J Oral MaxillofacSurg 50:1305-1309. 1992
Healing of Bony Defects in the Irradiated and Unirradiated Rat Mandible C.A.
LORENTE,
PHD, DMD,*
B.Z.
SONG,
DMD,t
AND
R.B.
DONOFF,
DMD,
MD*
A model system of the irradiated rat mandible has been developed and used in conjunction with a non-spontaneously healing mandibular defect. The contribution of the tissue components in the healing of bony defects was studied using demineralized bone powder (DBP) prepared from unirradiated or in vivo irradiated rat long bones. Better bony fill of the defects occurred in the irradiated beds filled with unirradiated DBP than in the unirradiated beds containing irradiated DBP. This suggests that, at least in the early postirradiation period, the bed is not the limiting factor in healing of bony defects and the osteogenic components of bone in the DBP may be most affected by irradiation. In the irradiated bed, the defects grafted 2 weeks after irradiation healed better than those grafted at 4 weeks. Thus, the timing of surgery after irradiation also plays a role in the healing process, with early surgery producing better results.
resistant to radiation damage,13 and osteocytes are the least sensitive.14 The goals of this study were development of a rat model system for the irradiated mandible; examination of contributions of the periosteum, soft-tissue bed, and bone to the healing of a bony defect in an irradiated bed; and study of the effect on healing of timing of surgery after irradiation.
The role of the various components in the healing of defects in irradiated bone has not yet been elucidated. Late soft-tissue changes include endarteritis and fibrosis, suggesting that early surgery after irradiation may be advantageous, as supported by our previous study’ and the work of others.* Studies of skin grafts and flaps in an irradiated field suggest that there is a window for optimal surgery between 4 and 14 days after irradiation.3,4 Both qualitative5-* and quantitative’ studies have demonstrated that periosteum and endosteum are the primary sources of osteogenic cells in bone grafts, and that irradiation decreases the regenerative capacity of bone.8,10*‘1The relative radiosensitivities of cells in the irradiated bed have been studied, with the migratory ameboid mesenchymal cells being the most sensitive,‘* followed by proliferating progenitor cells and sprouting capillaries. I3 Osteoblasts and chondroblasts are more
Materials and Methods Sixty retired male Sprague-Dawley outbred albino rats weighing 400 to 500 g and obtained from either Charles River Breeding Labs, Wilmington, MA, or Taconic Farms, Germantown, NY, were used for the study. All treatment of the rats followed the gulidelines of the Guide for the Care and Use of Laboratory Animals. ” Mandibular irradiation simulation was performed by the Department of Radiation Medicine and Biophysics, Massachusetts General Hospital. Cerroban blocks were constructed for right and left portals to limit the field of exposure to the mandible. The rats were anesthetized with 120 mg/kg body weight of ketamine intramuscularly, and a bite block was placed to remove the tongue and maxilla from the field of irradiation. A bolus (tissue-equivalent material) was placed over the mandible to ensure that the maximum dose of irradiation was at the level of the mandible and not at the skin. Thirty-two rats were irradiated from alternating right and left portals for a total dose of 45 Gy 6oCo. The initial group of six rats received 18, 25Gy
Received from the Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital and Harvard School of Dental Medicine, Boston, MA. * Instructor. t Fellow. $ Professor; Dean, School of Dental Medicine. Supported in part by the American Association of Oral and Maxillofacial Surgeons Education Foundation. Address correspondence and reprint requests to Dr Lorente: Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital, Boston, MA 022 15. 0 1992 American 0278-2391/92/501
Association l-001
of Oral and Maxillofacial
Surgeons
1$3.00/O
1305
1306
IRRADIATED
fractions and the subsequent group of 26 rats received 15, 3.0-Gy fractions. The change in protocol was initiated because the rats tolerated the irradiation well and we wished to limit the number of anesthetic exposures. Two weeks after completion of irradiation, 9 1% (3 1 of 32) of the rats were within 10% of their original weights. For the preparation of the irradiated demineralized bone powder (DBP), a group of rats had their limbs irradiated to a total dose of 30 Gy 6oCo in 12 2.5-Gy fractions. This dosage was chosen based on the work of Cooley and Goss,” which showed that 30 Gy completely inhibited fracture healing. The rats were killed 1 week after completion of the course of irradiation, and their long bones were removed and DBP was prepared according to the method of Reddi and Huggins.i6 Particles between 75 and 250 pm3 in size were used. Ten milligrams of the DBP moistened with normal saline was implanted per grail site. The surgical procedure used involved the model system of Kaban and Glowacki. l7 Rats were anesthetized with 160 mg/kg body weight of ketamine intramuscularly. A non-spontaneously healing, 4-mm diameter defect was made in the posterior-inferior aspect of the mandibular ramus from a lateral approach using a Hall drill with saline irrigation, and measured with a template. A separate periosteal closure was not performed, since the masseter was closely adherent to the lateral cortex of the mandible and a discrete periosteal envelope was not observed. On completion of the surgery, the masseter and skin were closed in separate layers and then a similar procedure was performed on the opposite lateral cortex. The unirradiated hemimandibles were divided into the following groups: 1) no surgery (2); 2) soft-tissue dissection on the lateral border of the mandible only (6); 3) mandibular defect left unfilled (27); 4) bony defect made in the mandible and filled with 10 mg unirradiated DBP (10); and 5) bony defect made in the mandible and filled with 10 mg of irradiated DBP ( 11). Groups 1 to 4 were also duplicated in the study of
1 Non-irradiated
0 n=
DBP (IO)
Defect Irrad.DBP (27)
(10)
Irradiated
1
DBP Defect (13)
(12)
DBP
Defect
(12)
(10)
FIGURE 1. Percentages of mandibular defects with 50% bone fill.
RAT MANDIBLE MODEL SYSTEM
FIGURE 2. Appearance of bone not subjected to surgery (hematoxylin-eosin stain, original magnification X70).
irradiated mandibles, with no surgery in 5, soft-tissue dissection in 6, unfilled defects in 22, and DBP-filled defects in 25. Surgery was performed 2 or 4 weeks after completion of irradiation. The rats were killed 4 weeks after surgery with an overdose of pentobarbital. After initial placement in Millonig’s fixative, the mandibles were cleaned and then photographed. The photographs were used to evaluate the size of the residual mandibular defects, using the measurement/estimate program of the Digital Paintbrush System (Jandel Corp, 1984, Sausalito, CA) interfaced with an IBM computer. The width of the mandible is approximately 1.O mm, so an estimate of the unfilled volume was made from the two dimensional measurements. Statistical analysis of the size of the unfilled areas was performed using analysis of variance. The mandibles were then decalcified with a formic acid-citrate solution, embedded, cut in crosssection, and stained with hematoxylin and eosin. Results The mandibles were evaluated clinically at the time of death, and the bony fill of the defects was described as either greater or less than 50% complete (Fig 1). There were very few mandibles with intermediate levels of bone fill; the majority of those with more than 50% had almost complete calcification, and those with less than 50% had a minimal amount of bony fill. When no surgery was performed, the cortical plates of both irradiated and unit-radiated mandibles were smooth, with no evidence of hyperostosis (Fig 2). In mandibles in which soft-tissue dissection alone was performed, hyperostosis of the lateral cortex was observed in all of the unirradiated (6 of 6) (Fig 3) and irradiated (9 of 9) jaws. The irradiated mandibles had less exuberant osteogenesis, but there was still a marked increase in bone formation in comparison with the jaws with which surgery was not performed. In the jaws in
LORENTE, SONG, AND DONOFF
FIGURE 3. Appearance of bone subjected to soft-tissue dissection (hematoxylin-eosin stain, original magnification X70).
which defects were made and left unfilled, only 4% ( 1 of 27) of the unirradiated (Fig 4) and 5% (1 of 22) of the irradiated mandibles had more than 50% fill. Thin fibrous tissue bridged the defect, which did not heal spontaneously in either irradiated or unirradiated animals. In the unirradiated mandibular defects filled with unirradiated DBP, 90% (9 of 10) had more than 50% solid bone fill (Fig 5) five had complete bone fill, and the rest had a combination of bone and incompletely calcified thick fibrous tissue. There was marked osteogenesis around the DBP, with bone bridging between the particles. When irradiated DBP was placed in an unit-radiated mandible, none (0 of 10) of the defects had more than 50% bone fill (Fig 6). Fibrous plugs were intermediate in density between those of unfilled defects and those containing unirradiated DBP. There was no evidence of osteoid being laid down on the DBP particles. The defects in irradiated mandibles filled with unirradiated DBP had more than 50% bone fill in 39% (5
FIGURE 4. Appearance of defect in unirradiated mandible (hematoxylin-eosin stain, original magnification X70).
1307
FIGURE 5. Appearance of defect with unirradiated DBP in unirradiated mandible (hematoxylin-eosin stain, original magnification X70).
of 13) when surgery was performed at 2 weeks (Fig 7) and in 17% (2 of 12) when surgery was performed 4 weeks after completion of irradiation. The remainder had more than 50% fill of the defect with partially calcified fibrous tissue. The histologic picture was intermediate between that seen with the unirradiated and irradiated DBP in the unit-radiated mandible, ie, new osteoid was present at the periphery of the defect, the DBP particles were intact in the central area, but the bony bridges between the particles were less dense than in the unirradiated situation. Thus, there was a timerelated decrease in osteogenesis in the irradiated mandibles; ie, less bone fill occurred at 4 weeks than at 2 weeks. However, all of the irradiated mandibles had better repair of the bony defect than did those filled with irradiated DBP in unit-radiated rats. The region without solid bony fill in the unirradiated mandibles filled with unirradiated DBP was signifi-
FIGURE 6. Appearance of defect with irradiated DBP in unirradiated mandible (hematoxylin-eosin stain, original magnification X70).
1308
IRRADIATED RAT MANDIBLE MODEL SYSTEM
FIGURE 7. Appearance of defecl with unirradiated DBP in irradiated mandible (hematoxylin-eosin stain, original magnification X70).
cantly less (P c -01) than that of the unfilled defect. (Table 1). Evaluation of the unfilled region and the percentage of fill showed that the irradiated DBP in the unirradiated mandible resulted in the largest residual defect, followed by the defects in both irradiated and unirradiated mandibles, irradiated mandibles with surgery at 4 and 2 weeks, respectively, and, finally, unirradiated mandibles with unirradiated DBP. Discussion Our previous study using a dog model’ suggested that periosteum plays a major role in osteogenesis in irradiated bone, and that early surgery after completion of irradiation results in better healing. The current study supports these observations. The majority of previous studies have used single large doses of irradiation and extrapolated the results to multiple divided doses, which may not be a valid assumption without having constructed a dose-response curve.” By specifically irradiating the mandible and shielding the rest of the head and neck region, as well as using a fractionated dosage regimen, we had minimal morbidity and mortality.
A continuity defect with a soft-tissue deficit would be most analogous to the clinical situation. However, a model system with this combined defect has not been described in the rat. The results of grafting inferiorborder defects in the irradiated dog2 and rabbitI were comparable to those for continuity defects in the dog. ’ Thus relevant information can be obtained from models that differ from the clinical defect. Because a distinct periosteal layer cannot be appreciated in the rat mandible, the ability of the unirradiated and irradiated beds to mount an osteogenic response without a periosteal contribution cannot be ascertained. However, the effect of periosteal disruption is well demonstrated by the mandibles that had softtissue dissection only. In the clinical situation, the periosteum is seldom left in the bed, although it is possible that the periosteum adjacent to the resected area may play a role in osteogenesis. DBP was first reported to be osteogenic by Huggins in 193 1.20Although DBP has been prepared and then irradiated in vitro21,22or implanted and then the host bed irradiated,23 DBP has not previously been prepared from long bones irradiated in vivo. In unpublished results, we showed that DBP prepared from irradiated and unirradiated dog mandibles did not induce osteogenesis in rats either subcutaneously or in bony defects. The multinucleated giant-cell reaction generated suggested that the xenogeneic DBP induced an immune response, a conclusion that is supported by the work of Sampath and Reddi.24 Thus, although bovine bone morphogenetic protein (BMP) is osteogenic in disparate species, 25,26the less-purified, xenogeneic DBP preparation in our model system is not. Our results showing decreased osteogenic potential of DBP irradiated in vivo support the work of Cooley and GOSS,” in which osteogenesis occurred with an unirradiated tibia1 transplant placed into a previously irradiated bed, but not with a previously irradiated bone placed in an unirradiated bed. Our results are also consistent with the work of Urist2’,23 and Weintroub and Reddi,22 who reported decreased osteoinductive act&ty of DBP with exposure to irradiation. It is postulated that irradiation denatures the protein responsible for osteoinduction (bone morphogenetic protein) and al-
Table 1. Size of Unfilled Area of Bone Irradiated Bed
Unirradiated Bed Defect Filled With
surgery at 2 weeks
surgery at 2 weeks
surgely at 4 weeks
surgery at 4 weeks
DBP-Filled
unmed Defect*
Irradiated DBP
DBP-Filled
Unfilled Defect
DBP-Filled
unfilled Defect
3.6 k 3.6 (10)
8.5 -c 3.9 (27)
11.2 + 8.1 (11)
5.6 + 3.1 (13)
9.7 +- 5.0 (12)
7.3 + 6.9 (11)
9.9 + 5.3 (10)
_
Results are millimeters squared +- SD; number of rats is in parentheses. * P < .O1compared with DBP in unirradiated bed.
1309
LORENTE, SONG, AND DONOFF
ters the collagen cross-linking necessary for new bone formation. Thus, irradiation is not the method of choice for sterilization of banked bone or DBP.*’
10.
11.
Summary 12.
We have been able to successfully develop a model system for an irradiated rat mandible with a nonspontaneously filling defect. The quality of repair of the bony defect from best to worst was 1) unirradiated DBP in the unirradiated mandible; 2) unirradiated DBP in the irradiated mandible; and 3) irradiated DBP in the unit-radiated mandible. Our studies suggest that healing in rats undergoing surgery at 2 weeks after completion of irradiation is better than that in rats at 4 weeks after completion of irradiation. It appears that irradiated beds are better able to support an osteogenic response than are unirradiated beds with irradiated DBP, and that irradiation affects the osteoinductive components of DBP more than the tissue bed.
13.
14.
15.
16.
17.
18.
References I. Altobelli DE, Lorente CA, Handren JH, Jr, et al: Free and microvascular bone grafting in the irradiated mandible. J Oral Maxillofac Surg 45:27, 87 2. Ostrup LT, Fredrickson JM: Reconstruction of mandibular defects after radiation, using a free, living bone graft transferred by microvascular anastomoses. An experimental study. Plast Reconstr Surg 55:563, 1975 3. Ueda M, Torii S, Oka T: An experimental study of skin autografts in irradiated tissue. J Oral Maxillofac Sung 40~74, 1982 4. Sumi Y, Ueda M, Kaneda T, et al: Postoperative irradiation after reconstructive surgery: Comparative study of radiosensitivity between free-skin grafts and skin flaps. Plast Reconstr Surg 74:385, 1984 5. DeBruyn PPH, Kabisch WT: Bone formation by fresh and frozen autogenous and homogenous transplants of bone, bone marrow and periosteum. Am J Anat 96:375, 1975 6. Bassett CAL, Creighton DK, Stinchfield FE: Contributions of endosteum, cortex, and soft tissue to osteogenesis. Surg Gynecol Obstet 112:145, 1961 7. Friedenstein A, Kuralesova AI: Osteogenic precursor cells of bone marrow in radiation chimeras. Transplantation 12:99, 197 1 8. Bras J, DeJonge HKT, van Merkesteyn JPR: Osteoradionecrosis of the mandible: Pathogenesis. Am J Otokuyngol 11:244, 1990 9. Gray JC. Elves MW: Donor cells’ contribution to osteogenesis
19.
20. 21. 22.
23.
24. 25.
26.
27.
in experimental cancellous bone grafts. Clin Orthop 163:26 1, 1982 Cooley LM, Goss RJ: The effects of transplantation and X-irradiation on the repair of fractured bones. Am J Anat 102: 167, 1958 Evans HB, Brown S, Hurst LN: The effects of early postoperative radiation on vascularized bone grafts. Ann Plast Surg 26:505, 1991 Craven PL, Urist MR: Osteogenesis by radioisotope labelled cell populations in implants of bone matrix under the influence of ionizing radiation. Clin Orthop 76:23 1. 197 1 Beumer J, Curtis T, Harrison RE: Radiation therapy of the oral cavity. Sequellae and management, parts I and 2. Head Neck Surg 1:301;392, 1979 Tonna EA, Pavelec M: Changes in the proliferative activity of young and old mouse skeletal tissues following Co 60 wholebody irradiation. J Gerontol25:9, 1970 Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources National Research Council, US Department of Health, Education and Welfare Public Health Service: Guide for the Care and Use of Laboratory Animals. Bethesda, MD, National Institutes of Health, NIH publication no. 80-23, 1978 Reddi AH, Huggins C: Biochemical sequences in the iransformation of normal fibroblasts in adolescent rats. Proc Nat1 Acad Sci USA 69: 160 1, 1972 Kaban LB, Glowacki J: Induced osteogenesis in the repair of experimental mandibular defects in rats. J Dent Res 60: 1356, 1981 Hayashi S, Suit HD: Effect of fractionation of radiation dose on callus formation at site of fracture. Radiology IO1:181, 197 I Nathanson A, Wersall J: Effects of 60 Co-gamma-irradiation on the early ingrowth of an autogenous bone inlay into an artificial defect in the rabbit mandibula. Stand J Plast Reconstr Surg 12:139, 1978 Huggins CB: The formation of bone under the influence of epithelium of the urinary tract. Arch Surg 22:377, 193 I Buring K, Urist MR: Effects of ionizing radiation on tbe bone induction principle in the matrix of bone implants. Clin Orthop 55:225, 1967 Weintroub S, Reddi AH: Influence of irradiation on the osteoinductive potential of demineralized bone matrix. Calcif Tissue Int 42:255, 1988 Craven PL, Urist MR: Osteogenesis by radioisotope labelled cell populations in implants of bone matrix under the influence of ionizing radiation. Clin Orthop 76:23 1, 197 1 Sampath TK, Reddi AH: Homology of bone-inductive proteins from human, monkey, bovine and rat extracellular~matrix. Proc Nat1 Acad Sci USA 80:659. 1983 Saito K, Urist MR: Induced regeneration of calvaria by bone morphogenetic protein (BMP) in dogs. Clin Orthop 197:301, 1985 Urist MR, Lietze A, Mizutani H, et al: A bovine low molecular weight bone morphogenetic protein (BMP) fraction. Chn Orthop 162:219, 1982 Urist MR, Mikulski A, Boyd SD: A chemosterilized antigenextracted autodigested alloimplant for bone banks. Arch Surg 110~416, 1975
Healing of Bony Defects in the Irradiated and Unirradiated Rat Mandible C.A.
LORENTE,
PHD, DMD,*
B.Z.
SONG,
DMD,t
AND
R.B.
DONOFF,
DMD,
MD*
A model system of the irradiated rat mandible has been developed and used in conjunction with a non-spontaneously healing mandibular defect. The contribution of the tissue components in the healing of bony defects was studied using demineralized bone powder (DBP) prepared from unirradiated or in vivo irradiated rat long bones. Better bony fill of the defects occurred in the irradiated beds filled with unirradiated DBP than in the unirradiated beds containing irradiated DBP. This suggests that, at least in the early postirradiation period, the bed is not the limiting factor in healing of bony defects and the osteogenic components of bone in the DBP may be most affected by irradiation. In the irradiated bed, the defects grafted 2 weeks after irradiation healed better than those grafted at 4 weeks. Thus, the timing of surgery after irradiation also plays a role in the healing process, with early surgery producing better results.
resistant to radiation damage,13 and osteocytes are the least sensitive.14 The goals of this study were development of a rat model system for the irradiated mandible; examination of contributions of the periosteum, soft-tissue bed, and bone to the healing of a bony defect in an irradiated bed; and study of the effect on healing of timing of surgery after irradiation.
The role of the various components in the healing of defects in irradiated bone has not yet been elucidated. Late soft-tissue changes include endarteritis and fibrosis, suggesting that early surgery after irradiation may be advantageous, as supported by our previous study’ and the work of others.* Studies of skin grafts and flaps in an irradiated field suggest that there is a window for optimal surgery between 4 and 14 days after irradiation.3,4 Both qualitative5-* and quantitative’ studies have demonstrated that periosteum and endosteum are the primary sources of osteogenic cells in bone grafts, and that irradiation decreases the regenerative capacity of bone.8,10*‘1The relative radiosensitivities of cells in the irradiated bed have been studied, with the migratory ameboid mesenchymal cells being the most sensitive,‘* followed by proliferating progenitor cells and sprouting capillaries. I3 Osteoblasts and chondroblasts are more
Materials and Methods Sixty retired male Sprague-Dawley outbred albino rats weighing 400 to 500 g and obtained from either Charles River Breeding Labs, Wilmington, MA, or Taconic Farms, Germantown, NY, were used for the study. All treatment of the rats followed the gulidelines of the Guide for the Care and Use of Laboratory Animals. ” Mandibular irradiation simulation was performed by the Department of Radiation Medicine and Biophysics, Massachusetts General Hospital. Cerroban blocks were constructed for right and left portals to limit the field of exposure to the mandible. The rats were anesthetized with 120 mg/kg body weight of ketamine intramuscularly, and a bite block was placed to remove the tongue and maxilla from the field of irradiation. A bolus (tissue-equivalent material) was placed over the mandible to ensure that the maximum dose of irradiation was at the level of the mandible and not at the skin. Thirty-two rats were irradiated from alternating right and left portals for a total dose of 45 Gy 6oCo. The initial group of six rats received 18, 25Gy
Received from the Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital and Harvard School of Dental Medicine, Boston, MA. * Instructor. t Fellow. $ Professor; Dean, School of Dental Medicine. Supported in part by the American Association of Oral and Maxillofacial Surgeons Education Foundation. Address correspondence and reprint requests to Dr Lorente: Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital, Boston, MA 022 15. 0 1992 American 0278-2391/92/501
Association l-001
of Oral and Maxillofacial
Surgeons
1$3.00/O
1305
1306
IRRADIATED
fractions and the subsequent group of 26 rats received 15, 3.0-Gy fractions. The change in protocol was initiated because the rats tolerated the irradiation well and we wished to limit the number of anesthetic exposures. Two weeks after completion of irradiation, 9 1% (3 1 of 32) of the rats were within 10% of their original weights. For the preparation of the irradiated demineralized bone powder (DBP), a group of rats had their limbs irradiated to a total dose of 30 Gy 6oCo in 12 2.5-Gy fractions. This dosage was chosen based on the work of Cooley and Goss,” which showed that 30 Gy completely inhibited fracture healing. The rats were killed 1 week after completion of the course of irradiation, and their long bones were removed and DBP was prepared according to the method of Reddi and Huggins.i6 Particles between 75 and 250 pm3 in size were used. Ten milligrams of the DBP moistened with normal saline was implanted per grail site. The surgical procedure used involved the model system of Kaban and Glowacki. l7 Rats were anesthetized with 160 mg/kg body weight of ketamine intramuscularly. A non-spontaneously healing, 4-mm diameter defect was made in the posterior-inferior aspect of the mandibular ramus from a lateral approach using a Hall drill with saline irrigation, and measured with a template. A separate periosteal closure was not performed, since the masseter was closely adherent to the lateral cortex of the mandible and a discrete periosteal envelope was not observed. On completion of the surgery, the masseter and skin were closed in separate layers and then a similar procedure was performed on the opposite lateral cortex. The unirradiated hemimandibles were divided into the following groups: 1) no surgery (2); 2) soft-tissue dissection on the lateral border of the mandible only (6); 3) mandibular defect left unfilled (27); 4) bony defect made in the mandible and filled with 10 mg unirradiated DBP (10); and 5) bony defect made in the mandible and filled with 10 mg of irradiated DBP ( 11). Groups 1 to 4 were also duplicated in the study of
1 Non-irradiated
0 n=
DBP (IO)
Defect Irrad.DBP (27)
(10)
Irradiated
1
DBP Defect (13)
(12)
DBP
Defect
(12)
(10)
FIGURE 1. Percentages of mandibular defects with 50% bone fill.
RAT MANDIBLE MODEL SYSTEM
FIGURE 2. Appearance of bone not subjected to surgery (hematoxylin-eosin stain, original magnification X70).
irradiated mandibles, with no surgery in 5, soft-tissue dissection in 6, unfilled defects in 22, and DBP-filled defects in 25. Surgery was performed 2 or 4 weeks after completion of irradiation. The rats were killed 4 weeks after surgery with an overdose of pentobarbital. After initial placement in Millonig’s fixative, the mandibles were cleaned and then photographed. The photographs were used to evaluate the size of the residual mandibular defects, using the measurement/estimate program of the Digital Paintbrush System (Jandel Corp, 1984, Sausalito, CA) interfaced with an IBM computer. The width of the mandible is approximately 1.O mm, so an estimate of the unfilled volume was made from the two dimensional measurements. Statistical analysis of the size of the unfilled areas was performed using analysis of variance. The mandibles were then decalcified with a formic acid-citrate solution, embedded, cut in crosssection, and stained with hematoxylin and eosin. Results The mandibles were evaluated clinically at the time of death, and the bony fill of the defects was described as either greater or less than 50% complete (Fig 1). There were very few mandibles with intermediate levels of bone fill; the majority of those with more than 50% had almost complete calcification, and those with less than 50% had a minimal amount of bony fill. When no surgery was performed, the cortical plates of both irradiated and unit-radiated mandibles were smooth, with no evidence of hyperostosis (Fig 2). In mandibles in which soft-tissue dissection alone was performed, hyperostosis of the lateral cortex was observed in all of the unirradiated (6 of 6) (Fig 3) and irradiated (9 of 9) jaws. The irradiated mandibles had less exuberant osteogenesis, but there was still a marked increase in bone formation in comparison with the jaws with which surgery was not performed. In the jaws in
LORENTE, SONG, AND DONOFF
FIGURE 3. Appearance of bone subjected to soft-tissue dissection (hematoxylin-eosin stain, original magnification X70).
which defects were made and left unfilled, only 4% ( 1 of 27) of the unirradiated (Fig 4) and 5% (1 of 22) of the irradiated mandibles had more than 50% fill. Thin fibrous tissue bridged the defect, which did not heal spontaneously in either irradiated or unirradiated animals. In the unirradiated mandibular defects filled with unirradiated DBP, 90% (9 of 10) had more than 50% solid bone fill (Fig 5) five had complete bone fill, and the rest had a combination of bone and incompletely calcified thick fibrous tissue. There was marked osteogenesis around the DBP, with bone bridging between the particles. When irradiated DBP was placed in an unit-radiated mandible, none (0 of 10) of the defects had more than 50% bone fill (Fig 6). Fibrous plugs were intermediate in density between those of unfilled defects and those containing unirradiated DBP. There was no evidence of osteoid being laid down on the DBP particles. The defects in irradiated mandibles filled with unirradiated DBP had more than 50% bone fill in 39% (5
FIGURE 4. Appearance of defect in unirradiated mandible (hematoxylin-eosin stain, original magnification X70).
1307
FIGURE 5. Appearance of defect with unirradiated DBP in unirradiated mandible (hematoxylin-eosin stain, original magnification X70).
of 13) when surgery was performed at 2 weeks (Fig 7) and in 17% (2 of 12) when surgery was performed 4 weeks after completion of irradiation. The remainder had more than 50% fill of the defect with partially calcified fibrous tissue. The histologic picture was intermediate between that seen with the unirradiated and irradiated DBP in the unit-radiated mandible, ie, new osteoid was present at the periphery of the defect, the DBP particles were intact in the central area, but the bony bridges between the particles were less dense than in the unirradiated situation. Thus, there was a timerelated decrease in osteogenesis in the irradiated mandibles; ie, less bone fill occurred at 4 weeks than at 2 weeks. However, all of the irradiated mandibles had better repair of the bony defect than did those filled with irradiated DBP in unit-radiated rats. The region without solid bony fill in the unirradiated mandibles filled with unirradiated DBP was signifi-
FIGURE 6. Appearance of defect with irradiated DBP in unirradiated mandible (hematoxylin-eosin stain, original magnification X70).
1308
IRRADIATED RAT MANDIBLE MODEL SYSTEM
FIGURE 7. Appearance of defecl with unirradiated DBP in irradiated mandible (hematoxylin-eosin stain, original magnification X70).
cantly less (P c -01) than that of the unfilled defect. (Table 1). Evaluation of the unfilled region and the percentage of fill showed that the irradiated DBP in the unirradiated mandible resulted in the largest residual defect, followed by the defects in both irradiated and unirradiated mandibles, irradiated mandibles with surgery at 4 and 2 weeks, respectively, and, finally, unirradiated mandibles with unirradiated DBP. Discussion Our previous study using a dog model’ suggested that periosteum plays a major role in osteogenesis in irradiated bone, and that early surgery after completion of irradiation results in better healing. The current study supports these observations. The majority of previous studies have used single large doses of irradiation and extrapolated the results to multiple divided doses, which may not be a valid assumption without having constructed a dose-response curve.” By specifically irradiating the mandible and shielding the rest of the head and neck region, as well as using a fractionated dosage regimen, we had minimal morbidity and mortality.
A continuity defect with a soft-tissue deficit would be most analogous to the clinical situation. However, a model system with this combined defect has not been described in the rat. The results of grafting inferiorborder defects in the irradiated dog2 and rabbitI were comparable to those for continuity defects in the dog. ’ Thus relevant information can be obtained from models that differ from the clinical defect. Because a distinct periosteal layer cannot be appreciated in the rat mandible, the ability of the unirradiated and irradiated beds to mount an osteogenic response without a periosteal contribution cannot be ascertained. However, the effect of periosteal disruption is well demonstrated by the mandibles that had softtissue dissection only. In the clinical situation, the periosteum is seldom left in the bed, although it is possible that the periosteum adjacent to the resected area may play a role in osteogenesis. DBP was first reported to be osteogenic by Huggins in 193 1.20Although DBP has been prepared and then irradiated in vitro21,22or implanted and then the host bed irradiated,23 DBP has not previously been prepared from long bones irradiated in vivo. In unpublished results, we showed that DBP prepared from irradiated and unirradiated dog mandibles did not induce osteogenesis in rats either subcutaneously or in bony defects. The multinucleated giant-cell reaction generated suggested that the xenogeneic DBP induced an immune response, a conclusion that is supported by the work of Sampath and Reddi.24 Thus, although bovine bone morphogenetic protein (BMP) is osteogenic in disparate species, 25,26the less-purified, xenogeneic DBP preparation in our model system is not. Our results showing decreased osteogenic potential of DBP irradiated in vivo support the work of Cooley and GOSS,” in which osteogenesis occurred with an unirradiated tibia1 transplant placed into a previously irradiated bed, but not with a previously irradiated bone placed in an unirradiated bed. Our results are also consistent with the work of Urist2’,23 and Weintroub and Reddi,22 who reported decreased osteoinductive act&ty of DBP with exposure to irradiation. It is postulated that irradiation denatures the protein responsible for osteoinduction (bone morphogenetic protein) and al-
Table 1. Size of Unfilled Area of Bone Irradiated Bed
Unirradiated Bed Defect Filled With
surgery at 2 weeks
surgery at 2 weeks
surgely at 4 weeks
surgery at 4 weeks
DBP-Filled
unmed Defect*
Irradiated DBP
DBP-Filled
Unfilled Defect
DBP-Filled
unfilled Defect
3.6 k 3.6 (10)
8.5 -c 3.9 (27)
11.2 + 8.1 (11)
5.6 + 3.1 (13)
9.7 +- 5.0 (12)
7.3 + 6.9 (11)
9.9 + 5.3 (10)
_
Results are millimeters squared +- SD; number of rats is in parentheses. * P < .O1compared with DBP in unirradiated bed.
1309
LORENTE, SONG, AND DONOFF
ters the collagen cross-linking necessary for new bone formation. Thus, irradiation is not the method of choice for sterilization of banked bone or DBP.*’
10.
11.
Summary 12.
We have been able to successfully develop a model system for an irradiated rat mandible with a nonspontaneously filling defect. The quality of repair of the bony defect from best to worst was 1) unirradiated DBP in the unirradiated mandible; 2) unirradiated DBP in the irradiated mandible; and 3) irradiated DBP in the unit-radiated mandible. Our studies suggest that healing in rats undergoing surgery at 2 weeks after completion of irradiation is better than that in rats at 4 weeks after completion of irradiation. It appears that irradiated beds are better able to support an osteogenic response than are unirradiated beds with irradiated DBP, and that irradiation affects the osteoinductive components of DBP more than the tissue bed.
13.
14.
15.
16.
17.
18.
References I. Altobelli DE, Lorente CA, Handren JH, Jr, et al: Free and microvascular bone grafting in the irradiated mandible. J Oral Maxillofac Surg 45:27, 87 2. Ostrup LT, Fredrickson JM: Reconstruction of mandibular defects after radiation, using a free, living bone graft transferred by microvascular anastomoses. An experimental study. Plast Reconstr Surg 55:563, 1975 3. Ueda M, Torii S, Oka T: An experimental study of skin autografts in irradiated tissue. J Oral Maxillofac Sung 40~74, 1982 4. Sumi Y, Ueda M, Kaneda T, et al: Postoperative irradiation after reconstructive surgery: Comparative study of radiosensitivity between free-skin grafts and skin flaps. Plast Reconstr Surg 74:385, 1984 5. DeBruyn PPH, Kabisch WT: Bone formation by fresh and frozen autogenous and homogenous transplants of bone, bone marrow and periosteum. Am J Anat 96:375, 1975 6. Bassett CAL, Creighton DK, Stinchfield FE: Contributions of endosteum, cortex, and soft tissue to osteogenesis. Surg Gynecol Obstet 112:145, 1961 7. Friedenstein A, Kuralesova AI: Osteogenic precursor cells of bone marrow in radiation chimeras. Transplantation 12:99, 197 1 8. Bras J, DeJonge HKT, van Merkesteyn JPR: Osteoradionecrosis of the mandible: Pathogenesis. Am J Otokuyngol 11:244, 1990 9. Gray JC. Elves MW: Donor cells’ contribution to osteogenesis
19.
20. 21. 22.
23.
24. 25.
26.
27.
in experimental cancellous bone grafts. Clin Orthop 163:26 1, 1982 Cooley LM, Goss RJ: The effects of transplantation and X-irradiation on the repair of fractured bones. Am J Anat 102: 167, 1958 Evans HB, Brown S, Hurst LN: The effects of early postoperative radiation on vascularized bone grafts. Ann Plast Surg 26:505, 1991 Craven PL, Urist MR: Osteogenesis by radioisotope labelled cell populations in implants of bone matrix under the influence of ionizing radiation. Clin Orthop 76:23 1. 197 1 Beumer J, Curtis T, Harrison RE: Radiation therapy of the oral cavity. Sequellae and management, parts I and 2. Head Neck Surg 1:301;392, 1979 Tonna EA, Pavelec M: Changes in the proliferative activity of young and old mouse skeletal tissues following Co 60 wholebody irradiation. J Gerontol25:9, 1970 Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources National Research Council, US Department of Health, Education and Welfare Public Health Service: Guide for the Care and Use of Laboratory Animals. Bethesda, MD, National Institutes of Health, NIH publication no. 80-23, 1978 Reddi AH, Huggins C: Biochemical sequences in the iransformation of normal fibroblasts in adolescent rats. Proc Nat1 Acad Sci USA 69: 160 1, 1972 Kaban LB, Glowacki J: Induced osteogenesis in the repair of experimental mandibular defects in rats. J Dent Res 60: 1356, 1981 Hayashi S, Suit HD: Effect of fractionation of radiation dose on callus formation at site of fracture. Radiology IO1:181, 197 I Nathanson A, Wersall J: Effects of 60 Co-gamma-irradiation on the early ingrowth of an autogenous bone inlay into an artificial defect in the rabbit mandibula. Stand J Plast Reconstr Surg 12:139, 1978 Huggins CB: The formation of bone under the influence of epithelium of the urinary tract. Arch Surg 22:377, 193 I Buring K, Urist MR: Effects of ionizing radiation on tbe bone induction principle in the matrix of bone implants. Clin Orthop 55:225, 1967 Weintroub S, Reddi AH: Influence of irradiation on the osteoinductive potential of demineralized bone matrix. Calcif Tissue Int 42:255, 1988 Craven PL, Urist MR: Osteogenesis by radioisotope labelled cell populations in implants of bone matrix under the influence of ionizing radiation. Clin Orthop 76:23 1, 197 1 Sampath TK, Reddi AH: Homology of bone-inductive proteins from human, monkey, bovine and rat extracellular~matrix. Proc Nat1 Acad Sci USA 80:659. 1983 Saito K, Urist MR: Induced regeneration of calvaria by bone morphogenetic protein (BMP) in dogs. Clin Orthop 197:301, 1985 Urist MR, Lietze A, Mizutani H, et al: A bovine low molecular weight bone morphogenetic protein (BMP) fraction. Chn Orthop 162:219, 1982 Urist MR, Mikulski A, Boyd SD: A chemosterilized antigenextracted autodigested alloimplant for bone banks. Arch Surg 110~416, 1975
