CLINICAL STUDY
Transport Distraction Osteogenesis With Recombinant Human Bone Morphogenic Protein-2 for Large Calvarial Defect Reconstruction Seung Yong Song, MD,* In Sik Yun, MD,* Chung Hun Kim, MD, PhD,† Dae Gon Woo, PhD,‡ and Yong Oock Kim, MD, PhD* Background: Transport distraction osteogenesis (TDO) has been used in attempts to treat large calvarial defects but has, until now, lacked consistency and reliability. To achieve sufficient bone formation, the effect of TDO was compared to the effect of TDO combined with recombinant human bone morphogenic protein-2 (rhBMP-2). Methods: Fourteen dogs were divided into 2 groups; 6 animals in the control group received TDO only, and 8 received TDO combined with rhBMP-2. A calvarial defect 33 35 mm in size was generated, and the drug-delivering internal distractor was applied. After a 5-day latency period, distraction with rhBMP-2 at 10 μg/day was initiated at a rate of 2 mm/day. This was followed by a consolidation period of 3 months, after which areas of osteogenesis and strength were measured and histologic examinations were conducted. Results: The average area of osteogenesis was higher in the experimental group (P < 0.01). Regenerated bone of the experimental group showed increased strength (P < 0.05). Histological examination showed typical mature bone in the experimental group. Prominent osteoblastic rimming was observed in the bone marrow of the experimental group. Conclusions: TDO with an internal distraction device delivering rhBMP-2 can enhance bone regeneration of large calvarial defects in a dog model. These results suggest the potential for human clinical testing of TDO combined with rhBMP-2. Key Words: Transport distraction osteogenesis, distraction osteogenesis, calvarial reconstruction, bone morphogenic protein-2 (J Craniofac Surg 2014;25: 502–508)
From the *Department of Plastic Reconstructive Surgery, Institute for Human Tissue Restoration, Yonsei University College of Medicine, Seoul; †Department of Plastic and Reconstructive Surgery, CHA Bundang Medical Center, CHA University School of Medicine, Gyeonggi-do; and ‡Cardiovascular Devices Division, Ministry of Food and Drugs, Osong, Republic of Korea. Received September 4, 2013. Accepted for publication December 28, 2013. Address correspondence and reprint requests to Yong Oock Kim, MD, PhD, Department of Plastic and Reconstructive Surgery, Institute for Human Tissue Restoration, Yonsei University College of Medicine, 250 Seongsan-no, Seodaemoon-gu, Seoul 120-752, Republic of Korea; E-mail: [email protected] The authors report no conflicts of interest. This work was supported by the Seoul R&BD program under Grant PA100085 (2010). Copyright © 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000000672
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ince first being reported by Bouletreau et al, calvarial defect repair by transport distraction osteogenesis (TDO) has been conducted in only a few animal models and one human as of 2010.1–10 One possible reason which makes this procedure not being widely used until now is the inconsistency of the results. Some studies reported TDO was effective for calvarial reconstruction.1,5,8 However, there was also a report that the volume of newly generated bone was not sufficient by this method.6 Another remarkable point is wide variability of the results. Even in the study that TDO had favorable effect on the treatment of cranial defect, one subject in the control group healed completely without DO, while the other 3 control animals showed no appreciable degree of healing.5 In another study using TDO for calvarial defect, wide variability of bone regeneration was also reported from 24% to 96% in TDO group and from 13% to 80% in control group.6 To promote bone regeneration by TDO, Yun et al used internal type of distraction device. Even in their study, bone formation from 26% to 97% in TDO group and from 0 to 82.9% in control group was reported. It had advantages of increased mechanical stability during distraction osteogenesis (DO), which resulted in more bone regeneration despite of a short consolidation period.7 However, variability of the results still remained to be overcome. Similar phenomena were seen in other reports.11 So, protocol for more predictable and sufficient bone formation is still necessary for clinical application. Two factors were prioritized in this study to overcome drawbacks of previous studies. One was the stability of the distractor. External distractors can generate a strong distraction force and permit easy handling of the distraction vector but suffer from inevitable instability because some of its structure is exposed outside of the skin, an issue that is of particular concern in young children and animals.12 These physical limitations can cause subacute or chronic inflammation, which may disrupt bone regeneration during the distraction period. In this regard, internal distractors have the advantage of reduced exposure to contaminants outside the skin. The other important factor in optimized bone formation by TDO is the administration of humoral factors. Among the various factors which can induce bone growth, bone morphogenic protein (BMP) is believed to be the primary facilitator of bone regeneration in DO.13–18 BMP belongs to the transforming growth factor β superfamily. It promotes vascular invasion, bone formation, bone remodeling, and bone marrow differentiation by directing cell differentiation of mesenchymal stem cells to become osteoblasts and osteocytes, which in turn build and renew the extracellular matrix.19–21 In this study, rhBMP-2 is used to supplement TDO. rhBMP-2 is known to enhance consolidation, shorten the period of distraction, and eventually accelerate bone regeneration.13,16,17,22,23 We used a specially designed drug-delivering distraction device for periodic injection of rhBMP-2 during TDO. Here we compare the bone regenerative effect of mechanical factors (TDO) alone to the combination of mechanical (TDO) and
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The Journal of Craniofacial Surgery • Volume 25, Number 2, March 2014
FIGURE 1. A newly designed drug-delivering internal distractor. A, Inlet of vehicle, sealed with silicone to prevent retrograde infection. B, Outlet of vehicle. C, Rotator rod. D, Guide rail
humoral (rhBMP-2) factors. This study will help demonstrate the feasibility and reliability of TDO for human clinical trials.
MATERIALS AND METHODS Experimental Animals and Groups Fourteen mongrels (female, 5 months gestational age) were divided into 2 groups, a control (n = 6) group receiving only TDO and an experimental group (n = 8) receiving TDO and rhBMP-2. All experimental animals were maintained at the animal facility at Yonsei University Medical College, and all experimental procedures were performed after approval by the Institutional Animal Care and Use Committee (2011-0032).
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with inhaling isoflurane (Foran; Choongwae Pharmaceutical Corporation, Hwaseong, Republic of Korea). To reduce cerebral edema, the concentration of isoflurane was maintained at 2% to 3%, and the CO2 concentration was reduced by inducing the hyperventilation state. Animal was placed in the dorsal position and sterilized with povidone iodine. A longitudinal incision was made in the midline of the scalp, and the calvarium was exposed. Ostectomy was performed with a burr to create the calvarial defect. Defect size was 33 35 (width length) mm and the long axis was centered on the midline of the cranium. A bone fragment of 30 8 (width length) mm was prepared from ostectomized bone to make the transport disc (TD). For easy movement of the transport disc, its width was smaller than that of the defect. The periosteum of the TD was removed. The internal bone distractor was specially designed with 2 guide rails for this experiment. For the injection of rhBMP-2 once a day during the distraction period, an inlet was located at the rotating part of the distractor. It was sealed with silicone to prevent retrograde infection. Injected rhBMP-2 flowed out to the distraction zone (Fig. 1). Microplates were used to fix the transport disc in place, and the disc was then connected to the distractor (Fig. 2). Muscles and the skin were sutured by #3-0 Vicryl and #4-0 nylon sutures. So, the whole device was placed under the soft tissue except rotator rod. After the surgery, the analgesic ketorolac (Tarasyn; Roche Pharmaceutical Company Ltd, Basel, Switzerland) and the antibiotic ceftriaxone (50 mg/kg; Donghwa Pharmaceutical Company Ltd, Ansan, Republic of Korea) were injected intramuscularly for 2 days.
Distraction Distraction was initiated after a 5-day latency period. The distraction rate was 2 mm/day, and it took 13 days to complete the entire defect length. rhBMP-2 (BioVision, Milpitas, CA) was prepared at a concentration of 10 μg/mL with phosphate buffered saline. It was injected at a rate of 1 mL/day after turning the rotating rod of distractor for the entire 13 days of distraction. This was followed by a consolidation period of 3 months (Fig. 3).
Operative Procedure All animals were anesthetized by intravenous injection of midazolam (0.2 mg/kg; Bukwang Pharmaceutical Company Ltd, Ansan, Republic of Korea) and propofol (3 mg/kg, Pofol V; Dongkook Pharmaceutical Company Ltd, Jincheon, Republic of Korea). In succession, endotracheal intubation was performed
Radiologic Studies A plain X-ray of the skull was taken in all experimental animals 5 days before starting the distraction. Another X-ray was taken near the end of the bone distraction period to check the status of the distraction device. Computed tomography (CT) images were obtained (Somatom Sensation 64; Siemens Medical Systems, Erlangen, Germany) at the end of the study after euthanizing the experimental animal (Fig. 3).
Assessment of Results Quantitative analysis of the area of regenerated bone was accomplished by measuring the area of the bone defect from the CT images. The newly formed bone area was measured by subtracting the bone defect area that remained in the 3-dimensional CT from the bone defect area created at the time of surgery (35 35 mm).
FIGURE 2. Intraoperative photograph. The newly designed drug-delivering internal distractor was applied to the calvaria of mongrel dogs. The size of calvarial defect and transport disc (TD) was 33 35 (width length) mm and 30 8 (width length) mm, respectively. Periosteum and dura mater were detached from TD.
FIGURE 3. Schedules of experiment. This study was designed with 5 days of latency, 13 days of distraction, and 3 months of consolidation. Radiologic studies included plain X-rays on postoperative 0 day and near the end of the consolidation period, with 3-dimensional computed tomography done at the end of the study.
© 2014 Mutaz B. Habal, MD
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TABLE 1. Area Ratio* of Regenerated Bone
No.
Control (Without rhBMP-2) (%)
Experimental Group (With rhBMP-2) (%)
95.21 68.87 72.47 54.00 49.33 68.38 68.04 ± 18.07
93.77 99.03 98.79 94.29 84.83 97.12 94.64 ± 5.29**
1 2 3 4 5 6 Mean ± SD †
*Area ratio: the ratio of regenerated area to the original defect. †
SD, standard deviation.
**P < 0.01.
We compared the calculated ratio (in percent) of the measured new bone formation area with the initial bone defect area. To measure bone strength, newly formed bone specimen was obtained from the TDO site. Each specimen was harvested with a full thickness of a bone. The strength of the harvested bone was estimated by compression strength measurements. These measurements were done using an Instron 5848 microtester (Instron, Norwood, MA). The strength value is presented in newtons per square meter. The strength of the experimental group and the control group was measured 3 times. Finally, the measured data were compared between the TDO regenerated bone and the normal calvarial bone by the strength ratio of regenerated bone to normal adjacent bone. For histologic examination, the piece of extracted bone was fixed in 4% phosphate buffered formalin (pH 7.73) solution for 14 days. After the dehydration process, the samples were placed in an alcohol and acetone solution. Then, they were further fixed with a methyl-methacrylate solution (Merck, Hohenbrunn, Germany) to prevent calcium demineralization. Next, the bone pieces were cut with a diamond saw at 30-μm intervals along the axis of distraction. The samples were then stained with hematoxylin and eosin, and a visual comparison was done under a microscope.
Statistical Analysis All data from our experimental results are represented as mean ± standard deviation (SD). Comparisons between groups were performed by the Student t test. A value of P less than 0.05 was
FIGURE 4. Photograph at the end of the consolidation period. A, The control group. White arrow indicated non-healed area at the end of distraction. B, The experimental group. Regenerated bone nearly covered the defect.
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considered significant. All data analyses were performed using the Statistical Package for the Social Sciences (SPSS v. 15.0; SPSS Inc, Chicago, IL).
RESULTS During the experiment, 2 dogs of the experimental group were excluded from this study due to infections. By the end of the study, data for 6 dogs in the control group and 6 dogs in the experimental group were used for analysis. The bone specimen for strength measurement was inadequate in 1 animal, so strength tests of the control group were performed in only 5 animals. Radiologic studies were performed according to a predetermined schedule with plain X-ray performed immediately postoperatively and again at the end of distraction, while 3-dimensional CT was completed at the end of consolidation.
Area of Osteogenesis The control group and the experimental group showed bone regeneration equal to 68.04 ± 18.07% and 94.64 ± 5.29% of the original defect, respectively. The area of osteogenesis was higher in the rhBMP-2-treated group with statistical significance (P < 0.01) (Table 1, Figs. 4–6).
Strength Regenerated bone of the control group and the experimental group showed strength values of 23.85 ± 6.19 N/mm2 and 53.75 ± 18.66 N/mm2 by compression test, respectively. We compared the 2 groups using the ratio of regenerated bone strength to normal bone strength. Regenerated bone by TDO combined with rhBMP-2 showed increased strength with statistical significance (P < 0.05) (Table 2, Fig. 7).
Histology Histologic examination revealed that normal bone components such as bone marrow, lamella, and osteocytes existed in the normal, control, and experimental group. However, bony specimen of the control group showed reduced lamella portion compared to normal bone and its lamella was not matured compared to the normal bone. So, it can be considered as woven bone rather than mature bone. The bony specimen of the experimental group had similar thickness and cortex portion with normal bone. These findings indicate that regenerated bone of the experimental group is more close to the normal cancellous bone (Fig. 8). In the bone marrow, osteoblasts rimming was observed in the control and the experimental group (Fig. 9). The number of osteoblasts was significantly higher in the experimental group compared to the control group (P < 0.01). On the other hand, the control
FIGURE 5. 3D CT images of the calvarial defects at the end of consolidation in each animal. Upper 1–6 images show remnant calvarial defects of control group. Lower 1–6 images show remnant calvarial defects of experimental group.
© 2014 Mutaz B. Habal, MD
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TABLE 2. Measurement of Bone Stiffness No.
Control (Without rhBMP-2) 2
1 2 3 4 5 6 Mean ± SD † †
Normal Bone (N/mm ) (N) 25.28 32.40 27.75 43.14 54.95 123.40 51.15 ± 37.06
Experimental Group (With rhBMP-2) 2
Regenerated Bone (N/mm ) (R) 15.26 20.21 Not measurable 25.41 31.00 27.39 23.85 ± 6.19
Ratio (R/N) 0.60 0.62 Not measurable 0.59 0.56 0.22 0.52
Normal Bone (N/mm2) (N) 60.87 23.21 46.70 42.82 76.18 84.48 55.71 ± 22.7
Regenerated Bone (N/mm2) (R) 29.06 34.83 75.74 55.20 70.31 57.36 53.75 ± 18.66
Ratio (R/N) 0.48 1.50 1.62 1.29 0.92 0.68 1.08*
SD, standard deviation.
*P < 0.05.
In TDO, regeneration of bone for large defect should be achieved using relatively small bare bone segment, so, it had less reliability on efficacy of bone formation and strength. This may be the reason that there were few reports about this technique since its first introduction. We believe that 3 factors are important for the successful repair of large calvarial defects using TDO. The first is the ratio of the transport disc size to bone defect size. In previous reports, the size ratio of the transport disc to calvarial defect ranged from 19% to 50%.1–10 In this study, we designed the transport disc size for a ratio of 18%, smaller than any previous study (Table 3). The length of our transport disc was 8 mm, which was the smallest size that permitted fixation to the distraction device. The defect size in this study was nearly one third of the whole cranium, a parameter that could provide useful information in the application to humans. However, the appropriate size of a transport disc has not been determined until now. Minimal transport disc size with maximal bone regeneration may be ideal in the clinical setting, and optimization of transport disc size according to defect size should be investigated for human application in the future.
Another important factor determining the success of TDO is the type of distraction device used. Nearly half of previous studies of TDO for calvarial defects used an external distractor; however, in recent years, internal distractors have been developed and are being used more frequently (Table 3). Device stability has a significant impact on DO success, especially in animal experiments. In this study, we used an internal distractor with guide rails. This allowed for increased durability and stability, so no extrusion or dysfunction of the device was observed in this study. There is some evidence to suggest it might result in increased bone regeneration.6,7 The last factor we believe to be important for TDO success is the use of humoral factors to facilitate or accelerate bone regeneration. DO with BMP treatment is one of the most well-known regimens capable of improving the results of bony regeneration. BMP is a strong osteoinducer and plays a pivotal role in the molecular signaling cascade leading to bone regeneration and remodeling.17 rhBMP-2 in particular is one of the most powerful growth factors for bone regeneration. In 2007, the Food and Drug Administration (FDA) approved the use of BMP-2 delivered with an absorbable collagen sponge carrier for clinical use in craniofacial deformities.24 DO combined with rhBMP-2 in a collagen sponge increased ossification and mineral content and decreased the consolidation period in the long bone.17,23 Further support for the use of BMP-2
FIGURE 6. Comparison of regenerated area. The experimental group showed significantly increased new bone formation compared to the control group (**P < 0.01).
FIGURE 7. Comparison of bone strength. Strength ratio was the ratio of regenerated bone to normal bone in each group. Regenerated bone from the experimental group showed significantly increased bone strength compared to the control group (*P < 0.05).
group also had significantly increased osteoblasts compared to normal bone (P < 0.01) (Fig. 10).
DISCUSSION
© 2014 Mutaz B. Habal, MD
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4 and TGF-β were increased in the DO model.27 From this, we can infer the important role of growth factors, including BMP, in bone regeneration with TDO. Icekson et al showed TDO without growth factor can be an effective tool for closing full-thickness calvarial defects in sheep. However, they used only 4 animals, the defect size was about 20 20 mm, a smaller defect than what was used in this study, and the size ratio of the transport disc to calvarial defect size was 50%, bigger than our study.5 In the comparison of osteogenesis area, the average ratio of regenerated bone is higher in the experimental group with statistical significance. This may be due to increased mechanical stability of newly designed distractor. The ratio of regeneration was 94.64% in the experimental group, and the magnitude of this result likely has significant clinical importance, especially because the size of the original defect was so large. Moreover, the variation seen in the results of the control group was much reduced after adding
FIGURE 8. Microscopic findings of normal (A), control (B), and experimental (C) group (original magnification 40). Normal bone components such as bone marrow (BM), lamella (L), and osteocytes are observed in all groups. However, cortex of regenerated bone in the control group is relatively small and consists of woven bone. Regenerated bone of the experimental group has similar thickness with normal bone and the amount of cortex is also similar with normal bone. It is more close to the histology of normal cancellous bone.
came in a study of membranous bone by Issa et al when they reported that rhBMP-2 with a monoolein gel carrier could enhance bone formation in the mandible and may potentially reduce the treatment period.14 Some researchers have also used mesenchymal stem cell–based gene therapy to continuously release BMP-2 during the distraction and consolidation periods in an effort to maximize bone regeneration.25 However, gene therapy has historically been difficult to use clinically due to unpredictable expression, variability in the effective period, complexity in gene transfection processes, and the unknown hazards of using an adenoviral vector.26 Fewer studies have been conducted in the TDO model using BMP-2. Cakir-Ozkan et al compared the effects of TDO and bone grafting in a sheep model and found that expression of BMP-2,
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FIGURE 9. Microscopic findings of normal (A), control (B), and experimental (C) group (original magnification 200). The marrow of the control and the experimental group reveals osteoblastic rimming (arrow).
© 2014 Mutaz B. Habal, MD
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The Journal of Craniofacial Surgery • Volume 25, Number 2, March 2014
FIGURE 10. Comparison of the number of osteoblasts in the normal, control, and experimental group (original magnification 100). The experimental group had significantly increased osteoblasts compared to normal and the control group (**P < 0.01). The control group also had increased number of osteoblasts compared to normal group (**P < 0.01).
rhBMP-2 to the treatment, as indicated by the small standard deviation. This could indicate that variable production and secretion of humoral factors in each organism explains the observed unpredictable bone defect healing. According to these results, TDO combined with rhBMP-2 may allow bone regeneration to proceed in a more consistent or predictable fashion. In this study, a new distractor for TDO was devised that allowed injection of liquid-type humoral factors like rhBMP-2. The use of a drug-delivering distractor for TDO has the advantage of simplicity, easy dose calculation, and increased predictability. The first drug-releasing distractor was devised by Grayson et al.28 Konas et al have also recently reported that DO with a drugreleasing internal distractor facilitated ossification of the femur in rats.11 However, their studies used DO rather than TDO and was conducted in the mandible or femur. Moreover, our distractor had guide rails on both sides of the device to allow stable movement of the transport disc. This design had also had the advantage of following a curved surface, unlike the traditional single-rod internal distractors. Because the calvarium has a round surface, transport discs should pass along a similarly curved area in most cases.9,10,29
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Two animals in the experimental group were excluded from the study because of infection at the site of distraction despite sealing the injection inlet with a silicone membrane in an effort to prevent retrograde infection. We guess that the exposed rod of distraction device may be an alternative route of the infection. Further research should be conducted to prevent perioperative and postoperative infection because internal distractors are placed in vulnerable locations deep within vital structures of the cranium.30 The distraction rate of this study was higher than is typical at 2 mm/day. As previously described, BMP-2 could shorten the distraction period,16 and in preliminary studies, we experienced similar results using TDO with rhBMP-2. So, the rate of the distraction was relatively faster compared to conventional DO. We detached periosteum and dura mater from the transport disc, but in some reports vascularized transport discs have been associated with improved success of DO.8 This is theoretically reasonable because ensuring disc viability is important for bone regeneration, and it is well known that dura mater is of paramount importance in calvarial bone regeneration31,32 However, preservation of dura mater with transport disc is not easy considering human application, so, the dura was detached from the transport disc in this study. This allowed a greater degree of freedom in movement of transport disc during distraction. In histologic examination, prominent osteoblastic rimming was observed in the bone marrow of the experimental group. This is the hallmark of bone regeneration. And the number of osteoblasts was increased in order of the normal, control, and experimental group. This finding reflects vigorous bony regeneration occurred by TDO itself. And it also indicates that application of rh-BMP-2 into distraction zone can augment this process additionally. It is reasonable because BMP promote bone formation, bone remodeling, and bone marrow differentiation by directing cell differentiation of mesenchymal stem cells to osteoblasts and osteocytes, as previously described.19–21 Non-steroidal anti-inflammatory drug (NSAIDs), ketorolac, was used to relieve pain of the experimental animals in this study. There were some reports that ketorolac could interfere negatively with new bone formation in animal and human.33–36 However, Martin et al reported that rhBMP-2 could overcome the detrimental effect of ketorolac in rabbit model.37 Because ketorolac was also used in the control group, the exact effect of NSAIDs in TDO for calvarial defect should be investigated by properly designed study in the future. TDO with administration of rhBMP-2 resulted in regeneration of about 94% of the original calvarial defect with a strength ratio nearly equal (1.08) to normal bone. The methods used in this study can also shorten the duration of treatment. Most importantly,
TABLE 3. Previous Literatures of Transport Distraction Osteogenesis for Calvarial Defect
Subject Distractor type Defect size (D) (mm) Transport disc size (T) (mm) Disc ratio (T/D) (%)
Bouletreau et al
Hirano et al
Kramer et al
Muller et al
Hong et al
Yun et al
Icekson et al
Cho-Lee GY et al
Durmus et al
Steinbacher et al
This Study
Rabbit External
Rabbit Internal
Sheep External
Sheep External
Dog External
Dog Internal
Sheep Internal
Human Internal
Sheep External
Rabbit Internal
Dog Internal
14 7
40 60
50 60
15 33
15 35
20 20
50 85
50 60
16 16
33 35
40 20
40 20
15 7
14 7
20 10
50 20
40 20
10 16a
30 7
33
27
21
19
50
24
27
38
18
15 15 15 10a
(7–10) 10 (trapezoid)*
40
36
*
Additional craniectomy was performed outside of the defect for preparation of transport disc.
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the results of this study further demonstrate proof of concept and reliability in support of human application.
CONCLUSIONS Transport disc distraction osteogenesis with an internal distraction device capable of delivering bone morphogenic protein-2 can enhance bone regeneration of calvarial defects in a dog model. The results on area of regeneration and strength indicate that clinical application to humans is possible. ACKNOWLEDGMENTS The authors thank Ji Hyung Park and Han Sung Kim (Department of Biomedical Engineering and Institute of Medical Engineering, Yonsei University, Wonju, Republic of Korea) for assistance with measurement of bone strengths.
REFERENCES 1. Bouletreau PJ, Warren SM, Paccione MF, et al. Transport distraction osteogenesis: a new method to heal adult calvarial defects. Plast Reconstr Surg 2002;109:1074–1084 2. Kramer FJ, Mueller M, Rahmstorf M, et al. Ortho- and heterotopic bone grafts in bifocal transport osteogenesis for craniofacial reconstruction—an experimental study in sheep. Int J Oral Maxillofac Surg 2004;33:575–583 3. Muller MC, Kramer FJ, Swennen GR, et al. A comparison of two types of free bone grafts as transport discs in segmental distraction for reconstruction of calvarial bone defects: an experimental study. Arch Orthop Trauma Surg 2004;124:665–674 4. Hirano N, Tanabe M, Watanabe T, et al. Novel approach to calvarial bone transport using a rabbit model. Neurol Med Chir (Tokyo) 2006;46:69–73; discussion 73–64 5. Icekson M, Regev E, Neuman R, et al. Transport distraction osteogenesis for closing full-thickness calvarial defects in sheep. Cleft Palate Craniofac J 2009;46:363–367 6. Hong JW, Song SY, Woo DG, et al. Transport disc distraction osteogenesis for the reconstruction of a calvarial defect. J Craniofac Surg 2009;20:790–796 7. Yun IS, Mun HY, Hong JW, et al. Transport disc distraction osteogenesis for the reconstruction of a calvarial defect. J Craniofac Surg 2011;22:690–693 8. Cho-Lee GY, Naval-Gias L, Gonzalez-Garcia R, et al. Bifocal transport osteogenesis for the reconstruction of adult calvarial defects: a new surgical technique. J Craniomaxillofac Surg 2010;38:368–373 9. Durmus M, Zor F, Ozturk S, et al. Calvarial reshaping using bifocal bidirectional transport distraction osteogenesis. J Oral Maxillofac Surg 2010;68:756–761 10. Koch FP, Yuhasz MM, Travieso R, et al. Targeted cranial bone transport distraction is enhanced using a novel device. Plast Reconstr Surg 2013;131:453e–455e 11. Konas E, Emin Mavili M, Korkusuz P, et al. Acceleration of distraction osteogenesis with drug-releasing distractor. J Craniofac Surg 2009;20:2041–2048 12. Pelo S, Gasparini G, Di Petrillo A, et al. Distraction osteogenesis in the surgical treatment of craniostenosis: a comparison of internal and external craniofacial distractor devices. Childs Nerv Syst 2007;23:1447–1453 13. Cheung LK, Zheng LW. Effect of recombinant human tissue inhibitor of matrix metalloproteinase-1 in mandibular distraction osteogenesis in rabbits: a computed tomographic study. Br J Oral Maxillofac Surg 2006;44:5–11 14. Issa JP, do Nascimento C, Lamano T, et al. Effect of recombinant human bone morphogenetic protein-2 on bone formation in the acute distraction osteogenesis of rat mandibles. Clin Oral Implants Res 2009;20:1286–1292 15. Shen Q, Zhu S, Hu J, et al. Recombinant human bone morphogenetic protein-4 (BMP-4)-stimulated cell differentiation and bone formation within the expanding calvarial suture in rats. J Craniofac Surg 2009;20:1561–1565
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16. Eguchi Y, Wakitani S, Naka Y, et al. An injectable composite material containing bone morphogenetic protein-2 shortens the period of distraction osteogenesis in vivo. J Orthop Res 2011;29:452–456 17. Lesaichot V, Leperlier D, Viateau V, et al. The influence of Bone Morphogenic Protein-2 on the consolidation phase in a distraction osteogenesis model. Injury 2011;42:1460–1466 18. Zhang WB, Zheng LW, Chua DT, et al. Treatment of irradiated mandibles with mesenchymal stem cells transfected with bone morphogenetic protein 2/7. J Oral Maxillofac Surg 2012;70:1711–1716 19. Reddi AH. Morphogenetic messages are in the extracellular matrix: biotechnology from bench to bedside. Biochem Soc Trans 2000;28:345–349 20. Vaibhav B, Nilesh P, Vikram S, et al. Bone morphogenic protein and its application in trauma cases: a current concept update. Injury 2007;38:1227–1235 21. Einhorn TA, Majeska RJ, Mohaideen A, et al. A single percutaneous injection of recombinant human bone morphogenetic protein-2 accelerates fracture repair. J Bone Joint Surg Am 2003;85-A:1425–1435 22. Li G, Bouxsein ML, Luppen C, et al. Bone consolidation is enhanced by rhBMP-2 in a rabbit model of distraction osteogenesis. J Orthop Res 2002;20:779–788 23. Sailhan F, Gleyzolle B, Parot R, et al. Rh-BMP-2 in distraction osteogenesis: dose effect and premature consolidation. Injury 2010;41:680–686 24. Razzouk S, Sarkis R. BMP-2: biological challenges to its clinical use. N Y State Dent J 2012;78:37–39 25. Long J, Li P, Du HM, et al. Effects of bone morphogenetic protein 2 gene therapy on new bone formation during mandibular distraction osteogenesis at rapid rate in rabbits. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:50–57 26. Varenne O, Gerard RD, Sinnaeve P, et al. Percutaneous adenoviral gene transfer into porcine coronary arteries: is catheter-based gene delivery adapted to coronary circulation? Hum Gene Ther 1999;10:1105–1115 27. Cakir-Ozkan N, Eyibilen A, Ozkan F, et al. Immunohistochemical analysis of reconstructed sheep mandibles: transport distraction osteogenesis versus autogenous bone grafting. J Oral Maxillofac Surg 2011;69:1248–1254 28. Grayson BH, Rowe NM, Hollier LH Jr, et al. Development of a device for the delivery of agents to bone during distraction osteogenesis. J Craniofac Surg 2001;12:19–25 29. Kwon SG, Kim YO, Rah DK. Anterior cranial base reconstruction with a reverse temporalis muscle flap and calvarial bone graft. Arch Plast Surg 2012;39:345–351 30. Cho JY, Jang YC, Hur GY, et al. One stage reconstruction of skull exposed by burn injury using a tissue expansion technique. Arch Plast Surg 2012;39:118–123 31. Greenwald JA, Mehrara BJ, Spector JA, et al. Biomolecular mechanisms of calvarial bone induction: immature versus mature dura mater. Plast Reconstr Surg 2000;105:1382–1392 32. Greenwald JA, Mehrara BJ, Spector JA, et al. Immature versus mature dura mater: II. Differential expression of genes important to calvarial reossification. Plast Reconstr Surg 2000;106:630–638; discussion 639 33. Ho ML, Chang JK, Wang GJ. Effects of ketorolac on bone repair: a radiographic study in modeled demineralized bone matrix grafted rabbits. Pharmacology 1998;57:148–159 34. Reikeraas O, Engebretsen L. Effects of ketoralac tromethamine and indomethacin on primary and secondary bone healing. An experimental study in rats. Arch Orthop Trauma Surg 1998;118:50–52 35. Mullis BH, Copland ST, Weinhold PS, et al. Effect of COX-2 inhibitors and non-steroidal anti-inflammatory drugs on a mouse fracture model. Injury 2006;37:827–837 36. Glassman SD, Rose SM, Dimar JR, et al. The effect of postoperative nonsteroidal anti-inflammatory drug administration on spinal fusion. Spine (Phila Pa 1976) 1998;23:834–838 37. Martin GJ Jr, Boden SD, Titus L. Recombinant human bone morphogenetic protein-2 overcomes the inhibitory effect of ketorolac, a nonsteroidal anti-inflammatory drug (NSAID), on posterolateral lumbar intertransverse process spine fusion. Spine (Phila Pa 1976) 1999;24:2188–2193; discussion 2193–2184
© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
Transport Distraction Osteogenesis With Recombinant Human Bone Morphogenic Protein-2 for Large Calvarial Defect Reconstruction Seung Yong Song, MD,* In Sik Yun, MD,* Chung Hun Kim, MD, PhD,† Dae Gon Woo, PhD,‡ and Yong Oock Kim, MD, PhD* Background: Transport distraction osteogenesis (TDO) has been used in attempts to treat large calvarial defects but has, until now, lacked consistency and reliability. To achieve sufficient bone formation, the effect of TDO was compared to the effect of TDO combined with recombinant human bone morphogenic protein-2 (rhBMP-2). Methods: Fourteen dogs were divided into 2 groups; 6 animals in the control group received TDO only, and 8 received TDO combined with rhBMP-2. A calvarial defect 33 35 mm in size was generated, and the drug-delivering internal distractor was applied. After a 5-day latency period, distraction with rhBMP-2 at 10 μg/day was initiated at a rate of 2 mm/day. This was followed by a consolidation period of 3 months, after which areas of osteogenesis and strength were measured and histologic examinations were conducted. Results: The average area of osteogenesis was higher in the experimental group (P < 0.01). Regenerated bone of the experimental group showed increased strength (P < 0.05). Histological examination showed typical mature bone in the experimental group. Prominent osteoblastic rimming was observed in the bone marrow of the experimental group. Conclusions: TDO with an internal distraction device delivering rhBMP-2 can enhance bone regeneration of large calvarial defects in a dog model. These results suggest the potential for human clinical testing of TDO combined with rhBMP-2. Key Words: Transport distraction osteogenesis, distraction osteogenesis, calvarial reconstruction, bone morphogenic protein-2 (J Craniofac Surg 2014;25: 502–508)
From the *Department of Plastic Reconstructive Surgery, Institute for Human Tissue Restoration, Yonsei University College of Medicine, Seoul; †Department of Plastic and Reconstructive Surgery, CHA Bundang Medical Center, CHA University School of Medicine, Gyeonggi-do; and ‡Cardiovascular Devices Division, Ministry of Food and Drugs, Osong, Republic of Korea. Received September 4, 2013. Accepted for publication December 28, 2013. Address correspondence and reprint requests to Yong Oock Kim, MD, PhD, Department of Plastic and Reconstructive Surgery, Institute for Human Tissue Restoration, Yonsei University College of Medicine, 250 Seongsan-no, Seodaemoon-gu, Seoul 120-752, Republic of Korea; E-mail: [email protected] The authors report no conflicts of interest. This work was supported by the Seoul R&BD program under Grant PA100085 (2010). Copyright © 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000000672
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ince first being reported by Bouletreau et al, calvarial defect repair by transport distraction osteogenesis (TDO) has been conducted in only a few animal models and one human as of 2010.1–10 One possible reason which makes this procedure not being widely used until now is the inconsistency of the results. Some studies reported TDO was effective for calvarial reconstruction.1,5,8 However, there was also a report that the volume of newly generated bone was not sufficient by this method.6 Another remarkable point is wide variability of the results. Even in the study that TDO had favorable effect on the treatment of cranial defect, one subject in the control group healed completely without DO, while the other 3 control animals showed no appreciable degree of healing.5 In another study using TDO for calvarial defect, wide variability of bone regeneration was also reported from 24% to 96% in TDO group and from 13% to 80% in control group.6 To promote bone regeneration by TDO, Yun et al used internal type of distraction device. Even in their study, bone formation from 26% to 97% in TDO group and from 0 to 82.9% in control group was reported. It had advantages of increased mechanical stability during distraction osteogenesis (DO), which resulted in more bone regeneration despite of a short consolidation period.7 However, variability of the results still remained to be overcome. Similar phenomena were seen in other reports.11 So, protocol for more predictable and sufficient bone formation is still necessary for clinical application. Two factors were prioritized in this study to overcome drawbacks of previous studies. One was the stability of the distractor. External distractors can generate a strong distraction force and permit easy handling of the distraction vector but suffer from inevitable instability because some of its structure is exposed outside of the skin, an issue that is of particular concern in young children and animals.12 These physical limitations can cause subacute or chronic inflammation, which may disrupt bone regeneration during the distraction period. In this regard, internal distractors have the advantage of reduced exposure to contaminants outside the skin. The other important factor in optimized bone formation by TDO is the administration of humoral factors. Among the various factors which can induce bone growth, bone morphogenic protein (BMP) is believed to be the primary facilitator of bone regeneration in DO.13–18 BMP belongs to the transforming growth factor β superfamily. It promotes vascular invasion, bone formation, bone remodeling, and bone marrow differentiation by directing cell differentiation of mesenchymal stem cells to become osteoblasts and osteocytes, which in turn build and renew the extracellular matrix.19–21 In this study, rhBMP-2 is used to supplement TDO. rhBMP-2 is known to enhance consolidation, shorten the period of distraction, and eventually accelerate bone regeneration.13,16,17,22,23 We used a specially designed drug-delivering distraction device for periodic injection of rhBMP-2 during TDO. Here we compare the bone regenerative effect of mechanical factors (TDO) alone to the combination of mechanical (TDO) and
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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 2, March 2014
FIGURE 1. A newly designed drug-delivering internal distractor. A, Inlet of vehicle, sealed with silicone to prevent retrograde infection. B, Outlet of vehicle. C, Rotator rod. D, Guide rail
humoral (rhBMP-2) factors. This study will help demonstrate the feasibility and reliability of TDO for human clinical trials.
MATERIALS AND METHODS Experimental Animals and Groups Fourteen mongrels (female, 5 months gestational age) were divided into 2 groups, a control (n = 6) group receiving only TDO and an experimental group (n = 8) receiving TDO and rhBMP-2. All experimental animals were maintained at the animal facility at Yonsei University Medical College, and all experimental procedures were performed after approval by the Institutional Animal Care and Use Committee (2011-0032).
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with inhaling isoflurane (Foran; Choongwae Pharmaceutical Corporation, Hwaseong, Republic of Korea). To reduce cerebral edema, the concentration of isoflurane was maintained at 2% to 3%, and the CO2 concentration was reduced by inducing the hyperventilation state. Animal was placed in the dorsal position and sterilized with povidone iodine. A longitudinal incision was made in the midline of the scalp, and the calvarium was exposed. Ostectomy was performed with a burr to create the calvarial defect. Defect size was 33 35 (width length) mm and the long axis was centered on the midline of the cranium. A bone fragment of 30 8 (width length) mm was prepared from ostectomized bone to make the transport disc (TD). For easy movement of the transport disc, its width was smaller than that of the defect. The periosteum of the TD was removed. The internal bone distractor was specially designed with 2 guide rails for this experiment. For the injection of rhBMP-2 once a day during the distraction period, an inlet was located at the rotating part of the distractor. It was sealed with silicone to prevent retrograde infection. Injected rhBMP-2 flowed out to the distraction zone (Fig. 1). Microplates were used to fix the transport disc in place, and the disc was then connected to the distractor (Fig. 2). Muscles and the skin were sutured by #3-0 Vicryl and #4-0 nylon sutures. So, the whole device was placed under the soft tissue except rotator rod. After the surgery, the analgesic ketorolac (Tarasyn; Roche Pharmaceutical Company Ltd, Basel, Switzerland) and the antibiotic ceftriaxone (50 mg/kg; Donghwa Pharmaceutical Company Ltd, Ansan, Republic of Korea) were injected intramuscularly for 2 days.
Distraction Distraction was initiated after a 5-day latency period. The distraction rate was 2 mm/day, and it took 13 days to complete the entire defect length. rhBMP-2 (BioVision, Milpitas, CA) was prepared at a concentration of 10 μg/mL with phosphate buffered saline. It was injected at a rate of 1 mL/day after turning the rotating rod of distractor for the entire 13 days of distraction. This was followed by a consolidation period of 3 months (Fig. 3).
Operative Procedure All animals were anesthetized by intravenous injection of midazolam (0.2 mg/kg; Bukwang Pharmaceutical Company Ltd, Ansan, Republic of Korea) and propofol (3 mg/kg, Pofol V; Dongkook Pharmaceutical Company Ltd, Jincheon, Republic of Korea). In succession, endotracheal intubation was performed
Radiologic Studies A plain X-ray of the skull was taken in all experimental animals 5 days before starting the distraction. Another X-ray was taken near the end of the bone distraction period to check the status of the distraction device. Computed tomography (CT) images were obtained (Somatom Sensation 64; Siemens Medical Systems, Erlangen, Germany) at the end of the study after euthanizing the experimental animal (Fig. 3).
Assessment of Results Quantitative analysis of the area of regenerated bone was accomplished by measuring the area of the bone defect from the CT images. The newly formed bone area was measured by subtracting the bone defect area that remained in the 3-dimensional CT from the bone defect area created at the time of surgery (35 35 mm).
FIGURE 2. Intraoperative photograph. The newly designed drug-delivering internal distractor was applied to the calvaria of mongrel dogs. The size of calvarial defect and transport disc (TD) was 33 35 (width length) mm and 30 8 (width length) mm, respectively. Periosteum and dura mater were detached from TD.
FIGURE 3. Schedules of experiment. This study was designed with 5 days of latency, 13 days of distraction, and 3 months of consolidation. Radiologic studies included plain X-rays on postoperative 0 day and near the end of the consolidation period, with 3-dimensional computed tomography done at the end of the study.
© 2014 Mutaz B. Habal, MD
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TABLE 1. Area Ratio* of Regenerated Bone
No.
Control (Without rhBMP-2) (%)
Experimental Group (With rhBMP-2) (%)
95.21 68.87 72.47 54.00 49.33 68.38 68.04 ± 18.07
93.77 99.03 98.79 94.29 84.83 97.12 94.64 ± 5.29**
1 2 3 4 5 6 Mean ± SD †
*Area ratio: the ratio of regenerated area to the original defect. †
SD, standard deviation.
**P < 0.01.
We compared the calculated ratio (in percent) of the measured new bone formation area with the initial bone defect area. To measure bone strength, newly formed bone specimen was obtained from the TDO site. Each specimen was harvested with a full thickness of a bone. The strength of the harvested bone was estimated by compression strength measurements. These measurements were done using an Instron 5848 microtester (Instron, Norwood, MA). The strength value is presented in newtons per square meter. The strength of the experimental group and the control group was measured 3 times. Finally, the measured data were compared between the TDO regenerated bone and the normal calvarial bone by the strength ratio of regenerated bone to normal adjacent bone. For histologic examination, the piece of extracted bone was fixed in 4% phosphate buffered formalin (pH 7.73) solution for 14 days. After the dehydration process, the samples were placed in an alcohol and acetone solution. Then, they were further fixed with a methyl-methacrylate solution (Merck, Hohenbrunn, Germany) to prevent calcium demineralization. Next, the bone pieces were cut with a diamond saw at 30-μm intervals along the axis of distraction. The samples were then stained with hematoxylin and eosin, and a visual comparison was done under a microscope.
Statistical Analysis All data from our experimental results are represented as mean ± standard deviation (SD). Comparisons between groups were performed by the Student t test. A value of P less than 0.05 was
FIGURE 4. Photograph at the end of the consolidation period. A, The control group. White arrow indicated non-healed area at the end of distraction. B, The experimental group. Regenerated bone nearly covered the defect.
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considered significant. All data analyses were performed using the Statistical Package for the Social Sciences (SPSS v. 15.0; SPSS Inc, Chicago, IL).
RESULTS During the experiment, 2 dogs of the experimental group were excluded from this study due to infections. By the end of the study, data for 6 dogs in the control group and 6 dogs in the experimental group were used for analysis. The bone specimen for strength measurement was inadequate in 1 animal, so strength tests of the control group were performed in only 5 animals. Radiologic studies were performed according to a predetermined schedule with plain X-ray performed immediately postoperatively and again at the end of distraction, while 3-dimensional CT was completed at the end of consolidation.
Area of Osteogenesis The control group and the experimental group showed bone regeneration equal to 68.04 ± 18.07% and 94.64 ± 5.29% of the original defect, respectively. The area of osteogenesis was higher in the rhBMP-2-treated group with statistical significance (P < 0.01) (Table 1, Figs. 4–6).
Strength Regenerated bone of the control group and the experimental group showed strength values of 23.85 ± 6.19 N/mm2 and 53.75 ± 18.66 N/mm2 by compression test, respectively. We compared the 2 groups using the ratio of regenerated bone strength to normal bone strength. Regenerated bone by TDO combined with rhBMP-2 showed increased strength with statistical significance (P < 0.05) (Table 2, Fig. 7).
Histology Histologic examination revealed that normal bone components such as bone marrow, lamella, and osteocytes existed in the normal, control, and experimental group. However, bony specimen of the control group showed reduced lamella portion compared to normal bone and its lamella was not matured compared to the normal bone. So, it can be considered as woven bone rather than mature bone. The bony specimen of the experimental group had similar thickness and cortex portion with normal bone. These findings indicate that regenerated bone of the experimental group is more close to the normal cancellous bone (Fig. 8). In the bone marrow, osteoblasts rimming was observed in the control and the experimental group (Fig. 9). The number of osteoblasts was significantly higher in the experimental group compared to the control group (P < 0.01). On the other hand, the control
FIGURE 5. 3D CT images of the calvarial defects at the end of consolidation in each animal. Upper 1–6 images show remnant calvarial defects of control group. Lower 1–6 images show remnant calvarial defects of experimental group.
© 2014 Mutaz B. Habal, MD
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TABLE 2. Measurement of Bone Stiffness No.
Control (Without rhBMP-2) 2
1 2 3 4 5 6 Mean ± SD † †
Normal Bone (N/mm ) (N) 25.28 32.40 27.75 43.14 54.95 123.40 51.15 ± 37.06
Experimental Group (With rhBMP-2) 2
Regenerated Bone (N/mm ) (R) 15.26 20.21 Not measurable 25.41 31.00 27.39 23.85 ± 6.19
Ratio (R/N) 0.60 0.62 Not measurable 0.59 0.56 0.22 0.52
Normal Bone (N/mm2) (N) 60.87 23.21 46.70 42.82 76.18 84.48 55.71 ± 22.7
Regenerated Bone (N/mm2) (R) 29.06 34.83 75.74 55.20 70.31 57.36 53.75 ± 18.66
Ratio (R/N) 0.48 1.50 1.62 1.29 0.92 0.68 1.08*
SD, standard deviation.
*P < 0.05.
In TDO, regeneration of bone for large defect should be achieved using relatively small bare bone segment, so, it had less reliability on efficacy of bone formation and strength. This may be the reason that there were few reports about this technique since its first introduction. We believe that 3 factors are important for the successful repair of large calvarial defects using TDO. The first is the ratio of the transport disc size to bone defect size. In previous reports, the size ratio of the transport disc to calvarial defect ranged from 19% to 50%.1–10 In this study, we designed the transport disc size for a ratio of 18%, smaller than any previous study (Table 3). The length of our transport disc was 8 mm, which was the smallest size that permitted fixation to the distraction device. The defect size in this study was nearly one third of the whole cranium, a parameter that could provide useful information in the application to humans. However, the appropriate size of a transport disc has not been determined until now. Minimal transport disc size with maximal bone regeneration may be ideal in the clinical setting, and optimization of transport disc size according to defect size should be investigated for human application in the future.
Another important factor determining the success of TDO is the type of distraction device used. Nearly half of previous studies of TDO for calvarial defects used an external distractor; however, in recent years, internal distractors have been developed and are being used more frequently (Table 3). Device stability has a significant impact on DO success, especially in animal experiments. In this study, we used an internal distractor with guide rails. This allowed for increased durability and stability, so no extrusion or dysfunction of the device was observed in this study. There is some evidence to suggest it might result in increased bone regeneration.6,7 The last factor we believe to be important for TDO success is the use of humoral factors to facilitate or accelerate bone regeneration. DO with BMP treatment is one of the most well-known regimens capable of improving the results of bony regeneration. BMP is a strong osteoinducer and plays a pivotal role in the molecular signaling cascade leading to bone regeneration and remodeling.17 rhBMP-2 in particular is one of the most powerful growth factors for bone regeneration. In 2007, the Food and Drug Administration (FDA) approved the use of BMP-2 delivered with an absorbable collagen sponge carrier for clinical use in craniofacial deformities.24 DO combined with rhBMP-2 in a collagen sponge increased ossification and mineral content and decreased the consolidation period in the long bone.17,23 Further support for the use of BMP-2
FIGURE 6. Comparison of regenerated area. The experimental group showed significantly increased new bone formation compared to the control group (**P < 0.01).
FIGURE 7. Comparison of bone strength. Strength ratio was the ratio of regenerated bone to normal bone in each group. Regenerated bone from the experimental group showed significantly increased bone strength compared to the control group (*P < 0.05).
group also had significantly increased osteoblasts compared to normal bone (P < 0.01) (Fig. 10).
DISCUSSION
© 2014 Mutaz B. Habal, MD
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4 and TGF-β were increased in the DO model.27 From this, we can infer the important role of growth factors, including BMP, in bone regeneration with TDO. Icekson et al showed TDO without growth factor can be an effective tool for closing full-thickness calvarial defects in sheep. However, they used only 4 animals, the defect size was about 20 20 mm, a smaller defect than what was used in this study, and the size ratio of the transport disc to calvarial defect size was 50%, bigger than our study.5 In the comparison of osteogenesis area, the average ratio of regenerated bone is higher in the experimental group with statistical significance. This may be due to increased mechanical stability of newly designed distractor. The ratio of regeneration was 94.64% in the experimental group, and the magnitude of this result likely has significant clinical importance, especially because the size of the original defect was so large. Moreover, the variation seen in the results of the control group was much reduced after adding
FIGURE 8. Microscopic findings of normal (A), control (B), and experimental (C) group (original magnification 40). Normal bone components such as bone marrow (BM), lamella (L), and osteocytes are observed in all groups. However, cortex of regenerated bone in the control group is relatively small and consists of woven bone. Regenerated bone of the experimental group has similar thickness with normal bone and the amount of cortex is also similar with normal bone. It is more close to the histology of normal cancellous bone.
came in a study of membranous bone by Issa et al when they reported that rhBMP-2 with a monoolein gel carrier could enhance bone formation in the mandible and may potentially reduce the treatment period.14 Some researchers have also used mesenchymal stem cell–based gene therapy to continuously release BMP-2 during the distraction and consolidation periods in an effort to maximize bone regeneration.25 However, gene therapy has historically been difficult to use clinically due to unpredictable expression, variability in the effective period, complexity in gene transfection processes, and the unknown hazards of using an adenoviral vector.26 Fewer studies have been conducted in the TDO model using BMP-2. Cakir-Ozkan et al compared the effects of TDO and bone grafting in a sheep model and found that expression of BMP-2,
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FIGURE 9. Microscopic findings of normal (A), control (B), and experimental (C) group (original magnification 200). The marrow of the control and the experimental group reveals osteoblastic rimming (arrow).
© 2014 Mutaz B. Habal, MD
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FIGURE 10. Comparison of the number of osteoblasts in the normal, control, and experimental group (original magnification 100). The experimental group had significantly increased osteoblasts compared to normal and the control group (**P < 0.01). The control group also had increased number of osteoblasts compared to normal group (**P < 0.01).
rhBMP-2 to the treatment, as indicated by the small standard deviation. This could indicate that variable production and secretion of humoral factors in each organism explains the observed unpredictable bone defect healing. According to these results, TDO combined with rhBMP-2 may allow bone regeneration to proceed in a more consistent or predictable fashion. In this study, a new distractor for TDO was devised that allowed injection of liquid-type humoral factors like rhBMP-2. The use of a drug-delivering distractor for TDO has the advantage of simplicity, easy dose calculation, and increased predictability. The first drug-releasing distractor was devised by Grayson et al.28 Konas et al have also recently reported that DO with a drugreleasing internal distractor facilitated ossification of the femur in rats.11 However, their studies used DO rather than TDO and was conducted in the mandible or femur. Moreover, our distractor had guide rails on both sides of the device to allow stable movement of the transport disc. This design had also had the advantage of following a curved surface, unlike the traditional single-rod internal distractors. Because the calvarium has a round surface, transport discs should pass along a similarly curved area in most cases.9,10,29
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Two animals in the experimental group were excluded from the study because of infection at the site of distraction despite sealing the injection inlet with a silicone membrane in an effort to prevent retrograde infection. We guess that the exposed rod of distraction device may be an alternative route of the infection. Further research should be conducted to prevent perioperative and postoperative infection because internal distractors are placed in vulnerable locations deep within vital structures of the cranium.30 The distraction rate of this study was higher than is typical at 2 mm/day. As previously described, BMP-2 could shorten the distraction period,16 and in preliminary studies, we experienced similar results using TDO with rhBMP-2. So, the rate of the distraction was relatively faster compared to conventional DO. We detached periosteum and dura mater from the transport disc, but in some reports vascularized transport discs have been associated with improved success of DO.8 This is theoretically reasonable because ensuring disc viability is important for bone regeneration, and it is well known that dura mater is of paramount importance in calvarial bone regeneration31,32 However, preservation of dura mater with transport disc is not easy considering human application, so, the dura was detached from the transport disc in this study. This allowed a greater degree of freedom in movement of transport disc during distraction. In histologic examination, prominent osteoblastic rimming was observed in the bone marrow of the experimental group. This is the hallmark of bone regeneration. And the number of osteoblasts was increased in order of the normal, control, and experimental group. This finding reflects vigorous bony regeneration occurred by TDO itself. And it also indicates that application of rh-BMP-2 into distraction zone can augment this process additionally. It is reasonable because BMP promote bone formation, bone remodeling, and bone marrow differentiation by directing cell differentiation of mesenchymal stem cells to osteoblasts and osteocytes, as previously described.19–21 Non-steroidal anti-inflammatory drug (NSAIDs), ketorolac, was used to relieve pain of the experimental animals in this study. There were some reports that ketorolac could interfere negatively with new bone formation in animal and human.33–36 However, Martin et al reported that rhBMP-2 could overcome the detrimental effect of ketorolac in rabbit model.37 Because ketorolac was also used in the control group, the exact effect of NSAIDs in TDO for calvarial defect should be investigated by properly designed study in the future. TDO with administration of rhBMP-2 resulted in regeneration of about 94% of the original calvarial defect with a strength ratio nearly equal (1.08) to normal bone. The methods used in this study can also shorten the duration of treatment. Most importantly,
TABLE 3. Previous Literatures of Transport Distraction Osteogenesis for Calvarial Defect
Subject Distractor type Defect size (D) (mm) Transport disc size (T) (mm) Disc ratio (T/D) (%)
Bouletreau et al
Hirano et al
Kramer et al
Muller et al
Hong et al
Yun et al
Icekson et al
Cho-Lee GY et al
Durmus et al
Steinbacher et al
This Study
Rabbit External
Rabbit Internal
Sheep External
Sheep External
Dog External
Dog Internal
Sheep Internal
Human Internal
Sheep External
Rabbit Internal
Dog Internal
14 7
40 60
50 60
15 33
15 35
20 20
50 85
50 60
16 16
33 35
40 20
40 20
15 7
14 7
20 10
50 20
40 20
10 16a
30 7
33
27
21
19
50
24
27
38
18
15 15 15 10a
(7–10) 10 (trapezoid)*
40
36
*
Additional craniectomy was performed outside of the defect for preparation of transport disc.
© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
507
The Journal of Craniofacial Surgery • Volume 25, Number 2, March 2014
Song et al
the results of this study further demonstrate proof of concept and reliability in support of human application.
CONCLUSIONS Transport disc distraction osteogenesis with an internal distraction device capable of delivering bone morphogenic protein-2 can enhance bone regeneration of calvarial defects in a dog model. The results on area of regeneration and strength indicate that clinical application to humans is possible. ACKNOWLEDGMENTS The authors thank Ji Hyung Park and Han Sung Kim (Department of Biomedical Engineering and Institute of Medical Engineering, Yonsei University, Wonju, Republic of Korea) for assistance with measurement of bone strengths.
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© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
