ORIGINAL ARTICLE
Effects of the Hyperbaric Oxygen on De Novo Bone Formation During Periosteal Distraction Berkay Tolga Suer, DDS, PhD,* Kerim Ortakoglu, DDS, PhD,† Yilmaz Gunaydin, DDS, PhD,‡ Metin Sencimen, DDS, PhD,§ Ibrahim Mutlu, DDS, PhD,∥ Necdet Dogan, DDS, PhD,§ and Ayper Kaya, MD¶ Objective: The purpose of this study was to investigate the effect of hyperbaric oxygen (HBO) therapy on de novo bone formation during periosteal distraction (PD). Materials and Methods: Periosteal distraction was performed in 24 mature male New Zealand rabbits using a custom-designed device placed on the lateral surface of the mandibular corpus. Twelve rabbits (group H) were given adjunctive HBO treatment, whereas 12 rabbits (group N) were kept in a normal environment (normobaric oxygen). After a 7-day latency period, the same distraction protocol was applied to both groups. However, the rabbits in group H were treated with pure oxygen at 2.4 atm absolute for 25 times. Both groups were further divided into 2 subgroups and killed after consolidation periods of 4 and 8 weeks. Photodensitometric and histologic analyses were performed to evaluate the newly formed bone. Results: There was no significant difference between the 4-week consolidated HBO group and the 8-week consolidated normobaric oxygen subgroup (P = 0.229). Moreover, there was better bone formation in the 8-week HBO group than in the 8-week control group. Conclusion: The results of this study indicate that PD with HBO could be used to increase the quality and the quantity of the bone newly formed by PD. Key Words: Hyperbaric oxygen treatment, periosteal distraction, densitometry, osteogenesis, angiogenesis (J Craniofac Surg 2014;25: 1740–1745)
B
ony deficiencies of the alveolar ridge can be surgically treated with augmentation procedures, guided bone regeneration, and vertically guided bone regeneration.1–3 Autogenous bone grafts, allogenic bone grafts, and various types of bone substitute materials are used to reconstruct such alveolar ridge deficiencies.4 The use of autogenous bone is still regarded as the criterion standard because
From the *Department of Oral and Maxillofacial Surgery, Gülhane Military Medical Academy (GMMA), Haydarpasa Teaching Hospital; †Service of Dental Medicine, Medicana Hospital, Istanbul; §Department of Oral and Maxillofacial Surgery, Gülhane Military Medical Academy (GMMA), Etlik; ∥Department of Dental Medicine, Tatvan Military Hospital, Tatvan; ‡Private Practice, Ankara; and ¶Kasimpasa Military Hospital, Kasimpasa, Istanbul, Turkey. Received November 13, 2013. Accepted for publication April 7, 2014. Address correspondence and reprint requests to Berkay Tolga Suer, DDS, PhD, Department of Oral and Maxillofacial Surgery, Gülhane Military Medical Academy (GMMA), Haydarpasa Teaching Hospital, Uskudar, 34668, Istanbul, Turkey; E-mail: [email protected] The authors report no conflicts of interest. Copyright © 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000000996
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of its osteogenic potential.5 However, such grafts have certain limitations such as limited quantity of donor bone, increased morbidity, requirement for a second surgical site, and inability to do simultaneous soft tissue augmentation.5,6 The use of allogenic and synthetic materials also has inherent disadvantages such as risk for infection, immunologic issues, and poorer structural integrity. To overcome these disadvantages and produce new bone, distraction osteogenesis (DO) techniques have become a widely accepted treatment for reconstructing bone defects in the maxillofacial region7 and treating vertical deficiencies in the alveolar bone.8–11 Creating a gap between the bony surface and the periosteum by gradually lifting the periosteum has recently been suggested as a way to create osteogenesis.12,13 Periosteal distraction osteogenesis (PDO), unlike DO, does not involve an osteotomy and thus is less traumatic. A custommade device lifts the periosteum gradually to induce supraosseous neogenesis.12 After the first description of PDO, many studies have been done to investigate the efficacy of this new technique.1,2,4,13–17 The amount of new bone obtained by controlled and guided elevation of the periosteum seems to be quick and massive.2 However, although application of this technique results in de novo bone formation, the quality and the maturity of the newly formed bone are less than ideal compared with that produced by DO.13 Therefore, methods for increasing the quality and the quantity of the de novo bone through PDO, such as adding mesenchymal stem cells, making perforations (decortication) in the cortical bone, or trying different latency periods, have been investigated.4,14,18 Hyperbaric oxygen (HBO) treatment increases the dissolved oxygen in the blood and results in high partial pressure of oxygen (PaO2) in the tissues. This promotes collagen formation, adenosine triphosphate synthesis, angiogenesis, and osteoblastic and osteoclastic activity.19–24 Recently, we have shown that 25 treatments of 90 minutes of HBO at 2.4 atmospheric absolute pressure (ATA) could enhance the quality and the quantity of bone regeneration gained by DO when compared with normobaric oxygen (NBO) controls using the rabbit model.20 This suggests that HBO has the potential to augment bony healing even under normal bone conditions.20 To the best of our knowledge, the effect of HBO on de novo bone gained by PD has not been studied. Therefore, the current investigation studied de novo bone formation after PD plus adjunctive HBO treatment in a rabbit mandible model.
MATERIALS AND METHODS Twenty-four adult New Zealand white male rabbits (Oryctolagus cuniculus) with a mean (SD) weight of 3.7 (0.550) kg were used as the animal model. The protocol for this study was approved by the Committee on Ethics of Experimental Animals of the Gulhane Military Medical Academy (GMMA), Ankara, Turkey, and was carried out in the academy's Experimental Animals Research and Development Center. During the study, animals were housed, fed, and handled according to international standards of animal welfare in this center with the help of a licensed veterinarian. The animals were
The Journal of Craniofacial Surgery • Volume 25, Number 5, September 2014
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery • Volume 25, Number 5, September 2014
allowed free access to food and water and cared for under the guidelines and personnel of this institution. All 24 rabbits received a PD device fixed to the lateral surface of the mandible. The animals were then divided into 2 groups of 12 rabbits each. The first group was given HBO (group H). The other 12 rabbits were kept in an NBO environment (group N). Both groups were then further divided into 2 subgroups of 6 rabbits each according to their consolidation periods that begin after the end of PD. Six animals in the HBO treatment group were killed at the end of the 4-week consolidation period (group H1), and the other 6 were killed at the end of an 8-week consolidation period (group H2). Similarly, the animals in the NBO control group were killed at the end of 4 weeks (group N1) and 8 weeks (group N2) (Table 1 parts A and B).
PD Device The custom-made periosteal distractor was designed in our unit and had been used effectively in various animal studies previously.13,18,20,21 It is made of stainless steel and consists of a reverse U–shaped body with 2 legs that can be rigidly fixed to the lateral surface of the rabbit's mandible using two 5-mm microscrews (Fig. 1). The device has a central distraction screw that is attached to a mesh plate with curved edges. This design can provide a 2-mm gap, as suggested by Schmidt et al.12 The mesh plate is attached to the distraction screw with a pin that allows free rotational movement. The mesh design of the plate facilitates attachment of the undifferentiated mesenchymal cells from the cambium layer of the periosteum. Rotation of the central screw results in the distraction of the mesh plate and the periosteum away from the bone surface (Fig. 1).
Surgical Protocol All surgical procedures were performed under general anesthesia. The rabbits were first sedated with an intramuscular injection of midazolam (2 mg/kg) and ketamine (40 mg/kg). After sedation, general anesthesia was initiated and maintained via inhalation with 5% to 6% sevoflurane using a custom-made mask. Operation sites were shaved and prepared with a 10% povidoneiodine (Betadine) solution and draped in a sterile surgical fashion to allow for PD device placement. Local anesthesia using 2% lidocaine with epinephrine (1:200,000) was infiltrated in the lateral ramus and TABLE 1. Experimental Protocol for HBO Test Groups (Part A) and Experimental Protocols for NBO Control Groups (Part B).
Hyperbaric Oxygen on Distraction
FIGURE 1. A, Diagram of the custom-designed PD device. B, Picture of the custom-designed PD device. Activation of the center rod distracted the subperiosteal mesh from the lateral ramus of the rabbit mandible.
submandibular regions. The lateral surface of the mandible was exposed through a linear submandibular incision approximately 2 cm long. After the skin incision, the subcutaneous tissue and the muscle layers were dissected down to the periosteum, and the periosteum at the level of the inferior border of the mandible was incised using a No. 15 blade. Having exposed the mandibular body, the distraction device was rigidly mounted perpendicular to the lateral surface of the mandible with 2 microscrews 1.3 5 mm in diameter (Fig. 2A). In both groups, while protecting the periosteum, the buccal cortex corresponding to the area covered with the distractor’s mesh plate was perforated 6 to 9 times with a round bur 0.5 mm in diameter to expose the trabecular bone. These perforations acted as an active source for osteoblast progenitor cells during active distraction, as described in earlier studies.14,17 The distraction devices were activated and deactivated to ensure that they were working properly. The surgical sites were then copiously irrigated with saline solution (0.9% NaCl). One small vertical incision penetrating all tissue layers was made to accommodate the activation screw of the device. Then, the periosteum was sutured back in place, covering the whole mesh. The subcutaneous tissues were closed in layers using 4-0 Vicryl sutures (Ethicon Inc, Somerville, NJ), and the skin was closed with 3-0 silk sutures (Fig. 2B). Immediately after the operation, the rabbits received Penicillin G (200.00 IU/mg, 0.04–0.06 mg/kg) intramuscularly, which was continued until postoperative day 3. During the study, the animals were kept in separate cages, and they were fed with commercial dry rabbit food and lettuce ad libitum.
HBO Treatment Protocol Two days after completion of the PD device placement, the animals in the HBO treatment group (groups H1 and H2) were placed in a pressure chamber (inner diameter of 80 cm, inner length of 220 cm) (ETC, Monoplace Hyperbaric Chamber, Southampton, PA) at the GMMA, Department of Undersea and Hyperbaric Medicine, and subjected to pure oxygen at 2.4 ATA for 90 minutes. During this period, the first 10 minutes was used for successive compression up to 2.4 ATA; the pressure was kept constant for 70 minutes, and decompression was continued for 10 minutes. The chamber temperature was
FIGURE 2. A, Intraoperative picture showing submandibular dissection and secure fixation of the custom-designed PD device to the lateral aspect of the mandibular body and showing perforations on the mesh. The mesh was lowered to the bone before the closure of the surgical wound. B, Photograph showing complete wound closure around the PD device on the lateral aspect of the mandible.
© 2014 Mutaz B. Habal, MD
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mineral density. Measurement of the pixel values of the PD area and the aluminum wedge was obtained. The amount of mineral density in the PD area in each radiograph was modified by the difference in the equivalent aluminum thickness (in millimeters), and the calculations were statistically evaluated (Fig. 3).
Specimen Preparation and Histologic Evaluation The animals were killed by an intravenous overdose of pentobarbital sodium 4 or 8 weeks after the completion of PD. After killing the animals, the PD areas, including peripheral soft tissues and PD devices, were carefully removed. Specimens were fixed in 10% neutral buffered formalin at room temperature for 6 days. Then, the PD device and the surrounding soft tissue were carefully removed. The specimens were decalcified in 10% formic acid, embedded in paraffin, and subsequently serially cut into 5-μm sections. Hematoxylin and eosin staining was performed on serial sections for morphologic evaluation of new bone formation in the PD gap under light microscopy. A board-certified pathologist in the Department of Pathology, GMMA, who was not a part of the study team, evaluated all slides stained with hematoxylin and eosin (Figs. 4 and 5). FIGURE 3. An occlusal radiograph of one specimen after the consolidation period. Note (white arrow) new bone formation in the gap area between the mesh and the lateral aspect of the ramus. This area was used for photodensitometry.
kept at +22°C by a water-cooling system. Produced CO2 was eliminated by constant flow of O2 with a flow rate of 0.9 L/min. The HBO treatment was given once daily for 25 days (Table 1 part A).
Distraction Protocol On the third day after the surgery, standardized occlusal radiographs were taken to determine the beginning position of the device and the cortical bone. After a 7-day latency period to allow for periosteal healing in both groups, distraction commenced at a rate of 0.25 mm twice a day. The activation screw was cleaned with 10% povidone-iodine before turning to avoid infection. One 180-degree turn of the activation screw yielded 0.25 mm of mesh elevation and lengthened the distraction gap by 0.25 mm. After 6 days, a periosteal expansion of 3 mm was obtained. Having started with a 2-mm gap at the time of placement because of the design of the device, 5 mm of periosteum distraction was obtained at the end of the distraction period. Before the animals were killed, standardized occlusal radiographs were taken. The rabbits were then killed after a consolidation period of 4 or 8 weeks, and the mandibles were surgically retrieved (Table 1 parts A and B).
Statistical Methods All the quantitative data were analyzed statistically with the Statistical Package for the Social Sciences (SPSS) software version 10.5 (SPSS, Inc, Chicago, IL). Statistical analyses were performed using the SPSS software version 10.5 (SPSS, Inc). Means and SDs were given in descriptive statistics. The statistical significance of differences between the groups and the consolidation periods was evaluated using the Mann-Whitney test. P values less than 0.05 (P < 0.05) were accepted as statistically significant.
RESULTS Clinical Evaluation All animals survived the surgery, the active PD period, and the HBO treatment. They resumed normal dietary habits during the
Standardization of Occlusal Radiographs We used photodensitometry analysis to measure bone density in the distraction gap. To standardize the measurements, we used the same standardization method described earlier.20 Occlusal radiographic images were obtained using Trophy Trex, 70 kilovolt (peak), 8 mA (Croissy Beaburg/France), x-ray tube, Novelix type 2.5 aluminum total filtration, with Ultraspeed D size 4 (Eastman Kodak Company, Rochester, NY) occlusal films. The exposure time (0.80 s) was kept the same for all animals. To achieve calibration and to minimize variations in the density of the radiographs, during exposure, all radiographs were taken with an aluminum wedge (step-wedge technique) of a known and stable thickness (equivalent aluminum thickness in millimeters). The aluminum step wedge was directly placed on the occlusal radiograph for each exposure. The radiographed aluminum step wedge comprised 9 steps (each 2 mm in thickness). All radiographs were developed in an automatic processing machine (Dent-X Model 410, Stamfort, CT). Unexposed radiographs were used to control the quality of the developing procedure. Densitometric analysis of the radiographs was done with a transmission densitometer (DT 1105 Ryparry Limited, Chatham, Kent, England) to calculate
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FIGURE 4. Macroscopic photomicrograph (2.5) of experimental 4-week consolidated (A) and 8-week consolidated (B) rabbit mandible specimens. The blue arrow shows newly formed cortical bone, and the black arrow shows the PD gap area. In high-power photomicrograph of the PD area (40), newly formed mature bone (marked by the red arrow), adipose tissue (marked by the green arrow), and neoangiogenesis in the PD gap area are observed. In the 8-week consolidated group, there were more lamellar bone and more neoangiogenesis but less adipose tissue observed compared with the 4-week consolidated groups. Compared with the control groups and the 4-week consolidated experimental group, more mature bone tissue was noted in the 8-week experimental group (group H2).
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The Journal of Craniofacial Surgery • Volume 25, Number 5, September 2014
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induced new cortex were few. Interstitial tissue between lamellar bony isles was filled with abundant fat cells, resembling normal mandibular marrow (Fig. 5A).
Group N2: 8-Week Consolidated Control Group Newly formed bone tissue was present in the PD gap area. A thin zone of cortical bone was observed under the entire mesh. There were more areas of lamellar bone with some interconnections between the PD-induced newly formed cortex and the lateral surface of the mandible in this group than in group N1. There was also an increase in the width of the de novo bone formation extending to the mesh. Newly formed capillary tissue was observed between the bony isles in the PD gap area. There were also fat cells present in the PD gap area, resembling normal mandibular marrow (Fig. 5B).
Group H1: 4-Week Consolidated Experimental Group FIGURE 5. Macroscopic photomicrograph (2.5) of the control 4-week consolidated (A) and 8-week consolidated (B) rabbit mandible specimens. The blue arrow shows newly formed cortical bone, and the black arrow shows the PD gap area. In high-power photomicrograph of the PD area (40), newly formed mature bone (marked by the red arrow), adipose tissue (marked by the green arrow), and neoangiogenesis in the PD distracted area are observed. In the 8-week consolidated group, there were more lamellar bone and more neoangiogenesis but less adipose tissue observed compared with the 4-week consolidated groups.
first 24 hours after the surgery. All of the devices remained rigidly fixed to the lateral surface of the mandible for the entire experimental period. During the time of the study, all animals remained healthy, and no weight loss, infection, or breakdown of the soft tissues was observed.
Radiologic Evaluation Radiologic examination of the PD gap area showed different amounts of newly formed bone in the HBO and NBO groups. The amount of the de novo bone formed in the area between the lateral bony surface of the mandible and the mesh was more in the group H experimental animals than in the group N control animals. To determine the effects of the HBO treatment on newly formed bone by PD, we measured the optical mineral density of the PD gap areas. The mean optical densities of de novo bone in the PD areas on the occlusal radiographs based on the equivalent aluminum thickness in millimeters are given in Table 2. Comparison of the mean optical density of the de novo bone areas between the subgroups within each group showed that the values in group H2 were higher than in group H1 (P < 0.004), but there was no statistically significant difference in the NBO control groups. Densitometric analysis of the occlusal radiographs also showed that there was no statistically significant difference between group H1 (4-week consolidated subgroup) and group N1 (4-week consolidated subgroup) or between group H1 and group N2 (8-week consolidated subgroup of the control group). However, radiographic densitometric measurements showed that all results in group H2 (8-week consolidated subgroup of the experimental group) were greater than in group N1 and N2, and these differences were statistically significant (P = 0.003, P = 0.004) (Table 2).
Gross Appearance and Histologic Evaluation Group N1: 4-Week Consolidated Control Group Newly formed bone tissue was seen in the PD gap area. A thin zone of newly formed cortical bone tissue was present just under the mesh and the periosteum, parallel to the mesh. In the PD gap, there were lamellar bony isles and new capillary formation. However, mature lamellar bone between the mandibular lateral cortex and PD-
De novo bone formation was observed in the PD gap area. There was also a thin zone of newly formed cortical bone just underneath the mesh. There was more new bone tissue between the mandibular lateral cortex and the PD-induced thin cortex in group H1 than in group N1. The bone in the PD gap area was in the form of small isles with a few interconnections, similar to that of group N2. The width of the newly formed bone under the mesh was increased compared with that of group N1 but somewhat similar to that of group N2. There was adipose tissue between newly formed thin cortex and the lateral cortex of the mandible, resembling normal mandibular marrow (Fig. 4A).
Group H2: 8-Week Consolidated Experimental Group There was abundant new bone formation present at the PD gap area. The newly formed cortical bone under the mesh was thicker compared with both NBO groups. In addition, there were more and longer isles of lamellar bone between the mandibular lateral cortex and the PD-induced new cortex under the mesh. Most of those isles had interconnections and were distributed homogenously along the distraction space. The lamellar bone mainly consisted of thick trabeculae. Newly formed capillaries and fat tissue also filled the interstitial tissue in the PD gap. The width of the regenerated bone in the PD gap was thicker in this group than in the rest of the groups (Fig. 4B).
DISCUSSION The cambium cell layer of the periosteum plays a major role in the formation of bone.22 Elevating the periosteum from the underlying bone yields a mechanical tension stimulus on the periosteum that triggers mesenchymal cells to differentiate into osteoblasts, resulting in TABLE 2. Descriptive Statistics of the Photodensitometry of Each Measured Area (Equivalent Aluminum Thickness in Millimeters) on Occlusal Radiographs and Comparison Between Subgroups Within Each and Between Groups Groups H1 H2 N1 N2
N1
N2
H1
H2
Mean (SD) υ P υ P υ P υ P 1.498 (0.164) 7.5 0.092 10.500 0.229 NA NA 0.000 0.004 2.970 (0.379) 0.000 0.003 0.000 0.004 0.000 0.004 NA NA 1.038 (0.540) NA NA 7.000 0.078 7.5 0.092 0.000 0.003 1.611 (0.180) 7.000 0.078 NA NA 10.500 0.229 0.000 0.004
The differences with P < 0.05 between subgroups were significant and are shown in bold. The HBO treatment subgroups were H1, HBO treatment group with PD killed at the end of the 4-week consolidation, and H2, HBO treatment group with PD killed at the end of the 8-week consolidation. The NBO control subgroups were N1, NBO control group with PD only killed at the end of the 4-week consolidation, and N2, NBO control group with PD only killed at the end of the 8-week consolidation. NA, not applicable.
© 2014 Mutaz B. Habal, MD
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subperiosteal bone formation.2,15,16,23 Dynamic PD is a relatively new technique, and its potential for producing new bone was first reported by Schmidt et al.12 Periosteal distraction without application of any chemical compounds, cells, or corticotomy has been reported to result in the generation of new bone tissue.1,2,12–14,23,24 However, some authors have reported that the degree of mineralization or the quality of the bone regenerate was not similar to the bone gained by DO and that the PD gap is abundantly filled with adipose tissue.12,13,18,21 On the basis of these studies, the effects of PD and improvements in the technique and the design of the devices call for more investigation to gain enhanced bone formation. On the basis of our previous promising findings with HBO treatment on DO,20 in this study, we tested whether HBO treatment has an effect on the amount of new bone formed through the PD procedure. We demonstrated that PD led to the formation of new bone between the perforated elevation mesh and the lateral cortex of the mandible in both groups. This result is in agreement with the results of studies published in the literature.1,2,12–15,17,18 However, we also showed that the administration of HBO during PD increased the maturation and the quantity of the newly formed bone compared with the control group that received only PD. This also verified the studies showing positive effects of HBO on new bone formation in various other experiments.19,20,25–27 The effect of HBO treatment on bone and soft tissues is well known.28,29 It increases the PaO2 in the blood and in the tissues and has been shown to increase capillary ingrowth and collagen synthesis,30 neovascularization, and osteogenesis.31,32 A considerable number of studies have shown enhanced osteogenic activity as a result of HBO treatment. An earlier union of autologous bone grafts in a rabbit model19 and improved bone formation around titanium implants were observed in vivo after exposure to HBO treatment,33 and recently, Pedersen et al25 showed that HBO treatment enhanced vascularization and bone formation in rat calvarial defects. Similar to their results, this study showed that HBO could make a positive difference in the quality and the quantity of the new bone gained by periosteum distraction. To the best of our knowledge, this is the first study combining PD and HBO treatment. Treatment protocols for HBO vary in the literature, and the most widely used and accepted protocol in animal and in vitro studies involves the delivery of 100% oxygen at 1.5 to 3.0 ATA for 60 to 90 minutes daily.19,26,28,34 As to the HBO treatment interval, it has been shown that once a day seems to accelerate bone repair and vessel ingrowth when compared with HBO treatment given twice a day.34 Our rabbit model and PD device design,13,18,21 as well as HBO treatment protocol,20 were all previously studied in our unit and reported in the literature and found reliable. We decided to use the same HBO treatment in which the HBO was given at 2.4 ATA for 90 minutes for 25 days once daily. Densitometric analysis showed no statistically significant difference between groups H1 and N2. This suggested that HBO treatment increases osteogenesis in PD and may result in a decrease in the consolidation period. This finding was in accordance with the results of an earlier study that evaluated the effect of HBO treatment in DO.20 These results also verify the enhanced bony mineralization by HBO.25 The density values of the de novo formed bone in the PD gap were significantly higher in group H2 than H1 (P = 0.004). Although densitometric values were higher in group N2 compared with N1, the difference was not statistically significant (P = 0.078). In this study, PD was performed in the range of widely used protocols for DO or PD in experimental animal studies,1,2,12–15,17,18,20,21 which usually allows a latency period of 5 to 10 days and distraction rate of 0.25 to 1.0 mm/d. In a previous study, we tested the DO protocol of 7 days of latency and 0.25-mm twice-a-day activation of the distractor in a rabbit model. Having obtained good results with that protocol, we adopted the same distraction protocol for the current study.
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Our main purpose in this experiment was to evaluate whether HBO was beneficial on the formation of new bone by PD. Therefore, 4 and 8 weeks were chosen as consolidation periods for this study. Furthermore, it should also be noted that observation of the maturation of de novo bone was beyond the scope of this study. The preferred consolidation periods in our study are also in accordance with the previous experimental studies on PDO in rabbits.12–14,17,18,20,21 In a comprehensive review by Swennen et al,35 it has been reported that varying consolidation periods ranging between 1.5 and 10 weeks were used in the several experimental DO studies in rabbits. In clinical studies, similarly, there are no rigidly specified consolidation parameters for DO, and significant variations exist between different craniofacial groups. As reported in another extensive review by Swennen et al,36 the consolidation period for maxillary and mandibular DO procedures in humans may vary between 2 weeks and 4 to 6 months with regard to a wide variety of clinical indications. The diversity in consolidation periods between clinical and experimental studies can be attributed to the different bone remodeling period in humans and rabbits. According to previous studies in the literature,10,13,32 with regard to an ideal consolidation period in DO, our findings seem to verify that a longer consolidation period can result in more dense bony structures versus a shorter consolidation period. The results of the densitometric analysis showed that there was a statistically significant difference between groups H2 and N1, groups H2 and N2, and groups H2 and H1 (P = 0.003, P = 0.004, and P = 0.004, respectively). According to these results, it can be concluded that the positive effects of HBO treatment are greater with longer consolidation periods. This finding is in accord with the results of our previous DO study.20 The use of an aluminum step wedge allowed us to transform the reading of light transmission in the radiographs into an equivalent thickness of aluminum.20 Therefore, it can be concluded that all occlusal radiographs were evaluated under the same conditions. The densitometric analysis in this study has shown that HBO treatment significantly increased the new bone density in the 8-week consolidated experimental group and also increased the new bone density in the 4-week consolidated group. Sato et al4 have argued that the difference in the quality of maturation of the new bone formed by PD compared with DO may be explained by several factors: the tissue may lack stimulatory stress, may lack a sufficient number of osteogenic cells, or may have an insufficient vascular supply. Recently, Altug et al18 compared different latency periods along with different consolidation periods in PD, and they reported that the newly formed bone by PD was mostly filled with fatty tissue, and they claimed that lack of bone marrow cells might play a role in the occurrence of fatty tissue. Sencimen et al13 also reported an abundance of adipose tissue and insufficient mature bone in the PD gap area, and therefore, they concluded that this newly formed bone is not suitable for occlusal forces, and it would be impossible to insert an endosteal implant into the area. They proposed that decortication of the bone surface to bring endosteal cells into the distraction area may increase the maturity of the woven bone. Lately, Oda et al14 also investigated the effect of using decorticating holes in the PD protocol for improving bone regeneration in a rabbit model. They suggested that the cortical bone might be a hindrance to bone formation, without access to the bone marrow, and they postulated that decorticating holes can be effective in improving the new bone regenerate in PD. On the basis of these studies, we used decortication holes to benefit from the undifferentiated mesenchymal cells and the osteoblastic activity originating from the endosteum to gain new bone. In this current study, more de novo bone tissue formation and more mature trabecular bony structure were observed in groups H2 and N2. These findings are in agreement with the reports that a longer © 2014 Mutaz B. Habal, MD
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consolidation period in PD or DO procedures results in the formation of more mature and mineralized bone.13,20,32 Nowadays, HBO therapy can be easily performed in singleperson chambers or chambers that can hold more than a dozen people at a time. However, HBO facilities are currently limited and treatment is expensive; hence, the clinical application is still limited to major craniofacial centers. In conclusion, we have demonstrated that HBO positively affects the PD process, leading to a more mature, mineralized bony regenerate. Further studies using higher animal models are anticipated to determine the most suitable HBO protocol for this purpose. ACKNOWLEDGMENT The authors thank Prof Daniel Laskin from the Department of Oral and Maxillofacial Surgery, Virginia Commonwealth University School of Dentistry, for constructive comments on the manuscript and editing assistance.
REFERENCES 1. Estrada JI, Saulacic N, Vazquez L, et al. Periosteal distraction osteogenesis: preliminary experimental evaluation in rabbits and dogs. Br J Oral Maxillofac Surg 2007;45:402–405 2. Kessler P, Bumiller L, Schlegel A, et al. Dynamic periosteal elevation. Br J Oral Maxillofac Surg 2007;45:284–287 3. Linde A, Thoren C, Dahlin C, et al. Creation of new bone by an osteopromotive membrane technique: an experimental study in rats. J Oral Maxillofac Surg 1993;51:892–897 4. Sato K, Haruyama N, Shimizu Y, et al. Osteogenesis by gradually expanding the interface between bone surface and periosteum enhanced by bone marrow stem cell administration in rabbits. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:32–40 5. Khojasteh A, Eslaminejad MB, Nazarian H. Mesenchymal stem cells enhance bone regeneration in rat calvarial critical size defects more than platelete-rich plasma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:356–362 6. Simion M, Jovanovic SA, Tinti C, et al. Long-term evaluation of osseointegrated implants inserted at the time or after vertical ridge augmentation. A retrospective study on 123 implants with 1–5 year follow-up. Clin Oral Implants Res 2001;12:35–45 7. Mofid MM, Manson PN, Robertson BC, et al. Craniofacial distraction osteogenesis: a review of 3278 cases. Plast Reconstr Surg 2001;108:1103–1114 8. Ortakoglu K, Suer BT, Ozyigit A, et al. Vertical distraction osteogenesis of fibula transplant for mandibular reconstruction: a case report. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:e8–e11 9. Jensen OT, Cockrell R, Kuhike L, et al. Anterior maxillary alveolar distraction osteogenesis: a prospective 5-year clinical study. Int J Oral Maxillofac Implants 2002;17:52–68 10. Rachmiel A, Srouji S, Peled M. Alveolar ridge augmentation by distraction osteogenesis. Int J Oral Maxillofac Surg 2001;30:510–517 11. Uckan S, Veziroglu F, Dayangac E. Alveolar distraction osteogenesis versus autogenous onlay bone grafting for alveolar ridge augmentation: technique, complications, and implant survival rates. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:511–515 12. Schmidt BL, Kung L, Jones C, et al. Induced osteogenesis by periosteal distraction. J Oral Maxillofac Surg 2002;60:1170–1175 13. Sencimen M, Aydintug YS, Ortakoglu K, et al. Histomorphometrical analysis of new bone obtained by distraction osteogenesis and osteogenesis by periosteal distraction in rabbits. Int J Oral Maxillofac Surg 2007;36:235–242 14. Oda T, Kinoshita K, Ueda M. Effects of cortical bone perforation on periosteal distraction: an experimental study in the rabbit mandible. J Oral Maxillofac Surg 2009;67:1478–1485 15. Lethaus B, Tudor C, Bumiller L, et al. Guided bone regeneration: dynamic procedures versus static shielding in an animal model. J Biomed Mater Res B Appl Biomater 2010;95:126–130
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16. Tudor C, Bumiller L, Birkholz T, et al. Static and dynamic periosteal elevation: a pilot study in a pig model. Int J Oral Maxillofac Surg 2010;39:897–903 17. Zakaria O, Madi M, Kasugai S. Induced osteogenesis using a new periosteal distractor. J Oral Maxillofac Surg 2012;70:e225–e234 18. Altug HA, Aydintug YS, Sencimen M, et al. Histomorphometric analysis of different latency periods effect on new bone obtained by periosteal distraction: an experimental study in the rabbit model. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:539–546 19. Sawai T, Niimi A, Takahashi H, et al. Histologic study of the effect of hyperbaric oxygen therapy on autogenous free bone grafts. J Oral Maxillofac Surg 1996;54:975–981 20. Mutlu I, Aydintug YS, Kaya A, et al. The evaluation of the effects of hyperbaric oxygen therapy on new bone formation obtained by distraction osteogenesis in terms of consolidation periods. Clin Oral Investig 2012;16:1363–1370 21. Bayar GR, Gunaydin Y, Ortakoglu K, et al. Histomorphometric analysis of new bone obtained by osteogenic periosteal distraction in ovariectomized rabbits. Oral Surg Oral Med Oral Pathol Oral Radiol 2012;113:472–479 22. Simon TM, Van Sickle DC, Kunishima DH, et al. Cambium cell stimulation from surgical release of the periosteum. J Orthop Res 2003;21:470–480 23. Kostopoulos L, Karring T. Role of periosteum in the formation of jaw bone. An experiment in the rat. J Clin Periodontol 1995;22:247–254 24. Casap N, Venezia NB, Wilensky A, et al. VEGF facilitates periosteal distraction-induced osteogenesis in rabbits: a micro-computerized tomography study. Tissue Eng Part A 2008;14:247–253 25. Pedersen TO, Xing Z, Finne-Wistrand A, et al. Hyperbaric oxygen stimulates vascularization and bone formation in rat calvarial defects. Int J Oral Maxillofac Surg 2013;42:907–914 26. Wu D, Malda J, Crawford R, et al. Effects of hyperbaric oxygen on proliferation and differentiation of osteoblasts from human alveolar bone. Connect Tissue Res 2007;48:206–213 27. Fok TC, Jan A, Peel SA, et al. Hyperbaric oxygen results in increased vascular endothelial growth factor (VEGF) protein expression in rabbit calvarial critical-sized defects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:417–422 28. Okubo Y, Bessho K, Fujimura K, et al. Preclinical study of recombinant human bone morphogenetic protein-2: application of hyperbaric oxygenation during bone formation under unfavourable condition. Int J Oral Maxillofac Surg 2003;32:313–317 29. Muhonen A, Muhonen J, Lindholm TC, et al. Osteodistraction of a previously irradiated mandible with or without adjunctive hyperbaric oxygenation: an experimental study in rabbits. Int J Oral Maxillofac Surg 2002;31:519–524 30. Yeh WL, Lin SS, Yuan LJ, et al. Effects of hyperbaric oxygen treatment on tendon graft and tendon-bone integration in bone tunnel: biochemical and histological analysis in rabbits. J Orthop Res 2007;25:636–645 31. Nilsson P, Albrektsson T, Granstrom G, et al. The effect of hyperbaric oxygen treatment on bone regeneration: an experimental study using the bone harvest chamber in the rabbit. Int J Oral Maxillofac Implants 1988;3:43–48 32. Dahlin C, Linde A, Rockert H. Stimulation of early bone formation by the combination of an osteopromotive membrane technique and hyperbaric oxygen. Scand J Plast Reconstr Surg Hand Surg 1993;27:103–108 33. Johnsson AA, Sawaii T, Jacobsson M, et al. A histomorphometric study of bone reactions to titanium implants in irradiated bone and the effect of hyperbaric oxygen treatment. Int J Oral Maxillofac Implants 1999;14:699–706 34. Barth E, Sullivan T, Berg E. Animal model for evaluating bone repair with and without adjunctive hyperbaric oxygen therapy (HBO): comparing dose schedules. J Invest Surg 1990;3:387–392 35. Swennen G, Dempf R, Schliephake H. Cranio-facial distraction osteogenesis: a review of the literature. Part II: experimental studies. Int J Oral Maxillofac Surg 2002;31:123–135 36. Swennen G, Schliephake H, Dempf R, et al. Craniofacial distraction osteogenesis: a review of the literature: part 1: clinical studies. Int J Oral Maxillofac Surg 2001;30:89–103
© 2014 Mutaz B. Habal, MD
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Effects of the Hyperbaric Oxygen on De Novo Bone Formation During Periosteal Distraction Berkay Tolga Suer, DDS, PhD,* Kerim Ortakoglu, DDS, PhD,† Yilmaz Gunaydin, DDS, PhD,‡ Metin Sencimen, DDS, PhD,§ Ibrahim Mutlu, DDS, PhD,∥ Necdet Dogan, DDS, PhD,§ and Ayper Kaya, MD¶ Objective: The purpose of this study was to investigate the effect of hyperbaric oxygen (HBO) therapy on de novo bone formation during periosteal distraction (PD). Materials and Methods: Periosteal distraction was performed in 24 mature male New Zealand rabbits using a custom-designed device placed on the lateral surface of the mandibular corpus. Twelve rabbits (group H) were given adjunctive HBO treatment, whereas 12 rabbits (group N) were kept in a normal environment (normobaric oxygen). After a 7-day latency period, the same distraction protocol was applied to both groups. However, the rabbits in group H were treated with pure oxygen at 2.4 atm absolute for 25 times. Both groups were further divided into 2 subgroups and killed after consolidation periods of 4 and 8 weeks. Photodensitometric and histologic analyses were performed to evaluate the newly formed bone. Results: There was no significant difference between the 4-week consolidated HBO group and the 8-week consolidated normobaric oxygen subgroup (P = 0.229). Moreover, there was better bone formation in the 8-week HBO group than in the 8-week control group. Conclusion: The results of this study indicate that PD with HBO could be used to increase the quality and the quantity of the bone newly formed by PD. Key Words: Hyperbaric oxygen treatment, periosteal distraction, densitometry, osteogenesis, angiogenesis (J Craniofac Surg 2014;25: 1740–1745)
B
ony deficiencies of the alveolar ridge can be surgically treated with augmentation procedures, guided bone regeneration, and vertically guided bone regeneration.1–3 Autogenous bone grafts, allogenic bone grafts, and various types of bone substitute materials are used to reconstruct such alveolar ridge deficiencies.4 The use of autogenous bone is still regarded as the criterion standard because
From the *Department of Oral and Maxillofacial Surgery, Gülhane Military Medical Academy (GMMA), Haydarpasa Teaching Hospital; †Service of Dental Medicine, Medicana Hospital, Istanbul; §Department of Oral and Maxillofacial Surgery, Gülhane Military Medical Academy (GMMA), Etlik; ∥Department of Dental Medicine, Tatvan Military Hospital, Tatvan; ‡Private Practice, Ankara; and ¶Kasimpasa Military Hospital, Kasimpasa, Istanbul, Turkey. Received November 13, 2013. Accepted for publication April 7, 2014. Address correspondence and reprint requests to Berkay Tolga Suer, DDS, PhD, Department of Oral and Maxillofacial Surgery, Gülhane Military Medical Academy (GMMA), Haydarpasa Teaching Hospital, Uskudar, 34668, Istanbul, Turkey; E-mail: [email protected] The authors report no conflicts of interest. Copyright © 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000000996
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of its osteogenic potential.5 However, such grafts have certain limitations such as limited quantity of donor bone, increased morbidity, requirement for a second surgical site, and inability to do simultaneous soft tissue augmentation.5,6 The use of allogenic and synthetic materials also has inherent disadvantages such as risk for infection, immunologic issues, and poorer structural integrity. To overcome these disadvantages and produce new bone, distraction osteogenesis (DO) techniques have become a widely accepted treatment for reconstructing bone defects in the maxillofacial region7 and treating vertical deficiencies in the alveolar bone.8–11 Creating a gap between the bony surface and the periosteum by gradually lifting the periosteum has recently been suggested as a way to create osteogenesis.12,13 Periosteal distraction osteogenesis (PDO), unlike DO, does not involve an osteotomy and thus is less traumatic. A custommade device lifts the periosteum gradually to induce supraosseous neogenesis.12 After the first description of PDO, many studies have been done to investigate the efficacy of this new technique.1,2,4,13–17 The amount of new bone obtained by controlled and guided elevation of the periosteum seems to be quick and massive.2 However, although application of this technique results in de novo bone formation, the quality and the maturity of the newly formed bone are less than ideal compared with that produced by DO.13 Therefore, methods for increasing the quality and the quantity of the de novo bone through PDO, such as adding mesenchymal stem cells, making perforations (decortication) in the cortical bone, or trying different latency periods, have been investigated.4,14,18 Hyperbaric oxygen (HBO) treatment increases the dissolved oxygen in the blood and results in high partial pressure of oxygen (PaO2) in the tissues. This promotes collagen formation, adenosine triphosphate synthesis, angiogenesis, and osteoblastic and osteoclastic activity.19–24 Recently, we have shown that 25 treatments of 90 minutes of HBO at 2.4 atmospheric absolute pressure (ATA) could enhance the quality and the quantity of bone regeneration gained by DO when compared with normobaric oxygen (NBO) controls using the rabbit model.20 This suggests that HBO has the potential to augment bony healing even under normal bone conditions.20 To the best of our knowledge, the effect of HBO on de novo bone gained by PD has not been studied. Therefore, the current investigation studied de novo bone formation after PD plus adjunctive HBO treatment in a rabbit mandible model.
MATERIALS AND METHODS Twenty-four adult New Zealand white male rabbits (Oryctolagus cuniculus) with a mean (SD) weight of 3.7 (0.550) kg were used as the animal model. The protocol for this study was approved by the Committee on Ethics of Experimental Animals of the Gulhane Military Medical Academy (GMMA), Ankara, Turkey, and was carried out in the academy's Experimental Animals Research and Development Center. During the study, animals were housed, fed, and handled according to international standards of animal welfare in this center with the help of a licensed veterinarian. The animals were
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Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery • Volume 25, Number 5, September 2014
allowed free access to food and water and cared for under the guidelines and personnel of this institution. All 24 rabbits received a PD device fixed to the lateral surface of the mandible. The animals were then divided into 2 groups of 12 rabbits each. The first group was given HBO (group H). The other 12 rabbits were kept in an NBO environment (group N). Both groups were then further divided into 2 subgroups of 6 rabbits each according to their consolidation periods that begin after the end of PD. Six animals in the HBO treatment group were killed at the end of the 4-week consolidation period (group H1), and the other 6 were killed at the end of an 8-week consolidation period (group H2). Similarly, the animals in the NBO control group were killed at the end of 4 weeks (group N1) and 8 weeks (group N2) (Table 1 parts A and B).
PD Device The custom-made periosteal distractor was designed in our unit and had been used effectively in various animal studies previously.13,18,20,21 It is made of stainless steel and consists of a reverse U–shaped body with 2 legs that can be rigidly fixed to the lateral surface of the rabbit's mandible using two 5-mm microscrews (Fig. 1). The device has a central distraction screw that is attached to a mesh plate with curved edges. This design can provide a 2-mm gap, as suggested by Schmidt et al.12 The mesh plate is attached to the distraction screw with a pin that allows free rotational movement. The mesh design of the plate facilitates attachment of the undifferentiated mesenchymal cells from the cambium layer of the periosteum. Rotation of the central screw results in the distraction of the mesh plate and the periosteum away from the bone surface (Fig. 1).
Surgical Protocol All surgical procedures were performed under general anesthesia. The rabbits were first sedated with an intramuscular injection of midazolam (2 mg/kg) and ketamine (40 mg/kg). After sedation, general anesthesia was initiated and maintained via inhalation with 5% to 6% sevoflurane using a custom-made mask. Operation sites were shaved and prepared with a 10% povidoneiodine (Betadine) solution and draped in a sterile surgical fashion to allow for PD device placement. Local anesthesia using 2% lidocaine with epinephrine (1:200,000) was infiltrated in the lateral ramus and TABLE 1. Experimental Protocol for HBO Test Groups (Part A) and Experimental Protocols for NBO Control Groups (Part B).
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FIGURE 1. A, Diagram of the custom-designed PD device. B, Picture of the custom-designed PD device. Activation of the center rod distracted the subperiosteal mesh from the lateral ramus of the rabbit mandible.
submandibular regions. The lateral surface of the mandible was exposed through a linear submandibular incision approximately 2 cm long. After the skin incision, the subcutaneous tissue and the muscle layers were dissected down to the periosteum, and the periosteum at the level of the inferior border of the mandible was incised using a No. 15 blade. Having exposed the mandibular body, the distraction device was rigidly mounted perpendicular to the lateral surface of the mandible with 2 microscrews 1.3 5 mm in diameter (Fig. 2A). In both groups, while protecting the periosteum, the buccal cortex corresponding to the area covered with the distractor’s mesh plate was perforated 6 to 9 times with a round bur 0.5 mm in diameter to expose the trabecular bone. These perforations acted as an active source for osteoblast progenitor cells during active distraction, as described in earlier studies.14,17 The distraction devices were activated and deactivated to ensure that they were working properly. The surgical sites were then copiously irrigated with saline solution (0.9% NaCl). One small vertical incision penetrating all tissue layers was made to accommodate the activation screw of the device. Then, the periosteum was sutured back in place, covering the whole mesh. The subcutaneous tissues were closed in layers using 4-0 Vicryl sutures (Ethicon Inc, Somerville, NJ), and the skin was closed with 3-0 silk sutures (Fig. 2B). Immediately after the operation, the rabbits received Penicillin G (200.00 IU/mg, 0.04–0.06 mg/kg) intramuscularly, which was continued until postoperative day 3. During the study, the animals were kept in separate cages, and they were fed with commercial dry rabbit food and lettuce ad libitum.
HBO Treatment Protocol Two days after completion of the PD device placement, the animals in the HBO treatment group (groups H1 and H2) were placed in a pressure chamber (inner diameter of 80 cm, inner length of 220 cm) (ETC, Monoplace Hyperbaric Chamber, Southampton, PA) at the GMMA, Department of Undersea and Hyperbaric Medicine, and subjected to pure oxygen at 2.4 ATA for 90 minutes. During this period, the first 10 minutes was used for successive compression up to 2.4 ATA; the pressure was kept constant for 70 minutes, and decompression was continued for 10 minutes. The chamber temperature was
FIGURE 2. A, Intraoperative picture showing submandibular dissection and secure fixation of the custom-designed PD device to the lateral aspect of the mandibular body and showing perforations on the mesh. The mesh was lowered to the bone before the closure of the surgical wound. B, Photograph showing complete wound closure around the PD device on the lateral aspect of the mandible.
© 2014 Mutaz B. Habal, MD
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mineral density. Measurement of the pixel values of the PD area and the aluminum wedge was obtained. The amount of mineral density in the PD area in each radiograph was modified by the difference in the equivalent aluminum thickness (in millimeters), and the calculations were statistically evaluated (Fig. 3).
Specimen Preparation and Histologic Evaluation The animals were killed by an intravenous overdose of pentobarbital sodium 4 or 8 weeks after the completion of PD. After killing the animals, the PD areas, including peripheral soft tissues and PD devices, were carefully removed. Specimens were fixed in 10% neutral buffered formalin at room temperature for 6 days. Then, the PD device and the surrounding soft tissue were carefully removed. The specimens were decalcified in 10% formic acid, embedded in paraffin, and subsequently serially cut into 5-μm sections. Hematoxylin and eosin staining was performed on serial sections for morphologic evaluation of new bone formation in the PD gap under light microscopy. A board-certified pathologist in the Department of Pathology, GMMA, who was not a part of the study team, evaluated all slides stained with hematoxylin and eosin (Figs. 4 and 5). FIGURE 3. An occlusal radiograph of one specimen after the consolidation period. Note (white arrow) new bone formation in the gap area between the mesh and the lateral aspect of the ramus. This area was used for photodensitometry.
kept at +22°C by a water-cooling system. Produced CO2 was eliminated by constant flow of O2 with a flow rate of 0.9 L/min. The HBO treatment was given once daily for 25 days (Table 1 part A).
Distraction Protocol On the third day after the surgery, standardized occlusal radiographs were taken to determine the beginning position of the device and the cortical bone. After a 7-day latency period to allow for periosteal healing in both groups, distraction commenced at a rate of 0.25 mm twice a day. The activation screw was cleaned with 10% povidone-iodine before turning to avoid infection. One 180-degree turn of the activation screw yielded 0.25 mm of mesh elevation and lengthened the distraction gap by 0.25 mm. After 6 days, a periosteal expansion of 3 mm was obtained. Having started with a 2-mm gap at the time of placement because of the design of the device, 5 mm of periosteum distraction was obtained at the end of the distraction period. Before the animals were killed, standardized occlusal radiographs were taken. The rabbits were then killed after a consolidation period of 4 or 8 weeks, and the mandibles were surgically retrieved (Table 1 parts A and B).
Statistical Methods All the quantitative data were analyzed statistically with the Statistical Package for the Social Sciences (SPSS) software version 10.5 (SPSS, Inc, Chicago, IL). Statistical analyses were performed using the SPSS software version 10.5 (SPSS, Inc). Means and SDs were given in descriptive statistics. The statistical significance of differences between the groups and the consolidation periods was evaluated using the Mann-Whitney test. P values less than 0.05 (P < 0.05) were accepted as statistically significant.
RESULTS Clinical Evaluation All animals survived the surgery, the active PD period, and the HBO treatment. They resumed normal dietary habits during the
Standardization of Occlusal Radiographs We used photodensitometry analysis to measure bone density in the distraction gap. To standardize the measurements, we used the same standardization method described earlier.20 Occlusal radiographic images were obtained using Trophy Trex, 70 kilovolt (peak), 8 mA (Croissy Beaburg/France), x-ray tube, Novelix type 2.5 aluminum total filtration, with Ultraspeed D size 4 (Eastman Kodak Company, Rochester, NY) occlusal films. The exposure time (0.80 s) was kept the same for all animals. To achieve calibration and to minimize variations in the density of the radiographs, during exposure, all radiographs were taken with an aluminum wedge (step-wedge technique) of a known and stable thickness (equivalent aluminum thickness in millimeters). The aluminum step wedge was directly placed on the occlusal radiograph for each exposure. The radiographed aluminum step wedge comprised 9 steps (each 2 mm in thickness). All radiographs were developed in an automatic processing machine (Dent-X Model 410, Stamfort, CT). Unexposed radiographs were used to control the quality of the developing procedure. Densitometric analysis of the radiographs was done with a transmission densitometer (DT 1105 Ryparry Limited, Chatham, Kent, England) to calculate
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FIGURE 4. Macroscopic photomicrograph (2.5) of experimental 4-week consolidated (A) and 8-week consolidated (B) rabbit mandible specimens. The blue arrow shows newly formed cortical bone, and the black arrow shows the PD gap area. In high-power photomicrograph of the PD area (40), newly formed mature bone (marked by the red arrow), adipose tissue (marked by the green arrow), and neoangiogenesis in the PD gap area are observed. In the 8-week consolidated group, there were more lamellar bone and more neoangiogenesis but less adipose tissue observed compared with the 4-week consolidated groups. Compared with the control groups and the 4-week consolidated experimental group, more mature bone tissue was noted in the 8-week experimental group (group H2).
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induced new cortex were few. Interstitial tissue between lamellar bony isles was filled with abundant fat cells, resembling normal mandibular marrow (Fig. 5A).
Group N2: 8-Week Consolidated Control Group Newly formed bone tissue was present in the PD gap area. A thin zone of cortical bone was observed under the entire mesh. There were more areas of lamellar bone with some interconnections between the PD-induced newly formed cortex and the lateral surface of the mandible in this group than in group N1. There was also an increase in the width of the de novo bone formation extending to the mesh. Newly formed capillary tissue was observed between the bony isles in the PD gap area. There were also fat cells present in the PD gap area, resembling normal mandibular marrow (Fig. 5B).
Group H1: 4-Week Consolidated Experimental Group FIGURE 5. Macroscopic photomicrograph (2.5) of the control 4-week consolidated (A) and 8-week consolidated (B) rabbit mandible specimens. The blue arrow shows newly formed cortical bone, and the black arrow shows the PD gap area. In high-power photomicrograph of the PD area (40), newly formed mature bone (marked by the red arrow), adipose tissue (marked by the green arrow), and neoangiogenesis in the PD distracted area are observed. In the 8-week consolidated group, there were more lamellar bone and more neoangiogenesis but less adipose tissue observed compared with the 4-week consolidated groups.
first 24 hours after the surgery. All of the devices remained rigidly fixed to the lateral surface of the mandible for the entire experimental period. During the time of the study, all animals remained healthy, and no weight loss, infection, or breakdown of the soft tissues was observed.
Radiologic Evaluation Radiologic examination of the PD gap area showed different amounts of newly formed bone in the HBO and NBO groups. The amount of the de novo bone formed in the area between the lateral bony surface of the mandible and the mesh was more in the group H experimental animals than in the group N control animals. To determine the effects of the HBO treatment on newly formed bone by PD, we measured the optical mineral density of the PD gap areas. The mean optical densities of de novo bone in the PD areas on the occlusal radiographs based on the equivalent aluminum thickness in millimeters are given in Table 2. Comparison of the mean optical density of the de novo bone areas between the subgroups within each group showed that the values in group H2 were higher than in group H1 (P < 0.004), but there was no statistically significant difference in the NBO control groups. Densitometric analysis of the occlusal radiographs also showed that there was no statistically significant difference between group H1 (4-week consolidated subgroup) and group N1 (4-week consolidated subgroup) or between group H1 and group N2 (8-week consolidated subgroup of the control group). However, radiographic densitometric measurements showed that all results in group H2 (8-week consolidated subgroup of the experimental group) were greater than in group N1 and N2, and these differences were statistically significant (P = 0.003, P = 0.004) (Table 2).
Gross Appearance and Histologic Evaluation Group N1: 4-Week Consolidated Control Group Newly formed bone tissue was seen in the PD gap area. A thin zone of newly formed cortical bone tissue was present just under the mesh and the periosteum, parallel to the mesh. In the PD gap, there were lamellar bony isles and new capillary formation. However, mature lamellar bone between the mandibular lateral cortex and PD-
De novo bone formation was observed in the PD gap area. There was also a thin zone of newly formed cortical bone just underneath the mesh. There was more new bone tissue between the mandibular lateral cortex and the PD-induced thin cortex in group H1 than in group N1. The bone in the PD gap area was in the form of small isles with a few interconnections, similar to that of group N2. The width of the newly formed bone under the mesh was increased compared with that of group N1 but somewhat similar to that of group N2. There was adipose tissue between newly formed thin cortex and the lateral cortex of the mandible, resembling normal mandibular marrow (Fig. 4A).
Group H2: 8-Week Consolidated Experimental Group There was abundant new bone formation present at the PD gap area. The newly formed cortical bone under the mesh was thicker compared with both NBO groups. In addition, there were more and longer isles of lamellar bone between the mandibular lateral cortex and the PD-induced new cortex under the mesh. Most of those isles had interconnections and were distributed homogenously along the distraction space. The lamellar bone mainly consisted of thick trabeculae. Newly formed capillaries and fat tissue also filled the interstitial tissue in the PD gap. The width of the regenerated bone in the PD gap was thicker in this group than in the rest of the groups (Fig. 4B).
DISCUSSION The cambium cell layer of the periosteum plays a major role in the formation of bone.22 Elevating the periosteum from the underlying bone yields a mechanical tension stimulus on the periosteum that triggers mesenchymal cells to differentiate into osteoblasts, resulting in TABLE 2. Descriptive Statistics of the Photodensitometry of Each Measured Area (Equivalent Aluminum Thickness in Millimeters) on Occlusal Radiographs and Comparison Between Subgroups Within Each and Between Groups Groups H1 H2 N1 N2
N1
N2
H1
H2
Mean (SD) υ P υ P υ P υ P 1.498 (0.164) 7.5 0.092 10.500 0.229 NA NA 0.000 0.004 2.970 (0.379) 0.000 0.003 0.000 0.004 0.000 0.004 NA NA 1.038 (0.540) NA NA 7.000 0.078 7.5 0.092 0.000 0.003 1.611 (0.180) 7.000 0.078 NA NA 10.500 0.229 0.000 0.004
The differences with P < 0.05 between subgroups were significant and are shown in bold. The HBO treatment subgroups were H1, HBO treatment group with PD killed at the end of the 4-week consolidation, and H2, HBO treatment group with PD killed at the end of the 8-week consolidation. The NBO control subgroups were N1, NBO control group with PD only killed at the end of the 4-week consolidation, and N2, NBO control group with PD only killed at the end of the 8-week consolidation. NA, not applicable.
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subperiosteal bone formation.2,15,16,23 Dynamic PD is a relatively new technique, and its potential for producing new bone was first reported by Schmidt et al.12 Periosteal distraction without application of any chemical compounds, cells, or corticotomy has been reported to result in the generation of new bone tissue.1,2,12–14,23,24 However, some authors have reported that the degree of mineralization or the quality of the bone regenerate was not similar to the bone gained by DO and that the PD gap is abundantly filled with adipose tissue.12,13,18,21 On the basis of these studies, the effects of PD and improvements in the technique and the design of the devices call for more investigation to gain enhanced bone formation. On the basis of our previous promising findings with HBO treatment on DO,20 in this study, we tested whether HBO treatment has an effect on the amount of new bone formed through the PD procedure. We demonstrated that PD led to the formation of new bone between the perforated elevation mesh and the lateral cortex of the mandible in both groups. This result is in agreement with the results of studies published in the literature.1,2,12–15,17,18 However, we also showed that the administration of HBO during PD increased the maturation and the quantity of the newly formed bone compared with the control group that received only PD. This also verified the studies showing positive effects of HBO on new bone formation in various other experiments.19,20,25–27 The effect of HBO treatment on bone and soft tissues is well known.28,29 It increases the PaO2 in the blood and in the tissues and has been shown to increase capillary ingrowth and collagen synthesis,30 neovascularization, and osteogenesis.31,32 A considerable number of studies have shown enhanced osteogenic activity as a result of HBO treatment. An earlier union of autologous bone grafts in a rabbit model19 and improved bone formation around titanium implants were observed in vivo after exposure to HBO treatment,33 and recently, Pedersen et al25 showed that HBO treatment enhanced vascularization and bone formation in rat calvarial defects. Similar to their results, this study showed that HBO could make a positive difference in the quality and the quantity of the new bone gained by periosteum distraction. To the best of our knowledge, this is the first study combining PD and HBO treatment. Treatment protocols for HBO vary in the literature, and the most widely used and accepted protocol in animal and in vitro studies involves the delivery of 100% oxygen at 1.5 to 3.0 ATA for 60 to 90 minutes daily.19,26,28,34 As to the HBO treatment interval, it has been shown that once a day seems to accelerate bone repair and vessel ingrowth when compared with HBO treatment given twice a day.34 Our rabbit model and PD device design,13,18,21 as well as HBO treatment protocol,20 were all previously studied in our unit and reported in the literature and found reliable. We decided to use the same HBO treatment in which the HBO was given at 2.4 ATA for 90 minutes for 25 days once daily. Densitometric analysis showed no statistically significant difference between groups H1 and N2. This suggested that HBO treatment increases osteogenesis in PD and may result in a decrease in the consolidation period. This finding was in accordance with the results of an earlier study that evaluated the effect of HBO treatment in DO.20 These results also verify the enhanced bony mineralization by HBO.25 The density values of the de novo formed bone in the PD gap were significantly higher in group H2 than H1 (P = 0.004). Although densitometric values were higher in group N2 compared with N1, the difference was not statistically significant (P = 0.078). In this study, PD was performed in the range of widely used protocols for DO or PD in experimental animal studies,1,2,12–15,17,18,20,21 which usually allows a latency period of 5 to 10 days and distraction rate of 0.25 to 1.0 mm/d. In a previous study, we tested the DO protocol of 7 days of latency and 0.25-mm twice-a-day activation of the distractor in a rabbit model. Having obtained good results with that protocol, we adopted the same distraction protocol for the current study.
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Our main purpose in this experiment was to evaluate whether HBO was beneficial on the formation of new bone by PD. Therefore, 4 and 8 weeks were chosen as consolidation periods for this study. Furthermore, it should also be noted that observation of the maturation of de novo bone was beyond the scope of this study. The preferred consolidation periods in our study are also in accordance with the previous experimental studies on PDO in rabbits.12–14,17,18,20,21 In a comprehensive review by Swennen et al,35 it has been reported that varying consolidation periods ranging between 1.5 and 10 weeks were used in the several experimental DO studies in rabbits. In clinical studies, similarly, there are no rigidly specified consolidation parameters for DO, and significant variations exist between different craniofacial groups. As reported in another extensive review by Swennen et al,36 the consolidation period for maxillary and mandibular DO procedures in humans may vary between 2 weeks and 4 to 6 months with regard to a wide variety of clinical indications. The diversity in consolidation periods between clinical and experimental studies can be attributed to the different bone remodeling period in humans and rabbits. According to previous studies in the literature,10,13,32 with regard to an ideal consolidation period in DO, our findings seem to verify that a longer consolidation period can result in more dense bony structures versus a shorter consolidation period. The results of the densitometric analysis showed that there was a statistically significant difference between groups H2 and N1, groups H2 and N2, and groups H2 and H1 (P = 0.003, P = 0.004, and P = 0.004, respectively). According to these results, it can be concluded that the positive effects of HBO treatment are greater with longer consolidation periods. This finding is in accord with the results of our previous DO study.20 The use of an aluminum step wedge allowed us to transform the reading of light transmission in the radiographs into an equivalent thickness of aluminum.20 Therefore, it can be concluded that all occlusal radiographs were evaluated under the same conditions. The densitometric analysis in this study has shown that HBO treatment significantly increased the new bone density in the 8-week consolidated experimental group and also increased the new bone density in the 4-week consolidated group. Sato et al4 have argued that the difference in the quality of maturation of the new bone formed by PD compared with DO may be explained by several factors: the tissue may lack stimulatory stress, may lack a sufficient number of osteogenic cells, or may have an insufficient vascular supply. Recently, Altug et al18 compared different latency periods along with different consolidation periods in PD, and they reported that the newly formed bone by PD was mostly filled with fatty tissue, and they claimed that lack of bone marrow cells might play a role in the occurrence of fatty tissue. Sencimen et al13 also reported an abundance of adipose tissue and insufficient mature bone in the PD gap area, and therefore, they concluded that this newly formed bone is not suitable for occlusal forces, and it would be impossible to insert an endosteal implant into the area. They proposed that decortication of the bone surface to bring endosteal cells into the distraction area may increase the maturity of the woven bone. Lately, Oda et al14 also investigated the effect of using decorticating holes in the PD protocol for improving bone regeneration in a rabbit model. They suggested that the cortical bone might be a hindrance to bone formation, without access to the bone marrow, and they postulated that decorticating holes can be effective in improving the new bone regenerate in PD. On the basis of these studies, we used decortication holes to benefit from the undifferentiated mesenchymal cells and the osteoblastic activity originating from the endosteum to gain new bone. In this current study, more de novo bone tissue formation and more mature trabecular bony structure were observed in groups H2 and N2. These findings are in agreement with the reports that a longer © 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery • Volume 25, Number 5, September 2014
consolidation period in PD or DO procedures results in the formation of more mature and mineralized bone.13,20,32 Nowadays, HBO therapy can be easily performed in singleperson chambers or chambers that can hold more than a dozen people at a time. However, HBO facilities are currently limited and treatment is expensive; hence, the clinical application is still limited to major craniofacial centers. In conclusion, we have demonstrated that HBO positively affects the PD process, leading to a more mature, mineralized bony regenerate. Further studies using higher animal models are anticipated to determine the most suitable HBO protocol for this purpose. ACKNOWLEDGMENT The authors thank Prof Daniel Laskin from the Department of Oral and Maxillofacial Surgery, Virginia Commonwealth University School of Dentistry, for constructive comments on the manuscript and editing assistance.
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© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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