrically. Decreasing in total suture area along three time intervals reaveld that there are continuous bone growths from both edges of suture, however, the hPRP groups showed higher reduction in total suture area than the control group. In addition, the histomorphometrical study revealed that the number of osteoblasts and, accordingly, the amount of new bone formation were increased in the hPRP group relative to the control group. At six weeks, the control group showed an increase in osteoblasts number and mineralized areas than the two and four weeks. This findings indicate that the application of hPRP accelerate new bone and connective tissue regeneration. It was noted by Haas (1970) that ossification of the expanded suture is completed in 60-90 days, while our study showed that applying hPRP gel stimulated suture bone formation and shortened the period of ossification to nearly 40 days with suture regeneration.
The histologically and histomorphometrically proved better suture regeneration after rapid expansion with application of hPRP gel could be attributed to the growth factors reservoir in the granules of the platelet. These growth factors are epidermal growth factor (EGF), insulin like growth factor (IGF), alpha and beta transforming growth factors (TGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and platelet factor (PF-4). Each of these growth factors has a role to play during wound healing and regeneration of soft and hard tissues. The EGF regulates cell proliferation, differentiation and survival (Savage et al.,1986). IGF is the key regulator of cell metabolism and growth and motivates the osteoblasts proliferation and differentiation (Karey et al.,1989). The PDGF is shown to be mitogenic for osteoblasts (Assoian et al.,1984) and stimulates migration of the mesenchymal progenitor cells (Fiedler et al., 2002). TGF promotes cellular proliferation and differentiation along with apoptosis.TGF also has the ability to activate osteoblasts to deposit collagen matrix and brings about mitogenesis of osteoblast precursors at the site of wound (Baylink et al., 1993). VEGF regulates angiogenesis (Wartiovaara et al., 1998).
The role of PRP in improving bone formation has shown great variability in animal studies. Some studies established a positive effect on bone regeneration. On the contrary, other studies have demonstrated that PRP has no benefit or even a negative effect on bone regeneration. The histological findings of our study come in agreement with many other studies.Torres et al.(2007) found that the histological and histomorphometric evaluation of rabbit calvarial defects augmented with PRP showed a significant bone regeneration at 4 weeks compared to non-treated control defects. Batista et al.(2011) evaluated histologically the beneficial effect of PRP on consolidation of defects in rabbit tibia at four weeks of application. Oryan et al.(2012) demonstrated histologically that hPRP-hydroxyapatite showed a better regeneration of 10mm long critical size defect of the rabbit radius than hydroxyapatite and control groups.
The findings of our study disagree with many other studies that didnot demonstrate any beneficial effect of PRP on bone healing. Aghaloo et al. (2002), evaluated autogenous bone graft healing with PRP, and failed to show any significant benefit when PRP was used in a rabbit model. Butterfield et al.(2003), performed a very similar study to that of Aghaloo et al.(2002) using the rabbit model and observed that the addition of PRP had no statistically significant effect on bone formation as measured with histomorphometric analysis and failed to find a direct effect of PRP on healing of autogenous bone grafts. Mooren et al. (2007) also found in a histological and histomorphometric examination that goat PRP was not able to enhance early or late bone healing of defects created in the skull of the goat after 1,2,6,and 12 weeks of implantation.
The explanation for this variation in the benefit of PRP in bone regeneration between diverse studies may be attributed to variations in experimental (animal, human) and bone defect models, differences in PRP biology among species, differences in PRP preparation techniques among studies and differences in investigated time points. In addition, growth factors may act at specific times and at proper concentrations and this may be another explanation for the distinction in the results of PRP studies
( Lacoste et al., 2003).
In the weekly- apart double application of hPRP, a remarkable increase in new bone formation was seen as compared to single application of hPRP, both histologically and histomorphometrically. The explanation for more beneficial effect of double hPRP application could be due to the fact that degradation of platelets and growth factors release accounted to be at first 3-5 days and ; therefore, growth factor activity is suggested to be at first 7-10 days (Raghoebar et al., 2005). It was also suggested that the direct effects of platelet-derived growth factors start to disappear gradually after 5-6 days ( Marx et al.,1998). In support to our findings, ??zdemir et al.(2012) found a histomorphologically remarkable bone formation of a critical-sized rabbit calvarial bone defect with double application of PRP. The more advantageous effect of double application of PRP was also found in soft tissue wound healing ( Driver et al., 2006; Kon et al.,2010).

5.2.4 Serum Markers of Bone Formation
Determination of the reference values of bone turnover parameters in rabbits could have special interest in experimental studies of bone healing research as they may provide complementary non-invasive information on the bone remodeling process. There is no report in the scientific literature about bone formation markers change during suture expansion of the rabbits, the reference data for serum calcium, phosphorous, alkaline phosphatase, bone alkaline phosphates, and osteocalcin are not available for rabbits. In this study, a group of non-treated non-expansion rabbits of the same age and sex to the expansion group were used to obtain the blood sample for measuring biochemical parameters of bone turnover to be used as a baseline for normal values of the rabbits.
After two, four and six weeks of retention of the expanded sagittal suture, the serum calcium and phosphorous are not significantly different in the control and hPRP groups with the means of the control group were nearly identical to those of hPRP groups.
The serum total alkaline phosphatase level showed no significant change after two, four, and six weeks among the three groups. The bone 'specific alkaline phosphatase (BALP) is significantly elevated in the double hPRP group than the other two groups at two weeks. At four weeks, the value of BALP started to decrease in control and double hPRP groups, while the single hPRP group showed no change in the value of BALP as compared to two weeks. At six weeks, the value of BALP showed further decrease in the three groups without any significant difference between the three groups.
The serum osteocalcin (OC) level was significantly higher in the double hPRP group than the single hPRP and control groups. At four weeks and six weeks, there was a tendency to a decrease in the OC level with no significant difference among the three groups. The serum BALP and OC level at six weeks showed lower values than the baseline means.
Total serum ALP, and more specifically, the serum activity of the bone-specific isoenzyme of BALP and serum bone gamma-carboxyglutamic acid-containing protein BGP or osteocalcin (OC) are widely accepted bone formation markers (Biver et al., 2012; Pagani et al., 2005). The total ALP level in serum is less sensitive and specific as a bone formation marker due to the contribution of alkaline phosphates from non-skeletal sources. Meanwhile, BALP, which is produced by immature osteoblasts, plays an essential role in the initiation of bone mineralization and is a more specific marker of bone formation (Whyte, 1994).
BALP is a sensitive bone formation marker during early osteogenesis. BALP functions as an ectoenzyme and is attached to the osteoblast cell membrane (Magnusson et al., 1997). It has been suggested that BALP could act as a plasma membrane transporter for inorganic phosphate, or an extracellular calcium-binding protein that stimulates calcium phosphate precipitation and orients mineral deposition into osteoid (Debernard et al., 1986). BALP may also be involved in the mineralization process by hydrolyzing organic phosphates to release free inorganic phosphate at sites of mineralization.
The observation of this study showed amarked increase in BALP as compared to baseline values. Leung et al. (1995) showed that BALP dropped in first few days after distraction osteogenesis in rabbits and then rose progressively until fifth week and resumed normal value gradually until tenth week. Lepage and Marcoux (1991) found that serum osteocalcin concentration elevated after 18 days of experimental fracture in foal. Mohamadnia et al. (2007) demonstrated that total ALP and BALP activities were significantly increased after experimental radius transaction in sheep,dogs and remained elevated throughout the four weeks study period. Thyese et al. (2006) concluded that serum osteocalcin is not efficacious as amarker of bone formation during osteogenesis in canine model.
Factors affecting the biological variability of bone serum markers are systemic disease, age, sex and exercise . In the present study, we used male rabbits of the same age range which minimized the influence of this factor. Clear diurnal and seasonal variation has however been demonstrated in various bone markers, both in humans and several animal species. Standardizing the time at which blood was sampled eliminated the diurnal variation. An issue that cannot be easily resolved is the fact that the direct comparison of the results between diverse studies is impeded by the inter-laboratory variation of bone marker assay (Seibel. et al., 2001).

5.3 Simvastatin Study
The statins include: atorvastatin, follistatin, lovastatin, vastatin series and simvastatin. Simvastatin was selected in this study, because many studies have shown that it has the most stimulating bone formation ability compared to the other statins (Park, 2009). Simvastatin, a specific competitive inhibitor of 3-hydroxy-2-methyl-glutaryl coenzyme A (HMG-CoA) reductase, is a widely-used anti-hyperlipidemia drug (Athul and Kumar, 2014; Mahmoud et al.,2014). In recent 15 years, interest has been shown in the possible effects of statins that appear to be diverse from those well-known on serum cholesterol. Among these, the potential effects of statins on bone tissue has received particular attention. Mundy et al. (1999) first reported that simvastatin in vivo enhanced bone formation in rodents and augment new bone volume in cultures from mouse calvaria.
The enzyme HMG-CoA reductase is one of the rate-limiting enzymes within the mevalonate pathway, through which cholesterol is biosynthesized. This enzyme is successfully inhibited by statins causing a reduction in blood-cholesterol levels. Other products of the mevalonate pathway are also essential for the prenylation of some kinds of small GTPases. Since small prenylated GTPases are important both for activating osteoclasts and inhibiting the synthesis of BMP-2, statins inhibit the prenylation of small GTPases and, as a result, they produce anabolic effect on bone by inducing BMP-2 and create anticatabolic effect by inhibition of osteoclast function (Garrett and Mundy, 2002). Human subjects with treated with statins have shown a high bone mineral density (Semisotova et al., 2012; Hernandez et al., 2014).
In the present study, local simvastatin delivery over the expanded sagittal suture was investigated to clarify its possible osteopromotory effect on bone formation. We didnot use systemic simvastatin although, several studies have been carried out to investigate the effects of systemically administered statins on bone healing and have found positive results (Saraf et al., 2007; Rivera et al., 2013). However, because of their high hepatic targeting property, statins do not accumulate in bone, and their effects after systemic administration would be minimal. extremely high doses of systemically applied statins can raise the risk of liver failure, kidney disease, and rhabdomyolysis (Guyton, 2006). This has encouraged the investigators to study the effects of locally applied statins.
In our study, local simvastatin was used with methylcellulose carrier. The successful employ of simvastatin to promote bone formation in vivo depends on the local concentration and there have been continuous attempts to find an appropriate delivery system (Wu et al., 2008).There are a number of advantages to an appropriate carrier, including localization and retention of the molecule to the site of application thus reducing the loading dose and providing a matrix for mesenchymal cell infiltration and a substrate for cell growth and differentiation (Schmidmaier et al.,2008). Methylcellulose gel has been used extensively in many oral and topical pharmaceutical preparations such as ophthalmic controlled-released gelling systems for ciprofloxacin and oral administration of methylcellulose particles (Al-Kassas and El-Khatib, 2009; Ravikumara et al.,2009). Methylcellulose is considered to be a non-toxic, non-sensitizing and safe material that can be used as a carrier for constitutive release of therapeutic drugs. As oral simvastatin primarily accumulate in the liver, it is difficult for it to apply its effects locally.Therefore, methylcellulose is used as a vehicle for local simvastatin application and in the performance of studies on local simvastatin application (Pradeep and Thorat, 2010).
Two doses of local simvastatin were used in the present study, 0.5mg and 1.0mg. Other authors also investigated the effects of locally applied simvastatin of variable doses for healing of surgically created defets: 2.2 mg (Thylin et al., 2002), 0.5 mg (Wong and Rabie, 2003; ??ze?? et al., 2007); 0.1, 0.5, 1.0, 1.5, and 2.2 mg (Stein et al., 2005)1mg (Rosselli et al.,2014)and 2.5 mg(Ishihara et al.,4014). The effect of local simvastatin was found to be dose-dependent, but local simvastatin installation in high doses can cause severe inflammatory reaction and could have a negative impact on bone repair. Stein et al.(2005) determined that reducing single dose of simvastatin from 2.2mg to 0.5mg reduced inflammation to a more clinically acceptable level without sacrificing bone-growth potential. In our study, the 0.5mg and 1.0mg simvastatin were well tolerated and there was no any sign of acute or chronic inflammation in the expanded sagittal suture.
5.3.1 Relapse Ratio of Expanded Sagittal Suture
The relapse ratio of the expanded sagittal suture was measured on digital radiograph of the animal cranium using image analysis software. The local simvastatin gel (0.5 mg and 1.0 mg) showed a less relapse of the expanded suture after one week of removing wire retainer. The relapse of the expanded suture in the control and methylellulose carrier gel was nearly identical (about 22%). The use of 0.5 mg simvastatin gel reduced the relapse ratio by 44% and the 0.1mg simvastatin gel reduced the relapse ratio by 64%. Up to our knowledge this is the first study of using local simvastatin in suture distraction model, therefore a direct comparison with other studies is not possible. The reduction in relapse ratio could be attributed to a more dense fibrous tissue and early bone formation which was observed histologically in the simvastatin treated group as compared to the negative and positive (methylcellulose carrier gel) control groups. The result of this study is a good evidence for osteopromotive activity of 0.5mg and 1.0 mg simvastatin on rapidly expanded suture and form a starting point in using this drug on patients with rapid maxillary expansion to reduce the relapse after expansion and to shorten the retention and overall treatment time in patients with posterior crossbite.
5.3.2 Radiographic Density of the Expanded Suture
The density of the expanded sagittal suture in the four groups was measured on digital radiograph of the cranial specimen of the rabbits after two, four, and six weeks of retention. The density of the suture was measured by image analysis software program (Image J). This software has been used in many studies to estimate bone density of healed tooth extraction socket (C??lio-Marino et al., 2012; Antonello et al., 2013). At the three time intervals, the density of the expanded suture was not significantly different between the control and methylcellulose gel group and indeed the density was nearly identical between the two groups. At the three time intervals, the density of the 0.5 mg and 1.0 mg simvastatin treated groups showed a significantly higher density of the expanded sagittal suture than the negative and positive (methylcellulose gel) groups. Although the suture density of the 1.0 mg simvastatin treated group was higher than 0.5 mg simvastatin treated group, the difference was not significant. The radiographic observations of the present study come in agreement with many other experimental studies which used simvastatin. Fukui et al. (2012), in an experimental study involving femoral fracture in rats, showed that 250 ??g local simvastatin gel injected locally at the site of fracture lead to a radiographically marked fracture union of 70% of the animals. Zhou et al. (2010) found that when 1 ??M simvastatin was locally applied to injectable tissue-engineered bone to restore the critical-sized calvarial defects in mice, radiographically denser bone was formed as compared to a control group. Saraf et al. (2007) found that simvastatin administered orally to rabbits led to significant radiographic union of experimental fracture of the femur as compared to control group. Ezirganli et al. (2013) found that 0.5 mg, 1.0 mg, and 1.5 mg simvastatin application to critical sized defects of diabetic rat led to a marked bone regeneration at one month of application. The results of our study disagree with those of Calixto et al.(2011) , who found that 0.5 mg and 2.2 mg simvastatin locally applied to 5 mm diameter bone defects in rat showed no any radiographic evidence of better healing than control after one and two months. Our results also disagree with those of Lima et al.(2011) who used 0.5mg simvastatin locally delivered to 5mm calvarial bone defects in rats and found that after 30 and 60 days, the treated defects revealed a lower radiographic density than control defects.
The explanation for the variation in the radiographic observation of local efficacy of simvastatin may be attributed to the dose of simvastatin, the carrier used and the nature of the defect. In our study, the sagittal suture is different from surgically created defects and fractures which heal by secondary intention, while the consolidation of the expanded suture is similar to distraction osteogenesis process.
5.3.3 Histology and Histomorphometry
The histological observations of the expanded sagittal suture showed regeneration in all study groups. At two, four and six weeks, the histological and histomorphometrical findings of the suture showed nearly the same features in the control and methylcellulose carrier gel group. This indicates that methylcellulose gel, which was used as a carrier for simvastatin, is biologically inert and didnot influence the regeneration process of the expanded suture. Therefore, the regenerative process of simvastatin-treated groups could not be attributed to the use of methylcellulose gel as a carrier.
The bone formation started early (at two weeks) in the 0.5mg and 0.1mg simvastatin-treated group as rounded islands of osteoid were noted in the middle of the suture, in addition to bone projections at the periphery of the suture. No bone mineralization was seen at this time in the control and methylcellulose gel groups. The total suture area at 2 weeks was significantly less in the two simvastatin-treated groups than the control and methylcellulose groups. This indicates that simvastatin has an early osteoinductive effect when applied locally which is supported by significantly higher number of osteoblasts and blood vessels in the simvastatin-treated groups than control groups. In addition, the 0.1mg simvastatin-treated group showed a greater number of osteoblasts and larger percentage of the osteoid to the total area of the suture.
At the fourth week, the beginning of bone formation was noted in the control groups with the cytological parameters and osteoid areas are still greater in the simvastatin groups than the control groups.
At the sixth week, the expanded suture gap in the 0.1mg simvastatin group was nearly completely ossified in comparison to the control groups. At the sixth week, the number of osteoblasts in 1.0mg simvastatin group was less than in the 0.5mg simvastatin group, this suggested that the new bone of the 1.0mg simvastatin group was more mature than that of the 0.5mg simvastatin group. This finding indicated that the local bone inductive effect of simvastatin is concentration dependent and not only restricted to the early stage of bone mineralization. Doubling the dose of simvastatin from 0.5mg to 1.0mg appeared to have an effect on bone regeneration.

Simvastatin affects the bone metabolism in different ways. According to Takenaka et al. (2003), simvastatin differentiate osteoblasts and enhance bone formation through affecting the release of VEGF. The results of Mundy et al. (1999) showed that statins increase bone formation by osteoblasts through elevation of BMP ' 2 which is an osteogenic growth factor involved in differentiation of osteoblasts and mineralization. As Bone formation depends mainly on the number of osteoblastic cells rather than the activity of the osteoblasts (Marie, 1995), the employment of osteoblastic cells plays an essential role in osteogenesis. This may be the explanation for greater number of osteoblasts and earlier bone formation in simvastatin groups in the current study. Simvastatin also regulates osteoblast function by increasing the expression of bone sialoprotein (BSP), osteocalcin (OC), and type I collagen (COL1), as well as suppressing the gene expression of collagenases such as MMP-1 and MMP-13 (Maeda et al., 2004). In addition, simvastatin prevents bone resorption through inhibiting the production of GTPase which is involved in activation of osteoclasts (Mundy et al.,1999). According to Lin et al. (2009) simvastatin prevents formation of Cyr61 protein through inhibiting the effect of TNF-alpha on osteoblasts. Consequently, chemotaxis of macrophages and bone resorption are reduced. Sakoda et al.(2006) found that simvastatin reduces the amount of IL-6 and 8 because of its anti-inflammatory effect. Since cytokine affects the activation of osteoclasts, it inhibits the inflammatory bone resorption.
The histological and histomorphometric findings of the present study agree with many other studies that used local simvastatin for healing of surgically created bone defects. Wang et al. (2007) used subcutaneous injection of 10mg/kg/day simvastatin for 5 days over the site of fractured tibia of ovariectomized rats and noted a significant increase in mineralization width, mineralization volume and mineral apposition rate. Chen et al. (2011) found that the intentionally created alveolar defects treated with 0.5, 1.5, and 2.2 mg/50 ??l simvastatin-methylcellulose gel, in miniature pigs, were completely filled with new bone and fibrous tissue as compared to control groups. They concluded that 0.5mg was the best dose of simvastatin to stimulate bone regeneration. Maciel-Oliveira et al. (2011) revealed that topical application of 2.5% simvastatin gel improve the quality of bone in 0.8mm defects of rat mandibular first molar. Esfahani et al. (2013) conducted a study to evaluate simvastatin effect on alveolar bone remodeling, root resorption and amount of tooth movement during orthodontic treatment in rats. They concluded that simvastatin decreased root resorption related to orthodontic tooth movement and recommended that patients and clinicians should be informed about a possible decrease in the amount of tooth movement and a prolonged period of orthodontic treatment. Sousa et al.(2014) who found that the local application of 0.1mg of simvastatin gel is effective in regenerating bone in rat calvariae created defects. The findings of the current study disagree with those of Calixto et al. (2011) who found that the use of 0.5 mg and 2.2 mg of simvastatin had a negative influence on healing of calvarial bone defects in rats, as observed by histometric analysis. The difference of our study with that of Calixto et al.(2011) may be attributed to the collagen sponge as a carrier for simvastatin. The collagen sponge they used is of bovine source and is highly antigenic that elicited an inflammatory reaction that lead to tissue destruction instead of bone formation. Our histological findings also disagree with those of ??ze?? et al. (2010), who found that the histological observations noted after single injection of 0.5mg simvastatin to a critical sized rat mandibular bone defects didnot make any significant improvement of new bone regeneration. The difference between our study and the study of ??ze?? et al (2010). is that they used simvastatin injection without carrier and this could be the possible negative effect on bone regeneration of 0.5mg simvastatin.
5.3.4 Serum Bone Formation Markers
Molecules associated with bone formation and turnover as well as their bi-products, have been widely utilized as markers of bone metabolism. Alkaline phosphatase and in particular BALP and OC are all associated with osteoblast activity and the serum levels of these proteins have been used to assess bone formation. The BALP is associated with early stages of osteogenesis and OC is associated with subsequent bone mineralization (Pagani et al., 2005; Biver et al., 2012).
The serum calcium and phosphorous levels were not influenced significantly by the use of simvastatin. This could be explained by the topical use of simvastatin which exerts its effect locally at the site of the expanded suture.
The serum levels of total ALP showed no significant differences between the four groups after 2, 4, and 6 weeks of retention of the expanded suture, indicating that total ALP is not a reliable marker of enhanced bone formation.
The serum level of BALP is significantly higher in the 0.5 mg and 1.0 mg simvastatin groups than the control ones. In all groups, the BALP showed a tendency to decrease at four weeks, but still significantly higher in the 1.0 mg simvastatin group, than the other three groups. At six weeks, the BALP showed further drop in the all four groups, with no significant difference among the groups.
The serum OC level is significantly higher in the simvastatin groups than the control groups, with higher value in the 1.0 mg group than 0.5 mg simvastatin group. At four and six weeks, the level of OC demonstrated a marked reduction. Although no significant difference was noted, its level in the simvastatin groups was higher than in the control.
It is known that BGP and BALP are produced exclusively by osteoblasts but at different developmental stages. The BALP is secreted by immature osteoblasts and BGP is produced by mature osteoblasts (Wang et al., 2006). The BALP activity has been reported to be necessary for the initiation of mineralization, but not for the continuation of the process (Hooper,1997). Indeed, reports indicating that statins promote osteocalcin expression by inhibiting the Rho and Rho-kinase pathway (Ohnaka et al., 2001), providing insights into the possible biological mechanism responsible for the increased serum levels of OC.
The present study demonstrated that serum level of biomarkers of bone formation namely BALP and OC are correlated with increased bone formation in orthopaedically expanded suture in rabbits treated with local application of simvastatin. As there is no data in the literature about bone formation markers of rabbits, so a direct comparison of the current results with other studies is not possible.

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