Abstract
This study evaluated the effect of a modified Hyrax device and platelet-rich fibrin (PRF) on osteogenic periosteal distraction (OPD). Twelve adult male New Zealand white rabbits were separated into two main groups (six in each) according to the duration of the consolidation period (4 or 8 weeks). In each main group, the animals underwent OPD of the left and right sides of the mandible and were divided into four subgroups (three animals per group): device vs. device + PRF, and PRF vs. sham. Radiographic, histological, histomorphometric, and micro-computed tomography (micro-CT) analyses were performed. New bone formation was observed on the lateral and vertical sides of the mandible of all groups. Micro-CT and histomorphometry showed that the device + PRF group presented the highest percentages of bone volume and bone area at 4 weeks (56.67 ± 12.67%, 41.37 ± 7.57%) and at 8 weeks (49.67 ± 8.33%, 55.46 ± 10.67%; significantly higher than the other groups, P < 0.001), followed by the device group at 4 weeks (33.00 ± 1.73%, 33.21 ± 11.00%) and at 8 weeks (30.00 ± 3.00%, 23.25 ± 5.46%). In conclusion, the modified Hyrax device was used successfully for OPD in a rabbit model to gain vertical ridge augmentation, and greater bone maturation was achieved with the addition of PRF.
Introduction
Reconstruction of the atrophic edentulous ridge always requires adequate bone height and width for ideal dental restoration of the edentulous area. Several methods have been used for alveolar ridge augmentation such as bone grafting, guided bone regeneration (GBR), and alveolar distraction osteogenesis (DO) . Bone grafts have some disadvantages such as morbidity of the donor site and surface resorption of the grafted bone, while the GBR technique is of use in a limited area of the alveolar ridge defect . DO is another technique to gain new bone and works by separating one portion of bone and gradually changing its position with respect to the bone direction, allowing for new bone to fill the space between the portion and the overall bone . DO also has some drawbacks, as it is technically complicated and may cause trauma to the patient . Osteogenic periosteal distraction (OPD) comprises a combination of guided bone regeneration, tissue expansion, and DO. It is proposed to produce new bone formation by osteogenic periosteal distraction without corticotomy . Newly formed bone is obtained from mesenchymal stem cells which are under tension and capable of differentiating into osteoblasts . In a previous study, OPD was performed on the lateral surface of the mandible in rabbits, either with or without cortical bone perforation; it was shown that more bone formation was induced in the group with decortication of the mandibular cortex than in the non-decorticated group .
The Hyrax, an orthodontic device, is normally used in children for widening of the upper jaw by separating the mid-palatal suture to treat crowding caused by a narrow arch form . Platelet-rich fibrin (PRF), introduced by Dohan et al. , is a source of autologous growth factors that improves the healing of soft and hard tissues . In a previous study it was shown that PRF enhances bone formation when used in combination with grafting material or when used alone.
It is hypothesized that by gradual periosteal lifting, in which the space can be maintained, osteogenesis would be induced and bone regeneration would be fostered by growth factors from PRF. The aim of this study was to evaluate the effect of a modified Hyrax device and PRF on supraosseous osteogenesis underneath the periosteal pouch in a rabbit mandible model, using micro-computed tomography (micro-CT) and histomorphometric analysis.
Materials and methods
This study was conducted with the approval of the university animal care centre research committee. The estimated sample size for a two-sample comparison of means was computed by using the assumption that the OPD group would gain at least 30 ± 5% of bone and the sham group around 15 ± 5% of bone; α was set at 0.05 and β was set at 0.1. The sample size for each group was three, with an estimated power of 0.96.
Animal preparations
Twelve adult male New Zealand white rabbits with a mean weight of 3.5 kg were used in this study. These rabbits were separated into two main groups of six rabbits each according to the duration of the consolidation period—either 4 or 8 weeks. The rabbits in each group were divided into four subgroups of three animals according to the experiment performed on the left and right mandibles: sham (right) vs. device + PRF (left), and device (right) vs. PRF (left). A device was used only on one side of the mandible in each animal and not used on both sides in order to avoid interference between devices. The device was activated to achieve 7 mm distraction, being moved 0.5 mm twice a day for 7 days.
Distractors
Hyrax devices (Leone S.p.A., Firenze, Italy) were modified for use as osteogenic periosteal distractors by welding a micro-titanium plate (Medicon eG, Tuttlingen, Germany) to one end (two rod arms) of the device, while the other end was bent into L-shaped rod arms ( Fig. 1 ). The device was designed to be fixed to the body of the rabbit mandible with the welded micro-titanium plate and microscrews (3 mm in length). The other end (L-shaped rod arms), being 3 cm in total length including the 7-mm length of the bottom part, was designed for placement underneath the periosteum over the alveolar crest, for lifting of the periosteum during device activation ( Fig. 1 ).
PRF preparation
Autologous PRF was prepared according to the PRF protocol developed by Dohan et al. . Ten millilitres of autologous whole blood was collected from the central ear artery of the rabbit before anaesthesia and placed into a 10-ml glass tube without anticoagulant. This was centrifuged immediately at 3000 rpm for 10 min using a table centrifuge (EBA20; Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany), separating the sample into three layers: a red blood cell layer at the bottom, PRF in the middle, and platelet-poor plasma at the top of the tube ( Fig. 2 ). The PRF in the middle part was removed from the tube using straight tissue forceps and cut from the red blood cells. This was then used underneath the periosteum in the PRF subgroup and the device + PRF subgroup.
Surgical procedure
General anaesthesia was induced by intramuscular injection of ketamine HCl 25 mg/kg (Calypsol 50 mg/ml; Gedeon Richter Ltd, Budapest, Hungary) and diazepam 5 mg/kg (10 mg/2 ml/ampoule), approximately 30 min prior to the administration of intravenous thiopental sodium l5 mg/kg (Anesthal 1 g/vial; Jagsonpal Pharmaceuticals Ltd, Haryana, India). Thiopental sodium was titrated at a rate of 2 mg/kg every 15 min with a maximum dose of 30 mg/kg to maintain unconsciousness.
The surgical field in the area of the ramus and body of the mandible was shaved and disinfected with 10% povidone–iodine (Polidine solution 10%, New Life Pharma Co., Ltd, Bangkok, Thailand). Local anaesthesia infiltration of 1% lidocaine HCl with 1:100,000 adrenalin 1.8 ml (Drocanil-A 1%; M&H Manufacturing Co., Ltd, Samutprakarn, Thailand) was administered to the surgical site for hemostasis and as an adjunct to control pain during and post surgery. A 5-cm sub-mandibular incision was made through the skin, the muscle, and the periosteum. A periosteal flap was then raised to expose the lateral aspect of the ramus. In all groups, defects were created at a site free of dentition, mesial to the mandibular molar tooth (7 mm in length and 2 mm in depth measured by periodontal probe); the mental nerve was avoided by identifying the nerve location. The reason for creating the defect in the area free of dentition was to gain enough room for vertical distraction so as not to interfere with the maxillary teeth during mastication. Reference points were established by creating a small hollow at the anterior and posterior corners of the defect and filling these with gutta percha ( Fig. 3 ). The device was fixed to the mandible with 3-mm titanium microscrews. The active arms of the device were placed on the defect underneath the periosteum.
Activation of the device was tested prior to wound closure. The periosteum, muscle, and skin were repositioned at the lower border of the mandible and sutured layer by layer using Vicryl 4–0. Local wound dressing was performed by applying chloramphenicol antibiotic ointment to the surgical site for 7 days (Cogetine ophthalmic ointment 1%; General Drugs House Co., Ltd, Bangkok, Thailand). Postoperatively, the animals were injected with penicillin G 100,000 IU/kg (Penicillin G 5000,000 units/vial; General Drugs House Co., Ltd, Bangkok, Thailand) and acetaminophen analgesic 200 mg/kg (Acetaphen injection 300 mg/2 ml/ampoule; Nida Pharma Incorporation Co., Ltd, Bangkok, Thailand) in the lateral thigh muscle for 3 days to prevent infection and pain during recovery. The rabbits were fed a standard rabbit diet, given water ad libitum, and kept in separate cages in the animal facility of the university. The animals were monitored in accordance with the guidelines of the institutional ethics committee. The clinical condition, wound condition, physical activity, food and water consumption, and weight of the animals were monitored closely.
In the sham group, the defect was created and used as a control. In the PRF group, the defect created was filled with PRF underneath the periosteum. In the device group, after the defect was created, the device was fixed rigidly on the lateral aspect of the mandible. In the device + PRF group, after the defect was created and the device was fixed, PRF was added under the active arms beneath the periosteum.
In the device and the device + PRF groups, the distraction period started after a latency period of 3 days. Distraction was performed by activating the distractor by 0.5 mm twice a day. A periosteal distraction of 7.0 mm was achieved after a distraction period of 7 days. After a consolidation period of 4 or 8 weeks, the animals were sacrificed with an overdose of 50 mg/ml thiopental sodium 5 ml (Anesthal 1 g/vial) administered intravenously via the marginal ear vein. The mandibles were harvested and fixed in 10% neutral-buffered formalin for 1 week. After decalcification with 10% formic acid, the distraction regions were sectioned and stained with hematoxylin and eosin.
Digital radiography
The mandibular specimens were radiographed using a hand-held portable X-ray device (NOMAD; Aribex Inc., Utah, USA) with digital sensor size 0 attached to a digital sensor holder (XCP-DS; Rinn, Dentsply, IL, USA). The setting for all exposures was 60 kVp, 2.3 mA, and 0.3 s at a distance of 10 cm. The digital image was taken in two views, a lateral–oblique view and a lateral view. The mean optical density (OD) of the defect was calculated and analyzed using Image-Pro Plus 7.0 software (Media Cybernetics Inc., Rockville, MD, USA).
Specimen processing
The specimen of the free alveolus containing the 7-mm defect was divided into two halves (each 3.5 mm in length); one half (distal half) underwent histology and histomorphometric analysis and the other (mesial half) underwent micro-CT analysis.
Micro-CT analysis
A high-resolution micro-CT system (Micro-CT80; Scanco Medical AG, Brüttisellen, Switzerland) was calibrated and the specimens were scanned perpendicular to the cranium vault at 55 kVp, 72 μA, and 4 W in high-resolution mode (18.5 μm 3 /voxel). Scanned data were reconstructed by built-in software.
After determination of the threshold values, the region of interest (ROI) was traced to specify the newly formed bone (BV). The percentage of new bone volume was calculated as the percentage of radio-opaque voxels in a bone threshold range divided by the total bone volume (TV): percentage of new bone volume = (new bone volume/total bone volume) x 100.
Histology processing
The distal half of the mandibular specimen was decalcified in 10% formic acid, trimmed, cut transversally, and then embedded in paraffin. Serial 5-μm sections were cut and stained with hematoxylin and eosin, and used for histomorphometric analysis.
Histomorphometric analysis
All slides were scanned via an Aperio ScanScope XT (Aperio ePathology Solutions, CA, USA) at 40× magnification. Digital histology images were captured with computer software (Aperio ImageScope 9.0; Aperio ePathology Solutions). The new bone formed was differentiated from the adjacent bone by identifying the defect line that was created during surgery. The area of newly formed bone (NB) was outlined and calculated as the percentage of newly formed bone area to the total defect area using Image-Pro Plus 7.0 (Media Cybernetics): percentage of new bone area = (new bone area/total defect area) x 100.
Statistical analysis
The statistical analysis was performed using SPSS version 15 statistical software (SPSS Inc., Chicago, IL, USA). Data were evaluated by one-way analysis of variance (ANOVA) and the post hoc Tukey HSD test. Significance was set at P < 0.05. Differences between the two times points for each group were analyzed using the paired t -test.
Materials and methods
This study was conducted with the approval of the university animal care centre research committee. The estimated sample size for a two-sample comparison of means was computed by using the assumption that the OPD group would gain at least 30 ± 5% of bone and the sham group around 15 ± 5% of bone; α was set at 0.05 and β was set at 0.1. The sample size for each group was three, with an estimated power of 0.96.
Animal preparations
Twelve adult male New Zealand white rabbits with a mean weight of 3.5 kg were used in this study. These rabbits were separated into two main groups of six rabbits each according to the duration of the consolidation period—either 4 or 8 weeks. The rabbits in each group were divided into four subgroups of three animals according to the experiment performed on the left and right mandibles: sham (right) vs. device + PRF (left), and device (right) vs. PRF (left). A device was used only on one side of the mandible in each animal and not used on both sides in order to avoid interference between devices. The device was activated to achieve 7 mm distraction, being moved 0.5 mm twice a day for 7 days.
Distractors
Hyrax devices (Leone S.p.A., Firenze, Italy) were modified for use as osteogenic periosteal distractors by welding a micro-titanium plate (Medicon eG, Tuttlingen, Germany) to one end (two rod arms) of the device, while the other end was bent into L-shaped rod arms ( Fig. 1 ). The device was designed to be fixed to the body of the rabbit mandible with the welded micro-titanium plate and microscrews (3 mm in length). The other end (L-shaped rod arms), being 3 cm in total length including the 7-mm length of the bottom part, was designed for placement underneath the periosteum over the alveolar crest, for lifting of the periosteum during device activation ( Fig. 1 ).
PRF preparation
Autologous PRF was prepared according to the PRF protocol developed by Dohan et al. . Ten millilitres of autologous whole blood was collected from the central ear artery of the rabbit before anaesthesia and placed into a 10-ml glass tube without anticoagulant. This was centrifuged immediately at 3000 rpm for 10 min using a table centrifuge (EBA20; Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany), separating the sample into three layers: a red blood cell layer at the bottom, PRF in the middle, and platelet-poor plasma at the top of the tube ( Fig. 2 ). The PRF in the middle part was removed from the tube using straight tissue forceps and cut from the red blood cells. This was then used underneath the periosteum in the PRF subgroup and the device + PRF subgroup.
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