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Effects of Timing of Extracorporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis: An Experimental Study in a Rat Model
Accepted Manuscript Effects of Timing of Extra Corporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis Jiriys George Ginini, DMD, MSc, Omri Emodi, DMD, Edmond Sabo, M.D, Gila Maor, PhD, Dekel Shilo, DMD, PhD, Adi Rachmiel, DMD, PhD PII:
S0278-2391(18)30838-3
DOI:
10.1016/j.joms.2018.07.018
Reference:
YJOMS 58393
To appear in:
Journal of Oral and Maxillofacial Surgery
Received Date: 10 January 2018 Revised Date:
9 June 2018
Accepted Date: 9 July 2018
Please cite this article as: Ginini JG, Emodi O, Sabo E, Maor G, Shilo D, Rachmiel A, Effects of Timing of Extra Corporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis, Journal of Oral and Maxillofacial Surgery (2018), doi: 10.1016/j.joms.2018.07.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of Timing of Extra Corporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis
Shilo, DMD, PhD ǁ, Adi Rachmiel, DMD, PhD¶
Resident, Department of Oral and Maxillofacial Surgery, Rambam Medical Care Center, Haifa, Israel;
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∗
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Jiriys George Ginini, DMD, MSc*, Omri Emodi, DMD†, Edmond Sabo, M.D‡, Gila Maor, PhD§, Dekel
Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Deputy Head, Department of Oral and Maxillofacial Surgery, Rambam Medical Care Center, Haifa;
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Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Division Head, Department of Pathology, Rambam Health Care Campus, Haifa, Israel
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Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel
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Resident, Department of Oral and Maxillofacial Surgery, Rambam Medical Care Center, Haifa, Israel
¶
Department Head, Department of Oral and Maxillofacial Surgery, Rambam Medical Care Center, Haifa;
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Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
Jiriys George Ginini
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Corresponding author:
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Rambam Health Care Campus
8 Ha’Aliyah Street, Haifa 35254, Israel Phone - +972-503330896 Fax - +972-7772557
[email protected]
Financial Disclosure Statement: The authors declare that they have no conflict of interest.
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Effects of Timing of Extra Corporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis: An Experimental Study in a Rat
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Model
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ACCEPTED MANUSCRIPT ABSTRACT Purpose: Distraction osteogenesis (DO) is an established method for bone lengthening in the craniofacial skeleton. Its major drawback is the long consolidation period with
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attendant morbidity and possible complications. Several methods have been suggested to shorten the consolidation period. In this study, we evaluate the effects and timing of extracorporeal shock wave therapy (ESWT) on bone mineralization and extra cellular
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bone matrix proteins during mandibular distraction osteogenesis.
Methods: Twenty-seven rats underwent mandibular DO (latency period, 3 days;
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distraction period, 10 days, 0.5 mm/day) and were divided into three groups according to the timing of ESWT application: group I (control) received no treatment, group II and III received (0.18 mJ/mm2) ESWT before and after the active distraction period, respectively. The distracted mandibles were harvested following four weeks of
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consolidation and analyzed radiographically, histologically and immunohistochemically.
Results: Group III showed significantly increased mineral density, enhanced bone
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formation, higher collagen orientation index and greater expression of collagen type-I and osteocalcin proteins.
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Conclusion: Application of ESWT following active distraction enhances bone maturation and mineralization.
Keywords: Distraction osteogenesis, Shock waves, ESWT, Collagen type 1, Osteocalcin, Fast Fourier Transformation.
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ACCEPTED MANUSCRIPT INTRODUCTION Distraction osteogenesis (DO) is an active process of bone elongation under gradual tension forces [1-3]. The controlled mechanical stimulation is converted into a cascade
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of molecular signals which promote bone regeneration without donor site morbidity [4]. This technique has gained widespread usage for the treatment of both congenital and
acquired deformity conditions secondary to oncology, trauma, severe atrophic bone and
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infections [5, 6].
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Although DO is recognized as an effective and safe technique, it has its limitations. The major drawback is the prolonged consolidation period, during which the newly regenerated bone and the surrounding tissues are vulnerable and thus prone to complications until mature bone is formed before the distractor device is removed [7].
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Therefore, many methods have been introduced to enhance bone maturation and improve the biomechanical properties of the regenerated bone. One of these methods is extracorporeal shock wave therapy (ESWT), which generates acoustic waves,
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transferred through tissues. This mechanical stimulation is translated into a biological effect. In the field of orthopedics, ESWT has been shown to induce neovascularization
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and promote tissue regeneration by upregulation of several osteogenesis growth factors and proliferating factors that stimulate differentiation of mesenchymal cells into the osteogenic lineage resulting in enhanced bone regeneration [8, 9]. These angiogenic and osteogenic growth factors that have been demonstrated to be up-regulated as a result of the biological effects of ESWT play a fundamental role in bone regeneration during DO.
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ACCEPTED MANUSCRIPT Our assumption is that ESWT enhances bone regeneration during DO and that these effects are mediated by modulating the expression of bone specific extracellular matrix proteins. The aim of the current study is to evaluate whether ESWT enhances bone
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regeneration and extracellular bone matrix proteins during mandibular DO and at which
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stage is it most effective.
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MATERIALS AND METHODS Ethics statement
All animal treatments in this experiment were approved by the Technion Committee for
Animal model
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the Supervision of Animal Experiments.
27 male Sprague Dawley rats, weighing between 300 and 400g underwent general
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anesthesia by ketamine (90mg/kg) and xylazine (10mg/kg). Systemic analgesia by buprenorphine (0.05mg/kg) and antibiotic prophylaxis by cephalexin (180mg/kg)
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subcutaneously were administered 20 minutes before the surgical procedure. A 2cm anterior-posterior incision along the inferior border of the mandible was performed using a scalpel. The skin and subcutaneous tissues were dissected in order to reveal the masseter muscle. The masseter was split and stripped, the mandibular angle and the entire mandibular body up to the symphysis was exposed with a periosteal incision. Using a micro sagittal saw (Medicon eG, Tuttlingen, Germany) activated at a rate of 18,000 rpm under intensive saline cooling, a vertical corticotomy was initiated at the
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ACCEPTED MANUSCRIPT inferior border of the mandible at the antegonial notch and continued coronally. Two holes 6 mm anterior and posterior to the marked site were drilled for the distraction device screws. A custom made distraction device was placed and fixed with mini
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titanium screws (two on each side). The activating rod was externalized posteriorly. The bone cut was completed using a thin osteotome through the vertical corticotomy line, and complete mobilization of the bone fragments was achieved. Activation of the
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distraction device was tested. The gap between the bone fragments was narrowed by reverse directed activation of the distractor (Figure 1). The periosteal flaps were
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repositioned and sutured by Vicryl 3-0 stitch.
After surgery, the rats were housed in separate cages and monitored by weighing twice a week. Physiology and behavior were monitored daily (observation of the surgical site,
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swelling, bleeding, secretion, eyes and fur). For three days postoperatively the animals were given buprenorphine for analgesia and cephalexin as prophylactic antibiotic therapy. Moreover, moistened food pellets and sweet jelly cubes were given for easier
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chewing and digestion.
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Following the osteotomy, and after three days of latency period the distractor devices were activated for 10 days at a rate of 0.5mm (two turns)/day. Following the distraction period, the rat mandibles were fixed for 4 weeks of consolidation.
The animals in the ESWT groups underwent a second general anesthesia procedure using ketamine and xylazine in order to administer the ESWT. A surgical lubricating gel was applied to the skin area that was in contact with the ESWT tube. ESWT was applied to
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ACCEPTED MANUSCRIPT groups II and III at the first day of distraction period and first day of the consolidation period, respectively. The treated groups received one episode of ESWT treatment of 500 impulses at energy level of 0.18mJ/mm², 1.0 Hz, to the distraction zone by using the
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Extracorporeal Pulse Treatment System (Dermagold 100, MTS, Konstanz, Germany) (Figure 2). The dosage of ESWT shockwave intensity was selected on the basis of
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previous reports with animal models [10-12].
Radiography
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Following four weeks of consolidation, the animals were euthanized by CO2 asphyxiation under general anesthesia of isoflurane. The mandibles were harvested, soft tissue was removed, and the specimens were washed in saline and fixed in 10% neutral buffered formalin for 48 hours. After tissue fixation radiographs were taken by X-ray
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source (Planmeca, Helsinki, Finland) at 60kV, 8mA for 0.032ms. The mandibles were placed directly on the digital X-ray sensor (Schick, Sirona Dental, Salzburg) and the Xray tube was placed at a distance of 10cm, tangent to the sagittal axis of the mandible.
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In order to assess bone mineral density (BMD), pixel intensity of each radiograph was standardized using Image-Pro-Plus image analysis software (Image Pro Plus 7.0, Media
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Cybernetics, Bethesda, MD). The distraction gap was divided into proximal, middle and distal regions of interest (ROI) with equal length[13, 14]. BMD percentage was calculated from the ratio of the BMD value of the middle area to the average BMD of the proximal and distal areas, in order to standardize each value (% BMD).
Histology
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ACCEPTED MANUSCRIPT The fixed mandible specimens were decalcified by treatment with 10% ethylenediaminetetraacetic acid (EDTA) for up to 4 weeks at room temperature until the tissue was soft and pliable. The decalcified mandibles were dehydrated through an
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ascending series of alcohol, cleared in xylene, embedded in paraffin and subsequently cut with a microtome (Leica, Nussloch, Germany) longitudinally into 5µm thick
sections. The sections were transferred onto glass slides. For histological evaluation
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tissues were deparaffinized by xylene and rehydrated through a descending alcohol
series and subsequently stained by hematoxylin eosin. Slides from each sample were
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examined under an Olympus BX51 microscope (Tokyo, Japan). Images were captured using a Retiga 2000R camera (QImaging, Vancouver, Canada). Images were analyzed using Image-Pro Plus image-analysis software (Image Pro Plus 7.0, Media Cybernetics,
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Bethesda, MD).
Collagen orientation index
In order to evaluate the level of bone organization and maturity, H&E sections imaged
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with polarized light were transformed from their space into their frequency domains, using a two-dimensional (2D) Fast Fourier Transform (FFT) algorithm. Eight bit
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digitized images are composed of 2D arrays of pixels, each having a light intensity value (gray level) ranging from 0 (black) through different shades of gray to 255 (white). Every row or column in this 2D array represents a signal within a range of coordinates defining a so-called space domain. Fourier Transform is an important image processing tool, which is used to decompose such 2D arrays (signals) into more basic components of sine and cosine functions [15]. FFT is an efficient computer algorithm for calculating the basic frequencies of these sinusoidal functions. The graphical output
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ACCEPTED MANUSCRIPT of such transformation is called the Fourier frequency domain of the image. In this Fourier frequency domain, each point represents a particular frequency contained in the spatial domain image. The 2D Fourier frequency plots are frequently used in image
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analysis in order to analyze pattern periodicity and orientation [16, 17]. To compute the orientation index, the Auto-Pro environment of the Image Pro-Plus 7
software was used. A macro algorithm was utilized that automatically traces thickness,
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and singles out the longest diameter of the fibers. Subsequently, the fast Fourier
transformation algorithm was applied to obtain two-dimensional power scattergrams of
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the histological images from four regions of interest within the distraction gap from each slide. A median filter was applied to remove noise. Woven bone with randomly oriented collagen fibers exhibited a circular scattergram, compared to lamellar bone with well-organized fibers, which showed an elongated scattergram. The larger the
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index value, the greater the degree of mature bone in the distraction gap.
Immunohistochemistry
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For immunohistochemistry (IHC) staining, paraffin-embedded tissues were sectioned onto poly-L-lysine coated slides. The specimens were incubated with 3% hydrogen
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peroxide (H2O2) in methanol for 20min to block endogenous peroxidases. Nonspecific binding was blocked by incubation in 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) for 30min and with BEAT-blocker kit (Invitrogen, Camarillo, CA) when using mouse primary antibody. For immunostaining, sections were incubated with primary antibodies rabbit anti-osteocalcin (1:200, Bioss Antibodies, Woburn, MA) and mouse anti-collagen type I (1:400, Abcam, Cambridge, UK) overnight at 4°C. The corresponding biotinylated secondary goat anti-mouse and
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ACCEPTED MANUSCRIPT goat anti-rabbit antibodies (Jackson ImmunoResearch) were incubated for 15min followed by 15min at RT with streptavidin-peroxidase conjugate (Jackson ImmunoResearch). Peroxidase substrate aminoethyl carbazole (AEC) (Invitrogen) was
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added until adequate color development appeared, then slides were mounted using Immu-Mount (Thermo Scientific) and coverslipped. Washing between stages was
performed by phosphate buffered saline (PBS) buffer pH 7.4 and all incubations were
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conducted in a moistened chamber.
For histomorphometric analysis the colored (red/orange) area from the distraction gap
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was measured at X100 magnification of two slides from each animal utilizing the Image Pro-Plus 7 software.
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RESULTS
Of the 27 animals that underwent the surgical procedure, there were two general
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anesthesia-related deaths during ESWT application (one animal from each group; II and III). One animal from the control group was excluded due to weight loss. Overall, we
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included 24 animals. No deep mucosal infection, pus, or other major adverse effects were observed.
Radiography Radiographs were taken four weeks after the consolidation period of all distracted mandibles. Control group showed radiolucency in the center of the gap indicating that the gap was not completely bridged (Figure 3). In addition, the radiopacity of the
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ACCEPTED MANUSCRIPT distracted mandibles in the treated groups was greater than in the control. The regenerated bone in group III was barely distinguishable from the native bone (Figure 3C). Group II appeared to be less radiodense than group III. Quantitatively, BMD
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percentage was significantly higher in Group III in comparison to the control group (p=0.01) (Figure 3D) with no significant difference between groups II and control
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(p>0.05).
Histology
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Histological evaluation showed that following four weeks of consolidation period, the distraction gaps in the treated groups were bridged with bony union, whereas the control group was partially bridged with tiny trabecular bone (Figure 4). The control group showed less immature bone and more fibrous tissue within the center of the distraction
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gap than the other groups. Group II showed greater and denser immature woven bone and less fibrous tissue than the control. In group III the bone marrow was mature, and the newly formed cancellous and cortical bone were more mature and thicker
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compared to the other groups.
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Collagen orientation index
H&E sections were visualized under polarized light microscopy to compare local tissue organization. Under polarized light, collagen fibers become birefringent and organized lamellar bone is distinguished from unorganized woven bone. Group III collagen fibers were more organized than in the control group and in group II which exhibited haphazard organization (Figure 5). Quantitatively, the collagen orientation index was
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ACCEPTED MANUSCRIPT significantly higher in group III (1.96 ± 0.42) which indicates more lamellar bone in group III than other groups which displayed woven bone (Figure 6).
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Immunohistochemical staining Immunohistochemical staining revealed that application of ESWT increased collagen-1 and osteocalcin (OCN) levels in the distraction gaps of the treated groups, as compared
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to the control group. The representative immunohistochemical staining photographs
showed collagen-1 and OCN positive staining, as indicated by the red/orange staining
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which is localized in both proteins to the extracellular matrix (Figure 7A-F). IHC histomorphometry analysis revealed significantly increased levels of collagen1 and OCN expressions in group III compared to the control (p<0.05) (Figure 7G, H). However, no statistically significant differences were observed between group II and
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DISCUSSION
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control, or between the two treated groups (p>0.05).
In DO, tension-stress is believed to be the key stimulator for recruitment of
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mesenchymal stem cells accompanied by expression of growth factors that increases as long as the distraction is maintained and decreases when the distraction is completed. Although many attempts have been made to shorten the consolidation period during DO, no efficient approach has been reported. ESWT is a novel non-surgical therapeutic method, which generates mechanical stimulation shown to be able to enhance bone regeneration by increasing the differentiation and vascularization.
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ACCEPTED MANUSCRIPT Publications in the field have shown that two weeks following the commencement of the distraction forces, progressive calcification of matrix collagen bundle oriented parallel to the distraction direction is observed. Continuing calcification of the
are deposited in the lamellar phase after 8 weeks [18, 19].
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distraction regenerate eventually leads to closure of the distraction gap and calcium salts
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In the current study on rat mandibles, radiographic examinations showed that the
distraction gap in the control group was radiolucent and did not completely bridge
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following four weeks of consolidation. However, the distraction gap in the treated groups appeared to be markedly radiodense and more mineralized. In addition, the radiopacity of the distracted mandible in group III was greater than that of group II. Radiographic examination showed faster calcification in the ESWT treated groups.
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Also, our histological findings showed accelerated new bone formation and less fibrous tissue in the treated groups compared to the control. The BMD percentage was higher in both treated groups compared to control, however, Group III showed markedly higher
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percentage when compared to the control group.
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The orientation of structures can be represented as a power plot of the fast Fourier transformation (FFT) of an image. Gregory et al., [20] applied the FFT to bone histological images. We used, for the first time to the best of our knowledge, the collagen ordination index obtained from De Vries et al., [21] to evaluate bone maturation following DO in ESWT treated and untreated rats. ESWT application at the consolidation period resulted in significantly higher orientation index compared to the
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ACCEPTED MANUSCRIPT control, indicating that ESWT at the consolidation period may lead to more mature and lamellar bone.
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DO considerably affects cellular gene expression and these effects result in the upregulation of bone specific extracellular matrix proteins. Type I fibrillary collagen is the primary extracellular matrix scaffold of bone [22]. Its heterotrimeric helix is reinforced
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with calcium hydroxyapatite providing infrastructural support and tensile strength to the skeleton. Collagens, the major component of the ECM appear to play a critical role in
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bone formation during distraction by facilitating the formation of early bone spicules extending from the osteotomy edges towards the center of the distraction gap [18]. The high level of type I collagen in group III (Figure 7) is consistent with mature bone demonstrated in the histological findings. Non-collagenous proteins like OCN have an
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important role in regulating ossification and bone remodeling [23]. OCN is a Glacontaining, vitamin K-dependent, calcium-binding protein expressed by well differentiated osteoblasts. Using a rabbit model of mandibular distraction, Meyer et
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al.[24] observed that hyperphysiological levels of strain resulted in a marked reduction in osteocalcin and osteonectin protein expression in the distraction gap compared to
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mandibles exposed to physiologic levels of strain. Moreover, the decrease in osteocalcin and osteonectin expression paralleled a loss of crystal formation suggesting that both proteins have an important role in mineralization during mechanical loading [24]. In an in vitro study, Tamma et al., [25] evaluated the direct effects of ESWT on murine osteoblasts. Results have shown increased expression of collagen type 1, OCN, bone sialoprotein and osteopontin, concluding that ESWT induces increased expression of extracellular matrix [25]. These findings correlate with ours, in which ESWT
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ACCEPTED MANUSCRIPT application at the consolidation period up-regulates the expression of OCN and Collagen type 1.
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Conclusion Our findings suggest that ESWT application following the distraction period enhances bone maturation and mineralization during DO and that these effects are mediated by
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modulating the expression of bone specific extracellular matrix proteins without side
effects. Despite the promising results, further studies are needed to investigate the exact
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mechanism through which ESWT affects DO.
REFERENCES
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1. Ilizarov GA: The tension-stress effect on the genesis and growth of tissues. Part I. The influence of stability of fixation and soft-tissue preservation. Clin Orthop Relat Res 238:249, 1989 2. Ilizarov GA: The tension-stress effect on the genesis and growth of tissues: Part II. The influence of the rate and frequency of distraction. Clin Orthop Relat Res 239:263, 1989 3. Ilizarov GA: Clinical application of the tension-stress effect for limb lengthening. Clin Orthop Relat Res 250:8, 1990 4. Rachmiel A, Leiser Y: The Molecular and Cellular Events That Take Place during Craniofacial Distraction Osteogenesis. Plast Reconstr Surg Glob Open 2:e98, 2014 5. Aronson J: Limb-lengthening, skeletal reconstruction, and bone transport with the Ilizarov method. J Bone Joint Surg Am 79:1243, 1997 6. Cohen SR, Burstein FD, Williams JK: The role of distraction osteogenesis in the management of craniofacial disorders. Ann Acad Med Singapore 28:728, 1999 7. Forriol F, Iglesias A, Arias M, Aquerreta D, Canadell J: Relationship between radiologic morphology of the bone lengthening formation and its complications. J Pediatr Orthop B 8:292, 1999 8. Chen YJ, Wurtz T, Wang CJ, Kuo YR, Yang KD, Huang HC, Wang FS: Recruitment of mesenchymal stem cells and expression of TGF-beta 1 and VEGF in the early stage of shock wave-promoted bone regeneration of segmental defect in rats. J Orthop Res 22:526, 2004 9. Wang CJ, Wang FS, Yang KD: Biological effects of extracorporeal shockwave in bone healing: a study in rabbits. Arch Orthop Trauma Surg 128:879, 2008 10. Lai JP, Wang FS, Hung CM, Wang CJ, Huang CJ, Kuo YR: Extracorporeal shock wave accelerates consolidation in distraction osteogenesis of the rat mandible. J Trauma 69:1252, 2010
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11. Narasaki K, Shimizu H, Beppu M, Aoki H, Takagi M, Takashi M: Effect of extracorporeal shock waves on callus formation during bone lengthening. J Orthop Sci 8:474, 2003 12. Wang FS, Wang CJ, Huang HJ, Chung H, Chen RF, Yang KD: Physical shock wave mediates membrane hyperpolarization and Ras activation for osteogenesis in human bone marrow stromal cells. Biochem Biophys Res Commun 287:648, 2001 13. Shimazaki A, Inui K, Azuma Y, Nishimura N, Yamano Y: Low-intensity pulsed ultrasound accelerates bone maturation in distraction osteogenesis in rabbits. J Bone Joint Surg Br 82:1077, 2000 14. Sakurakichi K, Tsuchiya H, Uehara K, Yamashiro T, Tomita K, Azuma Y: Effects of timing of low-intensity pulsed ultrasound on distraction osteogenesis. J Orthop Res 22:395, 2004 15. Translational College of Lex Tokyo: Who is Fourier? A Mathematical Adventure. Belmont, MA, Languate Research Foundation, 1995 16. Sabo E, Beck AH, Montgomery EA, Bhattacharya B, Meitner P, Wang JY, Resnick MB: Computerized morphometry as an aid in determining the grade of dysplasia and progression to adenocarcinoma in Barrett's esophagus. Lab Invest 86:1261, 2006 17. Har-Shai Y, Amar M, Sabo E: Intralesional cryotherapy for enhancing the involution of hypertrophic scars and keloids. Plast Reconstr Surg 111:1841, 2003 18. Karp NS, McCarthy JG, Schreiber JS, Sissons HA, Thorne CH: Membranous bone lengthening: a serial histological study. Ann Plast Surg 29:2, 1992 19. Califano L, Cortese A, Zupi A, Tajana G: Mandibular lengthening by external distraction: an experimental study in the rabbit. J Oral Maxillofac Surg 52:1179, 1994 20. Gregory JS, Junold RM, Undrill PE, Aspden RM: Analysis of trabecular bone structure using Fourier transforms and neural networks. IEEE Trans Inf Technol Biomed 3:289, 1999 21. de Vries HJ, Enomoto DN, van Marle J, van Zuijlen PP, Mekkes JR, Bos JD: Dermal organization in scleroderma: the fast Fourier transform and the laser scatter method objectify fibrosis in nonlesional as well as lesional skin. Lab Invest 80:1281, 2000 22. Valchanou VD, Michailov P: High energy shock waves in the treatment of delayed and nonunion of fractures. Int Orthop 15:181, 1991 23. Sandberg MM, Aro HT, Vuorio EI: Gene expression during bone repair. Clin Orthop Relat Res 292, 1993 24. Meyer U, Meyer T, Vosshans J, Joos U: Decreased expression of osteocalcin and osteonectin in relation to high strains and decreased mineralization in mandibular distraction osteogenesis. J Craniomaxillofac Surg 27:222, 1999 25. Tamma R, dell'Endice S, Notarnicola A, Moretti L, Patella S, Patella V, Zallone A, Moretti B: Extracorporeal shock waves stimulate osteoblast activities. Ultrasound Med Biol 35:2093, 2009
LEGENDS:
Figure 1. Intraoperative photograph showing the osteotomy line and the fixed distractor device.
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ACCEPTED MANUSCRIPT Figure 2. Schematic representation of the experimental study design
Figure 3. X-ray radiographs of the distracted mandibles in the control group (A), group
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II (B), and group III (C) following four weeks of consolidation period. Bone mineral density percentage analysis (D). The control group showed radiolucency at the center of the gap which did not completely bridge. The radiopacity of the distracted mandibles in
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the treated groups was greater than in the control. The regenerated bone in group III was barely distinguishable from native bone (C). Group II appeared to be less radiodense
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than Group III. (D) Group III showed significantly greater percentage of bone mineral density compared to the control group ("*" represents p value < 0.05).
Figure 4. Hematoxylin & Eosin sections of the distraction gap. Histological
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photomicrographs of the distracted mandibles in the control group (A), group II (B), and group III (C) at four weeks of consolidation period. The control group showed more fibrous tissue and immature bone at the center of the distraction gap than other groups.
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Group II showed more and denser immature woven bone and less fibrous tissue than the control. In group III the bone marrow was mature, and the newly formed cancellous and
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cortical (arrow) bone were more mature and thicker than other groups. Original magnification x20.
Figure 5. Collagen organization under polarized light microscopy (A-C) and fast Fourier transformation (D-F). The control group (A) showed less-oriented collagen fibers resulting in rounder frequency distribution (D) as opposed to group III with more
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ACCEPTED MANUSCRIPT well-oriented collagen in mature bone (C) providing more elliptic frequency distributions (F). Original magnification x200.
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Figure 6. Orientation index of the collagen fibers in the ESWT-treated groups and the control group using the fast Fourier transformation. In group III, the orientation indexes are significantly higher (1.92 +0.42) compared to the control group (1.43 +0.15) ("*"
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represents p value < 0.05).
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Figure 7. Representative immunohistochemical staining for Collagen1 (A-C) and OCN expressions in the distraction gaps (D-F). Original magnification x100. Histomorphometric evaluation for Collagen1(G) and OCN levels in the distraction gaps (H). Significantly increased levels of Collagen 1 and OCN in group III compared to the
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control ("*" represents p value < 0.05).
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S0278-2391(18)30838-3
DOI:
10.1016/j.joms.2018.07.018
Reference:
YJOMS 58393
To appear in:
Journal of Oral and Maxillofacial Surgery
Received Date: 10 January 2018 Revised Date:
9 June 2018
Accepted Date: 9 July 2018
Please cite this article as: Ginini JG, Emodi O, Sabo E, Maor G, Shilo D, Rachmiel A, Effects of Timing of Extra Corporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis, Journal of Oral and Maxillofacial Surgery (2018), doi: 10.1016/j.joms.2018.07.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of Timing of Extra Corporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis
Shilo, DMD, PhD ǁ, Adi Rachmiel, DMD, PhD¶
Resident, Department of Oral and Maxillofacial Surgery, Rambam Medical Care Center, Haifa, Israel;
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∗
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Jiriys George Ginini, DMD, MSc*, Omri Emodi, DMD†, Edmond Sabo, M.D‡, Gila Maor, PhD§, Dekel
Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Deputy Head, Department of Oral and Maxillofacial Surgery, Rambam Medical Care Center, Haifa;
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†
Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Division Head, Department of Pathology, Rambam Health Care Campus, Haifa, Israel
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Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel
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Resident, Department of Oral and Maxillofacial Surgery, Rambam Medical Care Center, Haifa, Israel
¶
Department Head, Department of Oral and Maxillofacial Surgery, Rambam Medical Care Center, Haifa;
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Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
Jiriys George Ginini
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Corresponding author:
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Rambam Health Care Campus
8 Ha’Aliyah Street, Haifa 35254, Israel Phone - +972-503330896 Fax - +972-7772557
[email protected]
Financial Disclosure Statement: The authors declare that they have no conflict of interest.
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Effects of Timing of Extra Corporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis: An Experimental Study in a Rat
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Model
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ACCEPTED MANUSCRIPT ABSTRACT Purpose: Distraction osteogenesis (DO) is an established method for bone lengthening in the craniofacial skeleton. Its major drawback is the long consolidation period with
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attendant morbidity and possible complications. Several methods have been suggested to shorten the consolidation period. In this study, we evaluate the effects and timing of extracorporeal shock wave therapy (ESWT) on bone mineralization and extra cellular
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bone matrix proteins during mandibular distraction osteogenesis.
Methods: Twenty-seven rats underwent mandibular DO (latency period, 3 days;
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distraction period, 10 days, 0.5 mm/day) and were divided into three groups according to the timing of ESWT application: group I (control) received no treatment, group II and III received (0.18 mJ/mm2) ESWT before and after the active distraction period, respectively. The distracted mandibles were harvested following four weeks of
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consolidation and analyzed radiographically, histologically and immunohistochemically.
Results: Group III showed significantly increased mineral density, enhanced bone
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formation, higher collagen orientation index and greater expression of collagen type-I and osteocalcin proteins.
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Conclusion: Application of ESWT following active distraction enhances bone maturation and mineralization.
Keywords: Distraction osteogenesis, Shock waves, ESWT, Collagen type 1, Osteocalcin, Fast Fourier Transformation.
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ACCEPTED MANUSCRIPT INTRODUCTION Distraction osteogenesis (DO) is an active process of bone elongation under gradual tension forces [1-3]. The controlled mechanical stimulation is converted into a cascade
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of molecular signals which promote bone regeneration without donor site morbidity [4]. This technique has gained widespread usage for the treatment of both congenital and
acquired deformity conditions secondary to oncology, trauma, severe atrophic bone and
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infections [5, 6].
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Although DO is recognized as an effective and safe technique, it has its limitations. The major drawback is the prolonged consolidation period, during which the newly regenerated bone and the surrounding tissues are vulnerable and thus prone to complications until mature bone is formed before the distractor device is removed [7].
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Therefore, many methods have been introduced to enhance bone maturation and improve the biomechanical properties of the regenerated bone. One of these methods is extracorporeal shock wave therapy (ESWT), which generates acoustic waves,
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transferred through tissues. This mechanical stimulation is translated into a biological effect. In the field of orthopedics, ESWT has been shown to induce neovascularization
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and promote tissue regeneration by upregulation of several osteogenesis growth factors and proliferating factors that stimulate differentiation of mesenchymal cells into the osteogenic lineage resulting in enhanced bone regeneration [8, 9]. These angiogenic and osteogenic growth factors that have been demonstrated to be up-regulated as a result of the biological effects of ESWT play a fundamental role in bone regeneration during DO.
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ACCEPTED MANUSCRIPT Our assumption is that ESWT enhances bone regeneration during DO and that these effects are mediated by modulating the expression of bone specific extracellular matrix proteins. The aim of the current study is to evaluate whether ESWT enhances bone
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regeneration and extracellular bone matrix proteins during mandibular DO and at which
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stage is it most effective.
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MATERIALS AND METHODS Ethics statement
All animal treatments in this experiment were approved by the Technion Committee for
Animal model
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the Supervision of Animal Experiments.
27 male Sprague Dawley rats, weighing between 300 and 400g underwent general
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anesthesia by ketamine (90mg/kg) and xylazine (10mg/kg). Systemic analgesia by buprenorphine (0.05mg/kg) and antibiotic prophylaxis by cephalexin (180mg/kg)
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subcutaneously were administered 20 minutes before the surgical procedure. A 2cm anterior-posterior incision along the inferior border of the mandible was performed using a scalpel. The skin and subcutaneous tissues were dissected in order to reveal the masseter muscle. The masseter was split and stripped, the mandibular angle and the entire mandibular body up to the symphysis was exposed with a periosteal incision. Using a micro sagittal saw (Medicon eG, Tuttlingen, Germany) activated at a rate of 18,000 rpm under intensive saline cooling, a vertical corticotomy was initiated at the
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ACCEPTED MANUSCRIPT inferior border of the mandible at the antegonial notch and continued coronally. Two holes 6 mm anterior and posterior to the marked site were drilled for the distraction device screws. A custom made distraction device was placed and fixed with mini
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titanium screws (two on each side). The activating rod was externalized posteriorly. The bone cut was completed using a thin osteotome through the vertical corticotomy line, and complete mobilization of the bone fragments was achieved. Activation of the
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distraction device was tested. The gap between the bone fragments was narrowed by reverse directed activation of the distractor (Figure 1). The periosteal flaps were
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repositioned and sutured by Vicryl 3-0 stitch.
After surgery, the rats were housed in separate cages and monitored by weighing twice a week. Physiology and behavior were monitored daily (observation of the surgical site,
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swelling, bleeding, secretion, eyes and fur). For three days postoperatively the animals were given buprenorphine for analgesia and cephalexin as prophylactic antibiotic therapy. Moreover, moistened food pellets and sweet jelly cubes were given for easier
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chewing and digestion.
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Following the osteotomy, and after three days of latency period the distractor devices were activated for 10 days at a rate of 0.5mm (two turns)/day. Following the distraction period, the rat mandibles were fixed for 4 weeks of consolidation.
The animals in the ESWT groups underwent a second general anesthesia procedure using ketamine and xylazine in order to administer the ESWT. A surgical lubricating gel was applied to the skin area that was in contact with the ESWT tube. ESWT was applied to
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ACCEPTED MANUSCRIPT groups II and III at the first day of distraction period and first day of the consolidation period, respectively. The treated groups received one episode of ESWT treatment of 500 impulses at energy level of 0.18mJ/mm², 1.0 Hz, to the distraction zone by using the
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Extracorporeal Pulse Treatment System (Dermagold 100, MTS, Konstanz, Germany) (Figure 2). The dosage of ESWT shockwave intensity was selected on the basis of
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previous reports with animal models [10-12].
Radiography
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Following four weeks of consolidation, the animals were euthanized by CO2 asphyxiation under general anesthesia of isoflurane. The mandibles were harvested, soft tissue was removed, and the specimens were washed in saline and fixed in 10% neutral buffered formalin for 48 hours. After tissue fixation radiographs were taken by X-ray
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source (Planmeca, Helsinki, Finland) at 60kV, 8mA for 0.032ms. The mandibles were placed directly on the digital X-ray sensor (Schick, Sirona Dental, Salzburg) and the Xray tube was placed at a distance of 10cm, tangent to the sagittal axis of the mandible.
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In order to assess bone mineral density (BMD), pixel intensity of each radiograph was standardized using Image-Pro-Plus image analysis software (Image Pro Plus 7.0, Media
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Cybernetics, Bethesda, MD). The distraction gap was divided into proximal, middle and distal regions of interest (ROI) with equal length[13, 14]. BMD percentage was calculated from the ratio of the BMD value of the middle area to the average BMD of the proximal and distal areas, in order to standardize each value (% BMD).
Histology
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ACCEPTED MANUSCRIPT The fixed mandible specimens were decalcified by treatment with 10% ethylenediaminetetraacetic acid (EDTA) for up to 4 weeks at room temperature until the tissue was soft and pliable. The decalcified mandibles were dehydrated through an
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ascending series of alcohol, cleared in xylene, embedded in paraffin and subsequently cut with a microtome (Leica, Nussloch, Germany) longitudinally into 5µm thick
sections. The sections were transferred onto glass slides. For histological evaluation
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tissues were deparaffinized by xylene and rehydrated through a descending alcohol
series and subsequently stained by hematoxylin eosin. Slides from each sample were
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examined under an Olympus BX51 microscope (Tokyo, Japan). Images were captured using a Retiga 2000R camera (QImaging, Vancouver, Canada). Images were analyzed using Image-Pro Plus image-analysis software (Image Pro Plus 7.0, Media Cybernetics,
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Bethesda, MD).
Collagen orientation index
In order to evaluate the level of bone organization and maturity, H&E sections imaged
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with polarized light were transformed from their space into their frequency domains, using a two-dimensional (2D) Fast Fourier Transform (FFT) algorithm. Eight bit
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digitized images are composed of 2D arrays of pixels, each having a light intensity value (gray level) ranging from 0 (black) through different shades of gray to 255 (white). Every row or column in this 2D array represents a signal within a range of coordinates defining a so-called space domain. Fourier Transform is an important image processing tool, which is used to decompose such 2D arrays (signals) into more basic components of sine and cosine functions [15]. FFT is an efficient computer algorithm for calculating the basic frequencies of these sinusoidal functions. The graphical output
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ACCEPTED MANUSCRIPT of such transformation is called the Fourier frequency domain of the image. In this Fourier frequency domain, each point represents a particular frequency contained in the spatial domain image. The 2D Fourier frequency plots are frequently used in image
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analysis in order to analyze pattern periodicity and orientation [16, 17]. To compute the orientation index, the Auto-Pro environment of the Image Pro-Plus 7
software was used. A macro algorithm was utilized that automatically traces thickness,
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and singles out the longest diameter of the fibers. Subsequently, the fast Fourier
transformation algorithm was applied to obtain two-dimensional power scattergrams of
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the histological images from four regions of interest within the distraction gap from each slide. A median filter was applied to remove noise. Woven bone with randomly oriented collagen fibers exhibited a circular scattergram, compared to lamellar bone with well-organized fibers, which showed an elongated scattergram. The larger the
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index value, the greater the degree of mature bone in the distraction gap.
Immunohistochemistry
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For immunohistochemistry (IHC) staining, paraffin-embedded tissues were sectioned onto poly-L-lysine coated slides. The specimens were incubated with 3% hydrogen
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peroxide (H2O2) in methanol for 20min to block endogenous peroxidases. Nonspecific binding was blocked by incubation in 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) for 30min and with BEAT-blocker kit (Invitrogen, Camarillo, CA) when using mouse primary antibody. For immunostaining, sections were incubated with primary antibodies rabbit anti-osteocalcin (1:200, Bioss Antibodies, Woburn, MA) and mouse anti-collagen type I (1:400, Abcam, Cambridge, UK) overnight at 4°C. The corresponding biotinylated secondary goat anti-mouse and
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ACCEPTED MANUSCRIPT goat anti-rabbit antibodies (Jackson ImmunoResearch) were incubated for 15min followed by 15min at RT with streptavidin-peroxidase conjugate (Jackson ImmunoResearch). Peroxidase substrate aminoethyl carbazole (AEC) (Invitrogen) was
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added until adequate color development appeared, then slides were mounted using Immu-Mount (Thermo Scientific) and coverslipped. Washing between stages was
performed by phosphate buffered saline (PBS) buffer pH 7.4 and all incubations were
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conducted in a moistened chamber.
For histomorphometric analysis the colored (red/orange) area from the distraction gap
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was measured at X100 magnification of two slides from each animal utilizing the Image Pro-Plus 7 software.
Animal model
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RESULTS
Of the 27 animals that underwent the surgical procedure, there were two general
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anesthesia-related deaths during ESWT application (one animal from each group; II and III). One animal from the control group was excluded due to weight loss. Overall, we
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included 24 animals. No deep mucosal infection, pus, or other major adverse effects were observed.
Radiography Radiographs were taken four weeks after the consolidation period of all distracted mandibles. Control group showed radiolucency in the center of the gap indicating that the gap was not completely bridged (Figure 3). In addition, the radiopacity of the
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ACCEPTED MANUSCRIPT distracted mandibles in the treated groups was greater than in the control. The regenerated bone in group III was barely distinguishable from the native bone (Figure 3C). Group II appeared to be less radiodense than group III. Quantitatively, BMD
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percentage was significantly higher in Group III in comparison to the control group (p=0.01) (Figure 3D) with no significant difference between groups II and control
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(p>0.05).
Histology
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Histological evaluation showed that following four weeks of consolidation period, the distraction gaps in the treated groups were bridged with bony union, whereas the control group was partially bridged with tiny trabecular bone (Figure 4). The control group showed less immature bone and more fibrous tissue within the center of the distraction
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gap than the other groups. Group II showed greater and denser immature woven bone and less fibrous tissue than the control. In group III the bone marrow was mature, and the newly formed cancellous and cortical bone were more mature and thicker
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compared to the other groups.
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Collagen orientation index
H&E sections were visualized under polarized light microscopy to compare local tissue organization. Under polarized light, collagen fibers become birefringent and organized lamellar bone is distinguished from unorganized woven bone. Group III collagen fibers were more organized than in the control group and in group II which exhibited haphazard organization (Figure 5). Quantitatively, the collagen orientation index was
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ACCEPTED MANUSCRIPT significantly higher in group III (1.96 ± 0.42) which indicates more lamellar bone in group III than other groups which displayed woven bone (Figure 6).
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Immunohistochemical staining Immunohistochemical staining revealed that application of ESWT increased collagen-1 and osteocalcin (OCN) levels in the distraction gaps of the treated groups, as compared
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to the control group. The representative immunohistochemical staining photographs
showed collagen-1 and OCN positive staining, as indicated by the red/orange staining
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which is localized in both proteins to the extracellular matrix (Figure 7A-F). IHC histomorphometry analysis revealed significantly increased levels of collagen1 and OCN expressions in group III compared to the control (p<0.05) (Figure 7G, H). However, no statistically significant differences were observed between group II and
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DISCUSSION
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control, or between the two treated groups (p>0.05).
In DO, tension-stress is believed to be the key stimulator for recruitment of
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mesenchymal stem cells accompanied by expression of growth factors that increases as long as the distraction is maintained and decreases when the distraction is completed. Although many attempts have been made to shorten the consolidation period during DO, no efficient approach has been reported. ESWT is a novel non-surgical therapeutic method, which generates mechanical stimulation shown to be able to enhance bone regeneration by increasing the differentiation and vascularization.
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ACCEPTED MANUSCRIPT Publications in the field have shown that two weeks following the commencement of the distraction forces, progressive calcification of matrix collagen bundle oriented parallel to the distraction direction is observed. Continuing calcification of the
are deposited in the lamellar phase after 8 weeks [18, 19].
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distraction regenerate eventually leads to closure of the distraction gap and calcium salts
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In the current study on rat mandibles, radiographic examinations showed that the
distraction gap in the control group was radiolucent and did not completely bridge
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following four weeks of consolidation. However, the distraction gap in the treated groups appeared to be markedly radiodense and more mineralized. In addition, the radiopacity of the distracted mandible in group III was greater than that of group II. Radiographic examination showed faster calcification in the ESWT treated groups.
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Also, our histological findings showed accelerated new bone formation and less fibrous tissue in the treated groups compared to the control. The BMD percentage was higher in both treated groups compared to control, however, Group III showed markedly higher
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percentage when compared to the control group.
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The orientation of structures can be represented as a power plot of the fast Fourier transformation (FFT) of an image. Gregory et al., [20] applied the FFT to bone histological images. We used, for the first time to the best of our knowledge, the collagen ordination index obtained from De Vries et al., [21] to evaluate bone maturation following DO in ESWT treated and untreated rats. ESWT application at the consolidation period resulted in significantly higher orientation index compared to the
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ACCEPTED MANUSCRIPT control, indicating that ESWT at the consolidation period may lead to more mature and lamellar bone.
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DO considerably affects cellular gene expression and these effects result in the upregulation of bone specific extracellular matrix proteins. Type I fibrillary collagen is the primary extracellular matrix scaffold of bone [22]. Its heterotrimeric helix is reinforced
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with calcium hydroxyapatite providing infrastructural support and tensile strength to the skeleton. Collagens, the major component of the ECM appear to play a critical role in
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bone formation during distraction by facilitating the formation of early bone spicules extending from the osteotomy edges towards the center of the distraction gap [18]. The high level of type I collagen in group III (Figure 7) is consistent with mature bone demonstrated in the histological findings. Non-collagenous proteins like OCN have an
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important role in regulating ossification and bone remodeling [23]. OCN is a Glacontaining, vitamin K-dependent, calcium-binding protein expressed by well differentiated osteoblasts. Using a rabbit model of mandibular distraction, Meyer et
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al.[24] observed that hyperphysiological levels of strain resulted in a marked reduction in osteocalcin and osteonectin protein expression in the distraction gap compared to
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mandibles exposed to physiologic levels of strain. Moreover, the decrease in osteocalcin and osteonectin expression paralleled a loss of crystal formation suggesting that both proteins have an important role in mineralization during mechanical loading [24]. In an in vitro study, Tamma et al., [25] evaluated the direct effects of ESWT on murine osteoblasts. Results have shown increased expression of collagen type 1, OCN, bone sialoprotein and osteopontin, concluding that ESWT induces increased expression of extracellular matrix [25]. These findings correlate with ours, in which ESWT
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ACCEPTED MANUSCRIPT application at the consolidation period up-regulates the expression of OCN and Collagen type 1.
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Conclusion Our findings suggest that ESWT application following the distraction period enhances bone maturation and mineralization during DO and that these effects are mediated by
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modulating the expression of bone specific extracellular matrix proteins without side
effects. Despite the promising results, further studies are needed to investigate the exact
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mechanism through which ESWT affects DO.
REFERENCES
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LEGENDS:
Figure 1. Intraoperative photograph showing the osteotomy line and the fixed distractor device.
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ACCEPTED MANUSCRIPT Figure 2. Schematic representation of the experimental study design
Figure 3. X-ray radiographs of the distracted mandibles in the control group (A), group
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II (B), and group III (C) following four weeks of consolidation period. Bone mineral density percentage analysis (D). The control group showed radiolucency at the center of the gap which did not completely bridge. The radiopacity of the distracted mandibles in
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the treated groups was greater than in the control. The regenerated bone in group III was barely distinguishable from native bone (C). Group II appeared to be less radiodense
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than Group III. (D) Group III showed significantly greater percentage of bone mineral density compared to the control group ("*" represents p value < 0.05).
Figure 4. Hematoxylin & Eosin sections of the distraction gap. Histological
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photomicrographs of the distracted mandibles in the control group (A), group II (B), and group III (C) at four weeks of consolidation period. The control group showed more fibrous tissue and immature bone at the center of the distraction gap than other groups.
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Group II showed more and denser immature woven bone and less fibrous tissue than the control. In group III the bone marrow was mature, and the newly formed cancellous and
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cortical (arrow) bone were more mature and thicker than other groups. Original magnification x20.
Figure 5. Collagen organization under polarized light microscopy (A-C) and fast Fourier transformation (D-F). The control group (A) showed less-oriented collagen fibers resulting in rounder frequency distribution (D) as opposed to group III with more
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ACCEPTED MANUSCRIPT well-oriented collagen in mature bone (C) providing more elliptic frequency distributions (F). Original magnification x200.
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Figure 6. Orientation index of the collagen fibers in the ESWT-treated groups and the control group using the fast Fourier transformation. In group III, the orientation indexes are significantly higher (1.92 +0.42) compared to the control group (1.43 +0.15) ("*"
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represents p value < 0.05).
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Figure 7. Representative immunohistochemical staining for Collagen1 (A-C) and OCN expressions in the distraction gaps (D-F). Original magnification x100. Histomorphometric evaluation for Collagen1(G) and OCN levels in the distraction gaps (H). Significantly increased levels of Collagen 1 and OCN in group III compared to the
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control ("*" represents p value < 0.05).
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