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Distraction osteogenesis after irradiation in a rabbit model
J Orthop Sci (2005) 10:627–633 DOI 10.1007/s00776-005-0945-1
Original article Distraction osteogenesis after irradiation in a rabbit model Hiroyuki Tsuchiya, Kenji Uehara, Keisuke Sakurakichi, Koji Watanabe, Hidenori Matsubara, and Katsuro Tomita Department of Orthopedic Surgery, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan
Abstract Background. The present study was performed to investigate the effects of preoperative irradiation on distraction osteogenesis, as little is known about how preoperative irradiation delays distraction osteogenesis. Methods. A single dose of irradiation was applied to the right rear legs of rabbits. This was followed by tibial lengthening at a rate of 0.5 mm/day, which was continued for 4 weeks. Bone regeneration was examined radiographically and histologically. Results. In the irradiation group, the radiographs showed little regeneration during the elongation phase. During the maturation phase, the callus appeared slowly, and its formation was spotty. Furthermore, regeneration was not completed until the fourth week of the maturation period. Histological examination at the end of distraction showed a gap in the distraction consisting of loose connective tissue, with part of the fibrous tissue oriented longitudinally. Four weeks after completion of distraction, the major part of the radiolucent region consisted of cartilage. The spotty osteogenesis was identified as enchondral ossification. Immunohistochemical examination of the regeneration area revealed that the blood vessels were extremely localized, and that the level of expression of vascular endothelial growth factor (VEGF) in the osteoblasts was high. Microangiography showed that vascularization at the distracted sites was poor. Distraction osteogenesis was decreased markedly by preoperative irradiation in terms of both rate and process. The results suggested that most of the osteoprogenitor cells were damaged immediately after irradiation. The high level of VEGF in the osteoblasts and the enchondral ossification also suggested a hypoxic state in the distracted region. Conclusions. Preoperative irradiation interferes with distraction osteogenesis by inducing a state of poor angiogenesis.
Introduction After induction of distraction osteogenesis1 or callotasis,2 most difficult orthopedic problems (e.g., limb length discrepancy, severe deformity, complicated nonunion, osteomyelitis, extensive bone loss) can be treated successfully.3–9 The biological advantage of distraction osteogenesis is the regeneration of living bone, with the regenerated tissue eventually having the same quality as native bone. Recently, distraction osteogenesis was also shown to provide durable reconstruction for skeletal defects after bone tumor excision for the restoration of natural limbs.9–11 In the treatment of cancer in children, irradiation is frequently indicated for patients with malignant lymphoma, Ewing’s sarcoma, rhabdomyosarcoma, leukemia, or other malignancies. When the epiphyseal growth plate is involved in the irradiation field, limblength inequality and deformities due to partial or entire growth arrest of the epiphyseal plate is anticipated.12 Limb lengthening and deformity correction using distraction osteogenesis is beneficial for these patients if feasible, although it has been reported that irradiation delays callus formation and maturation in fracture and bone defect models.13–15 This raised the question of how preoperative irradiation delays distraction osteogenesis. The effects of preoperative irradiation on distraction osteogenesis were investigated, as little is known about the mechanism involved. The results of the present study can lead to improvement of distraction osteogenesis in irradiated bones. Materials and methods Irradiation and lengthening procedure
Offprint requests to: H. Tsuchiya Received: April 20, 2005 / Accepted: July 4, 2005
Forty male Japanese White rabbits, weighing about 3 kg, were used in this study. Fifteen rabbits each were allotted to the control group and the 15-Gy group,
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H. Tsuchiya et al.: Distraction osteogenesis after irradiation
after completion of lengthening. The harvested tibial specimens were prepared for light microscopy by fixation in phosphate-buffered formaldehyde. Specimens were rinsed in buffer, decalcified, embedded in paraffin, and sectioned in the sagittal plane. Routine staining was performed with hematoxylin and eosin and immunohistochemical staining with an anti-vascular endothelial growth factor (VEGF) antibody. Microangiography Fig. 1. Preoperative irradiation. The irradiation is applied to the right leg in a medial-to-lateral direction at a focus–skin distance of 25 cm
respectively. Ten rabbits were used for radiological and histological analysis and five for microangiographic analysis in each group. Five rabbits each were used just for radiological analysis in both the 10-Gy and 20-Gy groups. Animals were anesthetized with an intramuscular injection of ketamine hydrochloride (50 mg/kg body weight) (Sankyo, Tokyo, Japan) and intravenous injection of pentobarbital sodium (50 mg/kg body weight) (Dainippon Pharmaceutical, Tokyo, Japan). A single dose of irradiation was administered in a medial-tolateral direction to the right rear leg at 150 kV and 20 mA at a focus–skin distance of 25 cm targeted for regeneration (Fig. 1). The doses were 10, 15, and 20 Gy. The control group underwent no irradiation. Just after irradiation, a 5-cm skin incision was made over the medial aspect of the right tibia. A unilateral dynamic external fixator of our design was fixed with four half-pins 2 mm in diameter (Howmedica, Geneva, Switzerland) inserted into the tibia. Osteotomy was performed on the tibia at the tibiofibular junction between the two inner half-pins using a thread wire saw 0.36 mm in diameter (Koshiya, Kanazawa, Japan). After a 7-day latency period, lengthening of the tibia was initiated at a rate of 0.5 mm/day and continued for 28 days with a total elongation of 14 mm. The regenerated area was allowed to consolidate for 4 weeks after completion of the distraction. Radiological analysis Radiographs of the leg were obtained once a week for 8 weeks after the initiation of lengthening. Five rabbits each in the 10- and 20-Gy groups were used only for radiological evaluation. Histological analysis Each of the five rabbits in the control and 15-Gy groups were examined. The animals were killed 0 and 4 weeks
Each of the five rabbits in the control and 15-Gy groups were killed 4 weeks after completion of lengthening by intraaortic injection of Microfil (Flow Tek, Carver, MA, USA). Using the Spalteholtz technique, the entire tibias were removed, stripped of soft tissues and periosteum, and fixed in 10% buffered formalin. After decalcification in 0.2 M EDTA, they were cleared with benzyl benzoate and the vascular patterns were examined. The average vascular occupation rates of the nine regions of interest (ROI) at the distracted site were measured using image analysis software (NIH image 1.61, Macintosh version). Differences in the vascular occupying rate between the two groups were analyzed using the unpaired Student’s t-test. All experiments were performed under the guidelines for animal experiments stipulated at Kanazawa University Graduate School of Medical Science.
Results Radiological analysis The radiographs of the control group showed good callus formation early during distraction with three distinct zones (proximal and distal sclerotic zones and a central fibrous zone). The distracted callus matured before the fourth week of the consolidation period (Fig. 2). In addition, the distracted callus formation of the 10-Gy group was similar to that of the control group (Fig. 3). In the 15-Gy group, however, the radiographs showed little regeneration during the elongation phase. The callus appeared slowly during the maturation phase and did not show three distinct zones but had only a spotty distribution. Furthermore, regeneration was not completed until the fourth week of the maturation period (Fig. 4). The radiographs of the 20-Gy group showed little regeneration until the fourth week of the maturation period (Fig. 5). Based on these results, further examinations of the control and 15-Gy groups were performed because it seemed to be adequate to use the 15-Gy group to investigate the effects of radiation.
H. Tsuchiya et al.: Distraction osteogenesis after irradiation
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Histological analysis In the control group, the distraction gap was seen to consist of three regions at the end of the lengthening period. In the radiolucent zone, parallel collagen bundles were arranged longitudinally. In the adjacent sclerotic zone, callus was formed by intramembranous ossification (Fig. 6). Four weeks after completion
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Fig. 2. Radiographs of tibias from the control group. a After 14 days of distraction. b After 28 days of distraction. c At 14 days after completion of distraction. d At 28 days after completion of distraction. The distracted callus is observed early during distraction with three distinct zones
c,d
Fig. 3. Radiographs of tibias from the 10-Gy irradiation group. a After 14 days of distraction. b After 28 days of distraction. c At 14 days after completion of distraction. d At 28 days after completion of distraction. The distracted callus formation of the 10-Gy group is similar to that of the control group
c,d
Fig. 4. Radiographs of tibias from the 15-Gy irradiation group. a After 14 days of distraction. b After 28 days of distraction. c At 14 days after completion of distraction. d At 28 days after completion of distraction. The callus appeared slowly during the maturation phase and showed spotty formation. Regeneration is not completed until the 28th day of the maturation period
of distraction, a mature trabecular network and neocorticalization were seen (Fig. 7). At the end of lengthening, the 15-Gy group showed a gap in the distraction consisting of loose connective tissue, with part of the fibrous tissue oriented longitudinally. There was no evidence of new mineralization (Fig. 8). Four weeks after completion of distraction, the major part of the radiolucent region consisted of cartilage. There was no
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c,d
a,b
Fig. 6. Longitudinal section from the control group after 28 days of distraction. In the radiolucent zone, parallel collagen bundles are arranged longitudinally (white arrow). In the adjacent sclerotic zone, the callus is formed by intramembranous ossification (black arrow). Bar 200 mm. Hematoxylin-eosin
evidence of the normal regeneration pattern. The spotty osteogenesis was identified as enchondral ossification (Fig. 9). Immunohistochemical staining of VEGF in the control group revealed diffuse vascularization with positive staining (Fig. 10). In the 15-Gy group, the blood vessels were scanty and localized, and the level of VEGF expression in the osteoblasts was stronger than in the control group (Fig. 11). Microangiography Microangiograms of the control group showed a diffuse, dense vascular network not only in the distracted region but also in the host bone. Those in the 15-Gy group showed that the vascularization was extremely thin in the whole tibia (Fig. 12). Neovasculization induced by distraction osteogenesis was inhibited in the 15-Gy
Fig. 5. Radiographs of tibias from the 20-Gy irradiation group. a After 14 days of distraction. b After 28 days of distraction. c At 14 days after completion of distraction. d At 28 days after completion of distraction. The radiographs showed little regeneration until 28 days after completion of distraction
a,b Fig. 7. Longitudinal section from the control group 28 days after completion of distraction. a Total longitudinal section. b Close-up of the boxed area in a. Histological examination showed a mature trabecular network and neocorticalization. Bar 200 mm. Hematoxylin-eosin
group. The average vascular occupying rates at the distracted site were 77.3% ± 3.5% in the control group and 46.1% ± 9.7% in the 15 Gy group. The rate of the 15 Gy group was significantly lower than that of the control group (P < 0.001).
Discussion This experiment demonstrated that distraction osteogenesis was markedly affected by preoperative irradiation. Gantous et al. reported that callus formation of the distracted site was not inhibited in a canine mandibular model when distraction was performed 6 months after 50-Gy irradiation in 20 fractions.16 In the fracture model, however, the length of time from irradiation to fracture is considered a major factor for union.15 That is,
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a,b Fig. 8. Longitudinal section from the 15-Gy irradiation group after 28 days of distraction. a Total longitudinal section. b Close-up of the boxed area in a. The gap in the distraction consists of loose connective tissue with part of the fibrous tissue oriented longitudinally. Bar 50 mm. Hematoxylin-eosin
Fig. 10. Immunohistochemical staining of vascular endothelial growth factor (VEGF) of the control group 28 days after completion of distraction. Diffuse vascularization with positive staining is observed (white arrows). The osteoblasts at the trabecula are also positive (partially strong) (black arrows). Most osteoblasts lining the trabeculae show relatively weak expression of VEGF
a,b Fig. 9. Longitudinal section from the 15-Gy irradiation group 28 days after completion of distraction. a Total longitudinal section. b Close-up of the boxed area in a. The major part of the radiolucent region consists of cartilage. Spotty osteogenesis is identified as enchondral ossification. OB, osteoblasts; CH, chondrocytes. Bar 100 mm. Hematoxylin-eosin
a longer time after irradiation is associated with fewer adverse effects of irradiation on callus formation. In the present study, distraction was performed immediately after irradiation. During the distraction phase, regeneration did not consist of new bone but of loose connective tissue. These observations suggested that most of the osteoprogenitor cells were damaged immediately after irradiation. Because the irradiation in our study was carried out at the time of the operation, there is little time for the damaged cells to recover during the waiting period (7 days) before distraction.
Fig. 11. Immunohistochemical staining for VEGF of the 15 Gy irradiation group 28 days after completion of distraction. The vascularization is much localized, and the presence of VEGF in the osteoblasts is stronger than of the control group (arrows). VEGF is strongly positive in almost all lining osteoblasts
However, some cells were able to proliferate and differentiate into chondroblasts because spotty enchondral ossification was observed during the consolidation period. Enchondral ossification of distraction osteogenesis is known to be species-specific,17,18 and to be due to mechanical instability19,20 and local hypoxia.21,22 It has also been reported that hypoxia induces chondroblastic differentiation of osteoprogenitor cells23 and stimulates
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were entirely different from those in the control group. Irregular regeneration might occur as distraction was completed before the repair of damage to osteoprogenitor cells. In addition, ischemic changes induced by irradiation modified regeneration. In the clinical situation, osteotomy for distraction osteogenesis would be carried out at a different time than the irradiation and at a different site at the present time. However, developments in bioengineering may overcome the adverse effects of irradiation on bone regeneration in terms of transplantation of osteoprogenitor stem cells, osteoblasts, or periosteal cells,29 injection of growth factors,30 implantation of bone matrix,31 or gene therapy using VEGF.32 In conclusion, preoperative irradiation interferes with distraction osteogenesis by inducing a state of poor angiogenesis.
References a,b Fig. 12. Microangiograms of the distracted site. a Control group. b The 15-Gy irradiation group. The control group shows a diffuse, dense vascular network. Vascularization is scanty in the irradiated group
VEGF expression in osteoblasts.24 Little enchondral ossification was observed in the control group, but significant levels of VEGF expression were found in osteoblasts during the maturation period in the irradiated group. These findings suggest that the distracted region had become hypoxic after irradiation because of the poor vascularization, as shown by microangiography, although dense vascular networks are usually found during normal regeneration in rabbits on microangiograms25 and the distraction osteogenesis procedure promotes neovascularization and increases blood flow.20,26 The disturbance of fracture healing is caused by ischemic changes resulting from vascular damage and inhibition of neovascularization induced by irradiation.27 However, more precise experiments on angiogenesis are needed for the accurate identification of hypoxia. The effects of irradiation and hyperbaric oxygen on the regenerated area have been investigated. Hyperbaric oxygen was not able to prevent suppression of osteogenesis caused by radiotherapy, but it might improve bone formation by prolonging high osteogenic activity.28 If inhibition of regeneration healing induced by 15 Gy of irradiation was merely the result of the delay in differentiation and proliferation of osteoprogenitor cells, regeneration would show delayed findings on radiography and histological examination. However, the rate and process of regeneration in the irradiation group
1. Ilizarov GA, Devyatov AA. Operative elongation of the leg with simultaneous correction of the deformities. Ortop Travmatol Protez 1969;30:32–7 (in Russian). 2. De Bastiani G, Aldegheri R, Renzi-Brivio L, Trivella G. Limb lengthening by callus distraction (callotasis). J Pediatr Orthop 1987;7:129–34. 3. Ilizarov GA. Basic principles of transosseous compression and distraction osteosynthesis. Ortop Travmatol Protez 1971;32:7–15 (in Russian). 4. Catagni MA, Guerreschi F, Holman JA, Cattaneo R. Distraction osteogenesis in the treatment of stiff hypertrophic nonunions using the Ilizarov apparatus. Clin Orthop 1994;301:159–63. 5. Paley D, Herzenberg JE, Tetsworth K, McKie J, Bhave A. Deformity planning for frontal and sagittal plane corrective osteotomies. Orthop Clin North Am 1994;25:425–65. 6. Uehara K, Tsuchiya H, Kabata T, Sakurakichi K, Shimozaki E, Tomita K. Ankle arthrodesis and tibial lengthening for congenital sensory neuropathy with anhidrosis. J Orthop Sci 2001;6:430–4. 7. Tsuchiya H, Uehara K, Abdel-Wanis ME, Sakurakichi K, Kabata T, Tomita K. Deformity correction followed by lengthening with the Ilizarov method. Clin Orthop 2002;402:176–83. 8. Sakurakichi K, Tsuchiya H, Uehara K, Kabata T, Yamashiro T, Tomita K. Ankle arthrodesis combined with tibial lengthening using the Ilizarov apparatus. J Orthop Sci 2003;8:20–5. 9. Tsuchiya H, Tomita K. Distraction osteogenesis for treatment of bone loss in the lower extremity. J Orthop Sci 2003;8:116–24. 10. Tsuchiya H, Tomita K, Minematsu K, Mori Y, Asada N, Kitano S. Limb salvage using distraction osteogenesis. J Bone Joint Surg Br 1997;79:403–11. 11. Tsuchiya H, Abdel-Wanis ME, Sakurakichi K, Yamashiro T, Tomita K. Osteosarcoma around the knee: intraepiphyseal excision and biological reconstruction with distraction osteogenesis. J Bone Joint Surg Br 2002;84:1162–6. 12. Robertson WW Jr, Butler MS, D’Angio GJ, Rate WR. Leg length discrepancy following irradiation for childhood tumors. J Pediatr Orthop 1991;11:284–7. 13. Arnold M, Kummermehr J, Trott KR. Radiation-induced impairment of osseous healing: quantitative studies using a standard drilling defect in rat femur. J Radiat Res 1995;143:77–84. 14. Pelker RR, Friedlaender GE. Fracture healing: radiation induced alterations. Clin Orthop 1997;341:267–82. 15. Takahashi S, Sugimoto M, Kotoura Y, Sasai K, Oka M, Yamamuro T. Long-term changes in the haversian systems fol-
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lowing high-dose irradiation: an ultrastructural and quantitative histomorphological study. J Bone Joint Surg Am 1994;76:722– 38. Gantous A, Phillips JH, Catton P, Holmberg D. Distraction osteogenesis in the irradiated canine mandible. Plast Reconstr Surg 1993;93:164–8. Kojimoto H, Yasui N, Goto T, Matsuda S, Shimomura Y. Bone lengthening in rabbits by callus distraction: the role of periosteum and endosteum. J Bone Joint Surg Br 1988;70:543–9. Li G, Simpson AHRW, Triffitt JT. The role of chondrocytes in intramembranous and endochondral ossification during distraction osteogenesis in the rabbit. Calcif Tissue Int 1999;64:310–7. Aronson J, Harrison BH, Stewart CL, Harp JH Jr. The histology of distraction osteogenesis using different external fixators. Clin Orthop 1989;241:106–16. Ilizarov GA. Clinical application of the tension-stress effect for limb lengthening. Clin Orthop 1990;250:8–26. Li G, Simpson AHRW, Kenwright J, Triffitt JT. Assessment of cell proliferation in regenerating bone during distraction osteogenesis at different distraction rates. J Orthop Res 1997;15:765– 72. Moro Robledo J, dela Varga Salto V, Guerado Parra E, De Santosdela Fuente J, Lopez Arevalo R, Espejo Baena A. Histologic analysis of distraction osteogenesis in the femoral shaft of the immature rabbit. Acta Orthop Belg 1998;64:125–30. Shaw JL, Bassett CAL. The effects of varying oxygen concentrations on osteogenesis and embryonic cartilage in vitro. J Bone Joint Surg Am 1967;49:73–80. Steinbrech DS, Mehrara BJ, Saadeh PB, Chin G, Dudziak ME, Gerrets RP, et al. Hypoxia regulates VEGF expression and cellu-
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lar proliferation by osteoblasts in vitro. Plast Reconstr Surg 1999; 104:738–47. Choi IH, Ahn JH, Chung CY, Cho TJ. Vascular proliferation and blood supply during distraction osteogenesis: a scanning electron microscopic observation. J Orthop Res 2000;18:698–705. Aronson J. Temporal and spatial increases in blood flow during distraction osteogenesis. Clin Orthop 1994;301:124–31. Takahashi S, Sugimoto M, Kotoura Y, Nishimatsu H, Shibamoto Y, Abe M, et al. The effects of intraoperative radiotherapy on bone-healing ability in relation to different doses and postradiotherapy intervals. Int J Radiat Oncol Biol Phys 1994;30:1147– 52. Muhonen A, Muhonen J, Minn H, Kulmala J, Klossner J, Teräs M, et al. The effects of irradiation and hyperbaric oxygen on bone formation during rabbit mandibular distraction. Arch Oral Biol 2002;47:701–7. Tsubota S, Tsuchiya H, Shinokawa Y, Tomita K, Minato H. Transplantation of osteoblast-like cells to the distracted callus in rabbits. J Bone Joint Surg Br 1999;81:125–9. Okazaki H, Kurokawa T, Nakamura K, Matsushita T, Mamada K, Kawaguchi H. Stimulation of bone formation by recombinant fibroblast growth factor-2 in callotasis bone lengthening of rabbits. Calcif Tissue Int 1999;64:542–6. Hagino T, Hamada Y. Accelerating bone formation and earlier healing after using demineralized bone matrix for limb lengthening in rabbits. J Orthop Res 1999;17:232–7. Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998;97:1114–23.
Original article Distraction osteogenesis after irradiation in a rabbit model Hiroyuki Tsuchiya, Kenji Uehara, Keisuke Sakurakichi, Koji Watanabe, Hidenori Matsubara, and Katsuro Tomita Department of Orthopedic Surgery, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan
Abstract Background. The present study was performed to investigate the effects of preoperative irradiation on distraction osteogenesis, as little is known about how preoperative irradiation delays distraction osteogenesis. Methods. A single dose of irradiation was applied to the right rear legs of rabbits. This was followed by tibial lengthening at a rate of 0.5 mm/day, which was continued for 4 weeks. Bone regeneration was examined radiographically and histologically. Results. In the irradiation group, the radiographs showed little regeneration during the elongation phase. During the maturation phase, the callus appeared slowly, and its formation was spotty. Furthermore, regeneration was not completed until the fourth week of the maturation period. Histological examination at the end of distraction showed a gap in the distraction consisting of loose connective tissue, with part of the fibrous tissue oriented longitudinally. Four weeks after completion of distraction, the major part of the radiolucent region consisted of cartilage. The spotty osteogenesis was identified as enchondral ossification. Immunohistochemical examination of the regeneration area revealed that the blood vessels were extremely localized, and that the level of expression of vascular endothelial growth factor (VEGF) in the osteoblasts was high. Microangiography showed that vascularization at the distracted sites was poor. Distraction osteogenesis was decreased markedly by preoperative irradiation in terms of both rate and process. The results suggested that most of the osteoprogenitor cells were damaged immediately after irradiation. The high level of VEGF in the osteoblasts and the enchondral ossification also suggested a hypoxic state in the distracted region. Conclusions. Preoperative irradiation interferes with distraction osteogenesis by inducing a state of poor angiogenesis.
Introduction After induction of distraction osteogenesis1 or callotasis,2 most difficult orthopedic problems (e.g., limb length discrepancy, severe deformity, complicated nonunion, osteomyelitis, extensive bone loss) can be treated successfully.3–9 The biological advantage of distraction osteogenesis is the regeneration of living bone, with the regenerated tissue eventually having the same quality as native bone. Recently, distraction osteogenesis was also shown to provide durable reconstruction for skeletal defects after bone tumor excision for the restoration of natural limbs.9–11 In the treatment of cancer in children, irradiation is frequently indicated for patients with malignant lymphoma, Ewing’s sarcoma, rhabdomyosarcoma, leukemia, or other malignancies. When the epiphyseal growth plate is involved in the irradiation field, limblength inequality and deformities due to partial or entire growth arrest of the epiphyseal plate is anticipated.12 Limb lengthening and deformity correction using distraction osteogenesis is beneficial for these patients if feasible, although it has been reported that irradiation delays callus formation and maturation in fracture and bone defect models.13–15 This raised the question of how preoperative irradiation delays distraction osteogenesis. The effects of preoperative irradiation on distraction osteogenesis were investigated, as little is known about the mechanism involved. The results of the present study can lead to improvement of distraction osteogenesis in irradiated bones. Materials and methods Irradiation and lengthening procedure
Offprint requests to: H. Tsuchiya Received: April 20, 2005 / Accepted: July 4, 2005
Forty male Japanese White rabbits, weighing about 3 kg, were used in this study. Fifteen rabbits each were allotted to the control group and the 15-Gy group,
628
H. Tsuchiya et al.: Distraction osteogenesis after irradiation
after completion of lengthening. The harvested tibial specimens were prepared for light microscopy by fixation in phosphate-buffered formaldehyde. Specimens were rinsed in buffer, decalcified, embedded in paraffin, and sectioned in the sagittal plane. Routine staining was performed with hematoxylin and eosin and immunohistochemical staining with an anti-vascular endothelial growth factor (VEGF) antibody. Microangiography Fig. 1. Preoperative irradiation. The irradiation is applied to the right leg in a medial-to-lateral direction at a focus–skin distance of 25 cm
respectively. Ten rabbits were used for radiological and histological analysis and five for microangiographic analysis in each group. Five rabbits each were used just for radiological analysis in both the 10-Gy and 20-Gy groups. Animals were anesthetized with an intramuscular injection of ketamine hydrochloride (50 mg/kg body weight) (Sankyo, Tokyo, Japan) and intravenous injection of pentobarbital sodium (50 mg/kg body weight) (Dainippon Pharmaceutical, Tokyo, Japan). A single dose of irradiation was administered in a medial-tolateral direction to the right rear leg at 150 kV and 20 mA at a focus–skin distance of 25 cm targeted for regeneration (Fig. 1). The doses were 10, 15, and 20 Gy. The control group underwent no irradiation. Just after irradiation, a 5-cm skin incision was made over the medial aspect of the right tibia. A unilateral dynamic external fixator of our design was fixed with four half-pins 2 mm in diameter (Howmedica, Geneva, Switzerland) inserted into the tibia. Osteotomy was performed on the tibia at the tibiofibular junction between the two inner half-pins using a thread wire saw 0.36 mm in diameter (Koshiya, Kanazawa, Japan). After a 7-day latency period, lengthening of the tibia was initiated at a rate of 0.5 mm/day and continued for 28 days with a total elongation of 14 mm. The regenerated area was allowed to consolidate for 4 weeks after completion of the distraction. Radiological analysis Radiographs of the leg were obtained once a week for 8 weeks after the initiation of lengthening. Five rabbits each in the 10- and 20-Gy groups were used only for radiological evaluation. Histological analysis Each of the five rabbits in the control and 15-Gy groups were examined. The animals were killed 0 and 4 weeks
Each of the five rabbits in the control and 15-Gy groups were killed 4 weeks after completion of lengthening by intraaortic injection of Microfil (Flow Tek, Carver, MA, USA). Using the Spalteholtz technique, the entire tibias were removed, stripped of soft tissues and periosteum, and fixed in 10% buffered formalin. After decalcification in 0.2 M EDTA, they were cleared with benzyl benzoate and the vascular patterns were examined. The average vascular occupation rates of the nine regions of interest (ROI) at the distracted site were measured using image analysis software (NIH image 1.61, Macintosh version). Differences in the vascular occupying rate between the two groups were analyzed using the unpaired Student’s t-test. All experiments were performed under the guidelines for animal experiments stipulated at Kanazawa University Graduate School of Medical Science.
Results Radiological analysis The radiographs of the control group showed good callus formation early during distraction with three distinct zones (proximal and distal sclerotic zones and a central fibrous zone). The distracted callus matured before the fourth week of the consolidation period (Fig. 2). In addition, the distracted callus formation of the 10-Gy group was similar to that of the control group (Fig. 3). In the 15-Gy group, however, the radiographs showed little regeneration during the elongation phase. The callus appeared slowly during the maturation phase and did not show three distinct zones but had only a spotty distribution. Furthermore, regeneration was not completed until the fourth week of the maturation period (Fig. 4). The radiographs of the 20-Gy group showed little regeneration until the fourth week of the maturation period (Fig. 5). Based on these results, further examinations of the control and 15-Gy groups were performed because it seemed to be adequate to use the 15-Gy group to investigate the effects of radiation.
H. Tsuchiya et al.: Distraction osteogenesis after irradiation
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a,b
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Histological analysis In the control group, the distraction gap was seen to consist of three regions at the end of the lengthening period. In the radiolucent zone, parallel collagen bundles were arranged longitudinally. In the adjacent sclerotic zone, callus was formed by intramembranous ossification (Fig. 6). Four weeks after completion
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Fig. 2. Radiographs of tibias from the control group. a After 14 days of distraction. b After 28 days of distraction. c At 14 days after completion of distraction. d At 28 days after completion of distraction. The distracted callus is observed early during distraction with three distinct zones
c,d
Fig. 3. Radiographs of tibias from the 10-Gy irradiation group. a After 14 days of distraction. b After 28 days of distraction. c At 14 days after completion of distraction. d At 28 days after completion of distraction. The distracted callus formation of the 10-Gy group is similar to that of the control group
c,d
Fig. 4. Radiographs of tibias from the 15-Gy irradiation group. a After 14 days of distraction. b After 28 days of distraction. c At 14 days after completion of distraction. d At 28 days after completion of distraction. The callus appeared slowly during the maturation phase and showed spotty formation. Regeneration is not completed until the 28th day of the maturation period
of distraction, a mature trabecular network and neocorticalization were seen (Fig. 7). At the end of lengthening, the 15-Gy group showed a gap in the distraction consisting of loose connective tissue, with part of the fibrous tissue oriented longitudinally. There was no evidence of new mineralization (Fig. 8). Four weeks after completion of distraction, the major part of the radiolucent region consisted of cartilage. There was no
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c,d
a,b
Fig. 6. Longitudinal section from the control group after 28 days of distraction. In the radiolucent zone, parallel collagen bundles are arranged longitudinally (white arrow). In the adjacent sclerotic zone, the callus is formed by intramembranous ossification (black arrow). Bar 200 mm. Hematoxylin-eosin
evidence of the normal regeneration pattern. The spotty osteogenesis was identified as enchondral ossification (Fig. 9). Immunohistochemical staining of VEGF in the control group revealed diffuse vascularization with positive staining (Fig. 10). In the 15-Gy group, the blood vessels were scanty and localized, and the level of VEGF expression in the osteoblasts was stronger than in the control group (Fig. 11). Microangiography Microangiograms of the control group showed a diffuse, dense vascular network not only in the distracted region but also in the host bone. Those in the 15-Gy group showed that the vascularization was extremely thin in the whole tibia (Fig. 12). Neovasculization induced by distraction osteogenesis was inhibited in the 15-Gy
Fig. 5. Radiographs of tibias from the 20-Gy irradiation group. a After 14 days of distraction. b After 28 days of distraction. c At 14 days after completion of distraction. d At 28 days after completion of distraction. The radiographs showed little regeneration until 28 days after completion of distraction
a,b Fig. 7. Longitudinal section from the control group 28 days after completion of distraction. a Total longitudinal section. b Close-up of the boxed area in a. Histological examination showed a mature trabecular network and neocorticalization. Bar 200 mm. Hematoxylin-eosin
group. The average vascular occupying rates at the distracted site were 77.3% ± 3.5% in the control group and 46.1% ± 9.7% in the 15 Gy group. The rate of the 15 Gy group was significantly lower than that of the control group (P < 0.001).
Discussion This experiment demonstrated that distraction osteogenesis was markedly affected by preoperative irradiation. Gantous et al. reported that callus formation of the distracted site was not inhibited in a canine mandibular model when distraction was performed 6 months after 50-Gy irradiation in 20 fractions.16 In the fracture model, however, the length of time from irradiation to fracture is considered a major factor for union.15 That is,
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a,b Fig. 8. Longitudinal section from the 15-Gy irradiation group after 28 days of distraction. a Total longitudinal section. b Close-up of the boxed area in a. The gap in the distraction consists of loose connective tissue with part of the fibrous tissue oriented longitudinally. Bar 50 mm. Hematoxylin-eosin
Fig. 10. Immunohistochemical staining of vascular endothelial growth factor (VEGF) of the control group 28 days after completion of distraction. Diffuse vascularization with positive staining is observed (white arrows). The osteoblasts at the trabecula are also positive (partially strong) (black arrows). Most osteoblasts lining the trabeculae show relatively weak expression of VEGF
a,b Fig. 9. Longitudinal section from the 15-Gy irradiation group 28 days after completion of distraction. a Total longitudinal section. b Close-up of the boxed area in a. The major part of the radiolucent region consists of cartilage. Spotty osteogenesis is identified as enchondral ossification. OB, osteoblasts; CH, chondrocytes. Bar 100 mm. Hematoxylin-eosin
a longer time after irradiation is associated with fewer adverse effects of irradiation on callus formation. In the present study, distraction was performed immediately after irradiation. During the distraction phase, regeneration did not consist of new bone but of loose connective tissue. These observations suggested that most of the osteoprogenitor cells were damaged immediately after irradiation. Because the irradiation in our study was carried out at the time of the operation, there is little time for the damaged cells to recover during the waiting period (7 days) before distraction.
Fig. 11. Immunohistochemical staining for VEGF of the 15 Gy irradiation group 28 days after completion of distraction. The vascularization is much localized, and the presence of VEGF in the osteoblasts is stronger than of the control group (arrows). VEGF is strongly positive in almost all lining osteoblasts
However, some cells were able to proliferate and differentiate into chondroblasts because spotty enchondral ossification was observed during the consolidation period. Enchondral ossification of distraction osteogenesis is known to be species-specific,17,18 and to be due to mechanical instability19,20 and local hypoxia.21,22 It has also been reported that hypoxia induces chondroblastic differentiation of osteoprogenitor cells23 and stimulates
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were entirely different from those in the control group. Irregular regeneration might occur as distraction was completed before the repair of damage to osteoprogenitor cells. In addition, ischemic changes induced by irradiation modified regeneration. In the clinical situation, osteotomy for distraction osteogenesis would be carried out at a different time than the irradiation and at a different site at the present time. However, developments in bioengineering may overcome the adverse effects of irradiation on bone regeneration in terms of transplantation of osteoprogenitor stem cells, osteoblasts, or periosteal cells,29 injection of growth factors,30 implantation of bone matrix,31 or gene therapy using VEGF.32 In conclusion, preoperative irradiation interferes with distraction osteogenesis by inducing a state of poor angiogenesis.
References a,b Fig. 12. Microangiograms of the distracted site. a Control group. b The 15-Gy irradiation group. The control group shows a diffuse, dense vascular network. Vascularization is scanty in the irradiated group
VEGF expression in osteoblasts.24 Little enchondral ossification was observed in the control group, but significant levels of VEGF expression were found in osteoblasts during the maturation period in the irradiated group. These findings suggest that the distracted region had become hypoxic after irradiation because of the poor vascularization, as shown by microangiography, although dense vascular networks are usually found during normal regeneration in rabbits on microangiograms25 and the distraction osteogenesis procedure promotes neovascularization and increases blood flow.20,26 The disturbance of fracture healing is caused by ischemic changes resulting from vascular damage and inhibition of neovascularization induced by irradiation.27 However, more precise experiments on angiogenesis are needed for the accurate identification of hypoxia. The effects of irradiation and hyperbaric oxygen on the regenerated area have been investigated. Hyperbaric oxygen was not able to prevent suppression of osteogenesis caused by radiotherapy, but it might improve bone formation by prolonging high osteogenic activity.28 If inhibition of regeneration healing induced by 15 Gy of irradiation was merely the result of the delay in differentiation and proliferation of osteoprogenitor cells, regeneration would show delayed findings on radiography and histological examination. However, the rate and process of regeneration in the irradiation group
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