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Effects of pre- and postoperative irradiation on the healing of bone grafts in the rabbit
J Oral Maxlllofac
Surg
45.34-41.1987
Effects of Pre- and Postoperative Irradiation on the Healing of Bone Grafts in the Rabbit MARCO
J. MORALES, CHARLES
DDS,*
ROBERT
F. GOTTLIEB,
E. MARX,
DDS,t
AND
PHD$
Healing of cellular bone grafts irradiated at various times in the postsurgical course was compared to the healing characteristics of bone grafts placed into already irradiated tissue and to controls of irradiated host mandible in a rabbit model. Physical graft consolidation was assessed by load stress characteristics and serial histologic examination. Results indicated that grafts placed into already irradiated tissues failed to form bone in both phases of expected regeneration resulting in structurally weakened and histologically deficient ossicles. Bone grafts irradiated after placement were tolerant of irradiation. Bone grafts irradiated after four weeks were found to be less affected by irradiation than those irradiated within the first four weeks, forming an ossicle structurally and histologically superior to that of bone ossicles developed from grafts placed into irradiated tissues.
Reconstruction of human jaws for cancer-related deformities and for osteoradionecrosis often involves the unpredictability of bone graft healing in tissues already irradiated or, the unknown response of a bone graft that may itself receive irradiation. Because of use of combined irradiation-surgery protocols for treatment of oral cancer, most jaw reconstruction today follows irradiation.1.2 Successful reconstruction of these patients has recently been improved with the use of adjunctive hyperbaric oxygen,3 pedicled myocutaneous flaps,4 and/ or microvascular bone periosteal flaps.5 Occasionally, however, the management of oral cancer also
involves early postsurgical irradiation to sterilize residual small tumor foci, or late postsurgical irradiation to salvage a surgical failure.6 Each of these possibilities poses the potential of irradiating a bone graft placed for jaw reconstruction. Work in several institutions has demonstrated improved functional and psychological responses to early reconstruction of cancer patients.7,8 Such interest in immediate or early delayed reconstruction makes it necessary to understand better the effects of irradiation on the type of bone grafts commonly used in oral and maxillofacial surgery. The purpose of this study was to determine the clinical and histologic changes, and to measure the strength, in bone grafts either placed into previously irradiated tissues or irradiated during the early phases of bone regeneration.
* Former Chief Resident and Research Fellow, Oral and Maxillofacial Surgery, University of Miami School of Medicine, Division of Oral and Maxillofacial Surgery. Presently Clinical Instructor. University of Miami School of Medicine. Division of Oral and Maxillofacial Surgery, Miami, Florida. t Associate Professor of Surgery and Director of Graduate Training and Research, University of Miami School of Medicine. Division of Oral and Maxillofacial Surgery, Miami, Florida. f Associate Professor of Radiology, Division of Radiation Therapy and Radiologic Sciences, Department of Radioloev. Univekty of Miami School of Medicine, Miami, Florida. -. Address correspondence and reprint requests to Dr. Marx: University of Miami School of Medicine, Division of Oral and Maxillofacial Surgery. I61 1 NW 12th Avenue..Miami. FL 33136.
Materials
and Methods
Ninety white New Zealand male rabbits (0~~tologus cuniculus) were divided into two groups of 45 animals each. The animals had their two mandibular incisor teeth and the regenerative germinal follicles removed to create a 3-cm edentulous space on each side of the midline. One additional animal served as a donor of allogeneic bone. Portions of rib, femur, and ilium were harvested in a sterile
0278-2391187 $0.00 + .25
34
MORALES ET AL.
FIGURE I (/cfi). Bone graft of autogenous particulate cancellous bone and marrow placed within an allogeneic bone crib. A measured I-cm graft was consistently placed into the 3-cm edentulous span. FIGURE 2 (ri&t). Specimen at point of fracture during steady rate loading of 1. lb/minute. A strain gauge records force and reflection, which are used to calculate Young’s modulus (E). modulus of flexion CM,.) and modules of force resistance Wf,), in addition to a measured yield point (f’).
fashion, frozen in liquid nitrogen. and freeze-dried for 17 days. Each specimen was separately packaged to serve as a sterile allogeneic bone crib. One experimental group received 5.5 Gy ‘j°Co 1 MEV external beam irradiation to the mandible in four fractions for a total of 22 Gy. Immobilization to accomplish controlled treatment was achieved by injecting ketamine, 25 mg/kg, and acepromazine, 0.25 mglkg, intramuscularly before irradiation. Each fractionation was separated by one week and was calculated to approximate a human dose of 640 Gy in 2 Gy fractions given daily, five days per week for six weeks. Irradiation began when the incisor sockets had complete mucosal covering (about six weeks). Recovery from acute irradiation damage to create the late chronic hypoxic tissue bed proceeded for three months. Each animal then underwent a transcutaneous resection and immediate bone graft reconstruction of a one cm length of the edentulous area on one side of the midline. The edentulous area on the opposite side served as a control. All bone grafts consisted of particulate autogenous cancellous bone and marrow harvested from the ilium and placed into an allogeneic bone crib from the donor animal fixed with a wire on each end (Fig. 1). No other form of fixation was necessary as the adult rabbit functions with a united midline suture imparting rigidity across the arch. The second group received identical surgery without preoperative irradiation. Instead, each animal received an equivalent irradiation protocol consisting of 8.5 Gy in two fractions spaced a week apart beginning at variable times in the postoperative period. A subgroup of five animals began the irradiation protocol immediately after surgery, an-
other at one week, another at two weeks, and other groups progressively at weekly intervals up to six weeks. All bone grafts were performed under monitored general anesthesia using intramuscularly injected ketamine. 25 mgikg, acepromazine. 0.25 mg/kg. and nembutal. 20 mg/kg. Each animal received 100,000 units of procaine penicillin G and 400,000 of benzanthene penicillin intramuscularly during surgery. The animals were fed Purina rabbit chow moistened in water and water ad libitum during the recovery phase. Each animal was given meperidine, 3 mgikg, twice-daily empirically for pain during the first two postsurgical days. Five animals in each postsurgical irradiation group were euthenized at weekly intervals beginning at postsurgical week two through week seven. For standardization, each postsurgical irradiation subgroup animal was killed two weeks after completion of irradiation. Therefore, each subgroup represented a one-week progressively later irradiation exposure. The grafts in 10 animals in this group were also allowed to heal a full 12 weeks. Five animals in the pregrafting irradiation group died prior to completion of the study. Three deaths were related to radiation toxicity and two to pasturella pneumonia. Three animals in the postgrafting irradiation group died before completing the study. One death was related to radiation toxicity, one to pasturella pneumonia, and the other to a nonspecific enterocolitis. TISSUESTUDYMETHODS
At necroscopy the entire mandible was harvested and cleaned of adherent tissue. Each mandible was split in the midline to create a grafted study spec-
36
EFFECT OF 1RRADlATION ON BONE GRAFT HEALING
L GRAFT SITE M-2 R-38 S-A. POST GRAFT IRR. 12 WEEKS
25
SLOPE = F/D = E = 375
20
FORCE (F) LSS.
25 /
L&S./IN.
20
15
P. E. MfY,.
L GRAFT M-4 R-13 PRE GRAFT IRR. 12 WEEKS
FORCE IF) LSS.
22.5 LSS. VOUNO’S MOD.. .575 LSS./IN.m RIGIDITY MOD. OF FLEXION. 39.1 -P/E MOD. OF RESISTANCE. P2/2Em 460
P. 3.2 LSS. E . VOUNO’S MOD. . 0.59 I RIGIDITY M,. MOD. OF FLEXION s 5.4 s P/E Yr= MOD. OF RESISTANCE P2/2E.8.7
1.5
L
5
10
15
20
5
25
FIGURE 3 (top lefr). Sample force (F) versus deflection (D) graph. This postgrafting irradiation specimen has a normal slope but a mid-range Young’s modulus (E) and a force resistance modulus (MR) less than ungrafted irradiated mandible. FIGURE 4 (top vighr). Sample force (F) versus deflection (D) graph. The slope of this pregrafted irradiation specimen is very flat. There is a lower Young’s modulus (E) than in the postgrafting irradiation animal in (Fig. 3) and a lower force resistance modulus (M,).
10
15
20
25
DEFLECTION (D) X 10-31N.
X 1O-3 IN. DEFLECTION (D) 5
4
PRE GRAFT IRR. -, POST QRAFT IRR. t CONTROL
/
3 CLINIC GRADING SCALE
FIGURE 5 (bottom rigl~t). Clinical grading score versus time. This graph demonstrates a slowed osteogenesis in the pregrafting irradiation group which peaks at four weeks and diminishes linearly thereafter. An improved osteogenesis in the postgrafting irradiation group that accelerates at four weeks and attains near normal bone formation by 12 weeks is also shown.
0123456789
10
11
12
TIME, WEEKS
imen and an ungrafted control specimen. All specimens were coded and analyzed in a blinded fashion in three ways. First, each specimen was examined clinically and described grossly by use of a scale from 1 to 5. Grade 1 represented no new bone formation; Grade 2, 1% to 25% new bone formation compared to the host bone size; Grade 3, 26% to 50% new bone formation compared to the host bone size; Grade 4, 51% to 75% new bone formation compared to the host bone size; and Grade 5, 76% to 100% new bone formation compared to the host bone size. The specimens were further radiographed at 90” to the occlusal plane. No specific grading of radiographs was undertaken but their interpretation was part of the clinical grading system. Physical consolidation of the grafts was measured by their load stress characteristics. Each fresh specimen was embedded in a holder with bone cement and loaded at a point 5 mm from the distal graft-host interface at a constant rate of 2
lb/minute until fracture occurred (Fig. 2). From strain gauge recordings, a graph comparing force to deflection at a yield point could be plotted (Figs. 3, 4 for each specimen). From each graph the following data were calculated or measured: P= E=
yield point; maximum force without fracture Young’s modulus; slope of force (F) v. deflection (D) (a measure of the graft rigidity and brittleness). Mf = modulus of flexion (Mf = P/E) (a measure of immature or osteoid bone formation and organic matrix) MR = modulus of resistance to load forces (Mr = P2/2Z$ (a measure of consolidation) After measuring the physical consolidation, each specimen was removed from the bone-holding apparatus and fixed in 10% neutral buffered formalin in a 2O:l volume-volume ratio for two weeks. All specimens were decalcified in 5% formic acid for
MORALES ET AL.
three weeks, embedded in paraffin, sectioned p_, and stained with hematoxylin and eosin.
at 6
Results CLINICAL
ASSESSMENT
A summary of the clinical findings is presented in Figure 5. In the pregraft irradiation group, bone formation was slowed but progressed linearly until week four. It then peaked at a 2.9 grade (about 25% of host bone size) and linearly declined to its lowest value (1.8) at 12 weeks (about 12% of host bone size) (Fig. 6). The mean clinical grade score in the postgrafting irradiation group also indicated early retardation of bone formation at two and three weeks. It then sharply improved at four weeks (3.9, or about .SO%of host bone size), reaching near control levels of bone formation by week five and 100% of control levels by week 12 (Figs. 5, 7). F’HYSICAL GRAFT CONSOLIDATION
A summary of the physical consolidation data is shown in Figure 8. The results indicate an inhibition of early bone production in the pregrafting irradiation group. They also strongly suggest a failure of the bone to mature. The graft reached a maximum resistance modulus of only 8.5% of controls at four weeks and lost further force resistance as each week progressed. In addition, the results show less inhibition of bone formation in grafts irradiated after placement. As indicated by the clinical grading scores. the fourth week seemed to be a critical time after which irradiation had less impact. The yield point and modulus of resistance each indicated an early production of osteoid that matured and consolidated to a resistance strength equal to 40-45% of ungrafted bone by five weeks and continued to at least 12 weeks. In contrast, the pregraft irradiation group also seemed to develop early osteoid. but the graft did not consolidate to develop any resistance strength, and it deteriorated even further with time. HISTOLOGIC
ASSESSMENT
The pregrafting irradiation group showed cellular loss in much of the transplanted bone. Osteocytes were absent in many lacunae and others showed nuclear changes consistent with cell death. Endosteal osteoblasts were also reduced in number. The stroma was fibrotic and poorly vascular. In the two-
FIGURE 6 (top). Pregrafting irradiation gross specimen (12 weeks) (above) and ungrafted irradiated mandible (below). Graft shows minimal new bone formation. as well as little structural integrity. FIGURE 7 (bottom). Postgrafting irradiation six-week specimen (above) and ungrafted irradiated mandible (below). Excellent bone formation and structural integrity values comparable to the ungrafted irradiated mandible is shown.
and three-week specimens osteoid production was sparse. In the four- and five-week specimens cellular osteoid production was evident but in a reduced quantity (Fig. 9). The later specimens, including the one at 12 weeks, showed osteoclastic activity and resorption of early osteoid without new bone replacement. In several areas the bone graft was replaced by a chondroid matrix (Fig. 10). The postgraft irradiation group also showed degenerative changes in the osteocytes. However, the endosteal osteoblasts were numerous, without evidence of nuclear changes. Osteoid production was sparse in the two- and three-week specimens but much more exuberant in the four- and five-week specimens (Fig. 11). In the later specimens, including the 12-week specimen, new bone formation could be observed arising from the host elements and the grafted elements. The osteoid tissue was
38
EFFECT
(IF
IKKAI>IAl‘ION
900
700 600
HONE
GRAFT
HEALING
l
800
STRENGTH: mogpJs
ON
I
l
*
* *
FIGLIKE
c
RESISTANCE MFbP2/2E
X. rt’\i\t;tnce
time.
I‘hc
strate\
* n
500 400
graph bone
cover\
with
of
vcr-\u\ demon-
a weakening
ungrafted
*
Modulu\
force
of the
which
time.
rt’-
and
a
PRE GRAFT IRR.
linear
r)
POST GRAFT IRR.
tegrity
l
CONTROL
irradiation
group
that
wa\
continuing
at through
the
gain in structural in the
I?-week
300 200
group
It
further
a failure
pregrafting
of
irradietion
to maintain
sufficient
100
postgrafting
point.
demon\trate5 the
in-
structural
or gain integ-
rity.
01234567
8
9
10
11
12
TIME, WEEKS
becoming more organized and less cellular, and was forming a larger mineralized ossicle (Fig. 12). In some specimens a periosteum could be identified surrounding the grafted bone. Discussion
Bone graft healing, as proposed by Axhausen9 and documented by Gray et al.,rO can be regarded as cellular bone regeneration occurring in two phases. In the first (cellular phase), bone forms from the transplanted cellular elements, especially those of endosteal origin. In the second (replacement-remodeling phase), the transplanted bone is physiologically resorbed and replaced by a more mature bone of host cell origin. Pertinent to this study is the probable inhibition of both phases of bone regeneration in the pregrafting irradiation group. The poorly vascular host tissue both reduces survival of transplanted cellular elements and inhibits the metabolic activity required actively to produce osteoid. In addition, the fibrotic stroma lacks the cellular elements capable of producing the host-derived second phase bone and hence the observed reduction in clinical grade score and modulus of force resistance after four weeks. In fact, the ongoing reduction in modulus of force resistance and observed absence of bone can be regarded as the result of the expected resorption of whatever phase-one bone was formed without its replacement by phase-two bone. The presence of chondroid matrix supports the supposition that the irradiation of the tissue bed is mostly responsible for
the lack of new bone formation or its subsequent disappearance. Marx has shown irradiated jaws to be hypovascular-hypocellular and measurably et al. have, in turn. shown bone hypoxic. ‘I Basset ~ precursor cells to undergo chondroblastic differention in hypoxic environments up to the point of forming mature cartilage.” It seems reasonable to view the pregrafting irradiated tissue bed as hypoxic and incapable of adequately supporting either cellular survival or host cell bone differentiation. The response of bone graft elements to irradiation in the postgrafting irradiation group can also be interpreted in light of the two phase concept of bone regeneration. The initial phase of cellular proliferation and osteoid production may have been slowed by the cellular killing and damage of irradiation. However, our observations and those in the studies by Hulth and Wesherborn’3 and by Melonotte and Follis.r4 indicate a relative radiation resistance to endosteal osteoblasts. These cells appear more sensitive to irradiation in the first three weeks after transplantation, but are capable of some survival and subsequent bone formation. When the graft is irradiated at four weeks or later. bone formation is minimally affected. This finding can be interpreted to mean that by four weeks the cellular phase-one bone has mostly formed and that its replacement by host-derived phase-two bone is not greatly affected by irradiation. The observation that even the postgrafting irradiation group showed a modulus of force resistance only 42% of controls by 12 weeks can be explained by either of two possible mechanisms. The first is
MORALES
ET AL.
FIGURE 9 crop). Photomicrograph of a four-week inflammation, and fibrous tissue ingrowth replacing FIGURE formation
pregrafting irradiation the transplanted bone.
10 (bottom). Chondroid matrix within an area of cellular may be secondary to postirradiation hypoxia. (Hematoxylin
specimen showing minimal new osteoid (Hematoxylin and eosin. Magnification.
bone in a I?-week pregrafting irradiation and eosin. Magnification, x IO.)
formation, x 10.)
specimen.
sevc :re
Chondroid
40
EFFECT
‘.
*.
OF IRRADIATION
ON BONE
GRAFT
HEALING
“_
FIGURE 11 (top). Exuberar __ .Magmhcation, X 10.)
It cellular
osteoid
formation
FIGURE 12 (bottom). A I?-week postgrafting irradiation tive tissue stroma. (Hematoxylin and eosin. Magnification.
in a four-week specimen ~4.)
postgrafting
demonstrating
irradiation a mature
specimen.
mineralized
(Hematoxylin
ossicle
and eosin.
and minimal
connec-
41
MORALES E:T AL.
that after four or five weeks bone grafts are somewhat resistant to irradiation damage or at least no more vulnerable than ungrafted host bone. The fact that force resistance was less than one-half that of host bone may merely be due to a greater than 12week requirement for the grafts to develop similar strength characteristics. Phase-two remodeling may continue at a slow pace and add structural integrity to a graft over many years. The slope of the control data in Figure 8 suggests this mechanism. A reduction in structural integrity of even ungrafted bone was seen in early irradiated mandibles. Although there was some individual variation, probably due to size differences between jaws, a redevelopment of measured structural integrity was seen. This observation implies that ungrafted bone is also weakened by irradiation and recovers slowly and incompletely. The postgrafting irradiated grafts were probably still in a recovery phase, although their clinical appearance was similar to ungrafted bone at 12:weeks. The second possible explanation for a reduced modulus of force resistance is that irradiation of a bone graft at four weeks or later may have a small or negligible effect on the graft itself but that it does lead to soft tissue changes of a hypovascular-hypocellular-hypoxic nature similar to those created in the pregrafting irradiated group, but of a lesser degree. Such an effect on the soft tissue envelope can inhibit full development of the graft by affecting later phase-two remodeling. Although much of the phase-two replacement and remodeling has occurred by the time the chronic hypoxic state develops from irradiation damage, late phase-two bone maintenance may be altered to create a weaker bone. Further studies with long-term follow-up are required to elucidate the correct explanation. Data obtained from animal models should be interpreted with caution. Bone physiology, structure, and healing rates vary between species and among members of the same species. The results obtained in this study, however, suggest a more detrimental effect on attempted bone formation in tissue previously altered by irradiation than to bone irradiated during its formative period. Bone grafts placed into irradiated tissue beds suffer loss in both phases of expected bone regeneration. The wellknown low success rates of this type of clinical reconstruction is attested to by the findings of this study. The hypovascular-hypoxic-hypocellular tissue bed seems to be the critical tissue effect. The current use of graft systems that circumvent this ir-
radiation effect, such as microvascular bone-periosteal grafts, osteomyocutaneous grafts. and hyperbaric oxygen-supported grafts, is supported by these findings. Bone grafts which are irradiated are, no doubt, affected by the cellular damage of high energy transfer. However, particulate bone and cancellous marrow grafts were found to be surprisingly tolerant of irradiation. A four-week period of phaseone bone initiation prior to irradiation appears to be the critical time for bone to develop in a near normal fashion. Such evidence establishes a scientific basis for the consideration of early jaw reconstruction in cancer patients who may require irradiation after grafting. It would seem that if irradiation were deferred for four weeks after jaw reconstruction, the clinical results of current bone graft systems could be as successful as nonirradiated grafts and certainly more successful than a graft placed into an unimproved irradiated tissue bed.
References 1. Perez CA, Lee FA, Ackerman LV, et al: Nonrandomized comparison of preoperative irradiation and surgery (vs) irradiation alone in the management of carcinoma of the tonsil. Am J Roentgen 126:248, 1976 2. Marx RE: A new concept in the treatment of osteoradionecrosis. J Oral Maxillofac Sum. 41:351. 1983 3. Marx RE, Ames JR: The use OFhyperbaric oxygen therapy in bone reconstruction of the irradiated and tissue-deficient patient. J Oral Maxillofac Surg 40:412, 1982 4. McGraw JB, Dibbell DC, Cart-away JH: Clinical definition of independent myocutaneous vascular territories. Plast Reconstr Surg 60:341, 1977 5. Donoff RB, May JW: Microvascular mandibular reconstruction. J Maxillofac Surg 40: 122, 1982 6. Fayas JV, Lampe I: Radiotherapy of squamous cell carcinoma of the oral portion of the tongue. Arch Surg 94:316, 1967 7. Bear SE, Green RK, Wentz WW: Stainless steel wire mesh -an aid in difficult oral surgery problems. J Oral Surg 29:27, 1971 8. Marx RE: Current Principles and Techniques in Major Jaw Reconstruction. Mini Lecture Series “A.” 66th Annual AAOMS Meeting, 14 September 1984. New York 9. Axhausen W: The osteogenetic phases of regeneration of bone. J Bone Joint Surg 38A:593, 1956 10. Gray J, Phil M, Elves M: Donor cells contribution to osteogenesis in experimental cancellous bone grafts. Clin Orthop 163:261, 1982 Il. Marx RE: Osteoradionecrosis: a new concept of its pathophysiology. J Oral Maxillofac Surg 41:283. 1983 12. Bassett CAL, et al: Clinical implications of cell function in bone grafting. Clin Orthop 87:49, 1972 13. Hulth A, Westerbom 0: Early changes of the growth zone in rabbit following roentgen irradiation. Acta Orthop Stand 30:155, 1960 14. Melanotte PL, Follis RH: Early effects of x-irradiation on cartilage and bone. Am J Pathol 39: 1, 1965
Surg
45.34-41.1987
Effects of Pre- and Postoperative Irradiation on the Healing of Bone Grafts in the Rabbit MARCO
J. MORALES, CHARLES
DDS,*
ROBERT
F. GOTTLIEB,
E. MARX,
DDS,t
AND
PHD$
Healing of cellular bone grafts irradiated at various times in the postsurgical course was compared to the healing characteristics of bone grafts placed into already irradiated tissue and to controls of irradiated host mandible in a rabbit model. Physical graft consolidation was assessed by load stress characteristics and serial histologic examination. Results indicated that grafts placed into already irradiated tissues failed to form bone in both phases of expected regeneration resulting in structurally weakened and histologically deficient ossicles. Bone grafts irradiated after placement were tolerant of irradiation. Bone grafts irradiated after four weeks were found to be less affected by irradiation than those irradiated within the first four weeks, forming an ossicle structurally and histologically superior to that of bone ossicles developed from grafts placed into irradiated tissues.
Reconstruction of human jaws for cancer-related deformities and for osteoradionecrosis often involves the unpredictability of bone graft healing in tissues already irradiated or, the unknown response of a bone graft that may itself receive irradiation. Because of use of combined irradiation-surgery protocols for treatment of oral cancer, most jaw reconstruction today follows irradiation.1.2 Successful reconstruction of these patients has recently been improved with the use of adjunctive hyperbaric oxygen,3 pedicled myocutaneous flaps,4 and/ or microvascular bone periosteal flaps.5 Occasionally, however, the management of oral cancer also
involves early postsurgical irradiation to sterilize residual small tumor foci, or late postsurgical irradiation to salvage a surgical failure.6 Each of these possibilities poses the potential of irradiating a bone graft placed for jaw reconstruction. Work in several institutions has demonstrated improved functional and psychological responses to early reconstruction of cancer patients.7,8 Such interest in immediate or early delayed reconstruction makes it necessary to understand better the effects of irradiation on the type of bone grafts commonly used in oral and maxillofacial surgery. The purpose of this study was to determine the clinical and histologic changes, and to measure the strength, in bone grafts either placed into previously irradiated tissues or irradiated during the early phases of bone regeneration.
* Former Chief Resident and Research Fellow, Oral and Maxillofacial Surgery, University of Miami School of Medicine, Division of Oral and Maxillofacial Surgery. Presently Clinical Instructor. University of Miami School of Medicine. Division of Oral and Maxillofacial Surgery, Miami, Florida. t Associate Professor of Surgery and Director of Graduate Training and Research, University of Miami School of Medicine. Division of Oral and Maxillofacial Surgery, Miami, Florida. f Associate Professor of Radiology, Division of Radiation Therapy and Radiologic Sciences, Department of Radioloev. Univekty of Miami School of Medicine, Miami, Florida. -. Address correspondence and reprint requests to Dr. Marx: University of Miami School of Medicine, Division of Oral and Maxillofacial Surgery. I61 1 NW 12th Avenue..Miami. FL 33136.
Materials
and Methods
Ninety white New Zealand male rabbits (0~~tologus cuniculus) were divided into two groups of 45 animals each. The animals had their two mandibular incisor teeth and the regenerative germinal follicles removed to create a 3-cm edentulous space on each side of the midline. One additional animal served as a donor of allogeneic bone. Portions of rib, femur, and ilium were harvested in a sterile
0278-2391187 $0.00 + .25
34
MORALES ET AL.
FIGURE I (/cfi). Bone graft of autogenous particulate cancellous bone and marrow placed within an allogeneic bone crib. A measured I-cm graft was consistently placed into the 3-cm edentulous span. FIGURE 2 (ri&t). Specimen at point of fracture during steady rate loading of 1. lb/minute. A strain gauge records force and reflection, which are used to calculate Young’s modulus (E). modulus of flexion CM,.) and modules of force resistance Wf,), in addition to a measured yield point (f’).
fashion, frozen in liquid nitrogen. and freeze-dried for 17 days. Each specimen was separately packaged to serve as a sterile allogeneic bone crib. One experimental group received 5.5 Gy ‘j°Co 1 MEV external beam irradiation to the mandible in four fractions for a total of 22 Gy. Immobilization to accomplish controlled treatment was achieved by injecting ketamine, 25 mg/kg, and acepromazine, 0.25 mglkg, intramuscularly before irradiation. Each fractionation was separated by one week and was calculated to approximate a human dose of 640 Gy in 2 Gy fractions given daily, five days per week for six weeks. Irradiation began when the incisor sockets had complete mucosal covering (about six weeks). Recovery from acute irradiation damage to create the late chronic hypoxic tissue bed proceeded for three months. Each animal then underwent a transcutaneous resection and immediate bone graft reconstruction of a one cm length of the edentulous area on one side of the midline. The edentulous area on the opposite side served as a control. All bone grafts consisted of particulate autogenous cancellous bone and marrow harvested from the ilium and placed into an allogeneic bone crib from the donor animal fixed with a wire on each end (Fig. 1). No other form of fixation was necessary as the adult rabbit functions with a united midline suture imparting rigidity across the arch. The second group received identical surgery without preoperative irradiation. Instead, each animal received an equivalent irradiation protocol consisting of 8.5 Gy in two fractions spaced a week apart beginning at variable times in the postoperative period. A subgroup of five animals began the irradiation protocol immediately after surgery, an-
other at one week, another at two weeks, and other groups progressively at weekly intervals up to six weeks. All bone grafts were performed under monitored general anesthesia using intramuscularly injected ketamine. 25 mgikg, acepromazine. 0.25 mg/kg. and nembutal. 20 mg/kg. Each animal received 100,000 units of procaine penicillin G and 400,000 of benzanthene penicillin intramuscularly during surgery. The animals were fed Purina rabbit chow moistened in water and water ad libitum during the recovery phase. Each animal was given meperidine, 3 mgikg, twice-daily empirically for pain during the first two postsurgical days. Five animals in each postsurgical irradiation group were euthenized at weekly intervals beginning at postsurgical week two through week seven. For standardization, each postsurgical irradiation subgroup animal was killed two weeks after completion of irradiation. Therefore, each subgroup represented a one-week progressively later irradiation exposure. The grafts in 10 animals in this group were also allowed to heal a full 12 weeks. Five animals in the pregrafting irradiation group died prior to completion of the study. Three deaths were related to radiation toxicity and two to pasturella pneumonia. Three animals in the postgrafting irradiation group died before completing the study. One death was related to radiation toxicity, one to pasturella pneumonia, and the other to a nonspecific enterocolitis. TISSUESTUDYMETHODS
At necroscopy the entire mandible was harvested and cleaned of adherent tissue. Each mandible was split in the midline to create a grafted study spec-
36
EFFECT OF 1RRADlATION ON BONE GRAFT HEALING
L GRAFT SITE M-2 R-38 S-A. POST GRAFT IRR. 12 WEEKS
25
SLOPE = F/D = E = 375
20
FORCE (F) LSS.
25 /
L&S./IN.
20
15
P. E. MfY,.
L GRAFT M-4 R-13 PRE GRAFT IRR. 12 WEEKS
FORCE IF) LSS.
22.5 LSS. VOUNO’S MOD.. .575 LSS./IN.m RIGIDITY MOD. OF FLEXION. 39.1 -P/E MOD. OF RESISTANCE. P2/2Em 460
P. 3.2 LSS. E . VOUNO’S MOD. . 0.59 I RIGIDITY M,. MOD. OF FLEXION s 5.4 s P/E Yr= MOD. OF RESISTANCE P2/2E.8.7
1.5
L
5
10
15
20
5
25
FIGURE 3 (top lefr). Sample force (F) versus deflection (D) graph. This postgrafting irradiation specimen has a normal slope but a mid-range Young’s modulus (E) and a force resistance modulus (MR) less than ungrafted irradiated mandible. FIGURE 4 (top vighr). Sample force (F) versus deflection (D) graph. The slope of this pregrafted irradiation specimen is very flat. There is a lower Young’s modulus (E) than in the postgrafting irradiation animal in (Fig. 3) and a lower force resistance modulus (M,).
10
15
20
25
DEFLECTION (D) X 10-31N.
X 1O-3 IN. DEFLECTION (D) 5
4
PRE GRAFT IRR. -, POST QRAFT IRR. t CONTROL
/
3 CLINIC GRADING SCALE
FIGURE 5 (bottom rigl~t). Clinical grading score versus time. This graph demonstrates a slowed osteogenesis in the pregrafting irradiation group which peaks at four weeks and diminishes linearly thereafter. An improved osteogenesis in the postgrafting irradiation group that accelerates at four weeks and attains near normal bone formation by 12 weeks is also shown.
0123456789
10
11
12
TIME, WEEKS
imen and an ungrafted control specimen. All specimens were coded and analyzed in a blinded fashion in three ways. First, each specimen was examined clinically and described grossly by use of a scale from 1 to 5. Grade 1 represented no new bone formation; Grade 2, 1% to 25% new bone formation compared to the host bone size; Grade 3, 26% to 50% new bone formation compared to the host bone size; Grade 4, 51% to 75% new bone formation compared to the host bone size; and Grade 5, 76% to 100% new bone formation compared to the host bone size. The specimens were further radiographed at 90” to the occlusal plane. No specific grading of radiographs was undertaken but their interpretation was part of the clinical grading system. Physical consolidation of the grafts was measured by their load stress characteristics. Each fresh specimen was embedded in a holder with bone cement and loaded at a point 5 mm from the distal graft-host interface at a constant rate of 2
lb/minute until fracture occurred (Fig. 2). From strain gauge recordings, a graph comparing force to deflection at a yield point could be plotted (Figs. 3, 4 for each specimen). From each graph the following data were calculated or measured: P= E=
yield point; maximum force without fracture Young’s modulus; slope of force (F) v. deflection (D) (a measure of the graft rigidity and brittleness). Mf = modulus of flexion (Mf = P/E) (a measure of immature or osteoid bone formation and organic matrix) MR = modulus of resistance to load forces (Mr = P2/2Z$ (a measure of consolidation) After measuring the physical consolidation, each specimen was removed from the bone-holding apparatus and fixed in 10% neutral buffered formalin in a 2O:l volume-volume ratio for two weeks. All specimens were decalcified in 5% formic acid for
MORALES ET AL.
three weeks, embedded in paraffin, sectioned p_, and stained with hematoxylin and eosin.
at 6
Results CLINICAL
ASSESSMENT
A summary of the clinical findings is presented in Figure 5. In the pregraft irradiation group, bone formation was slowed but progressed linearly until week four. It then peaked at a 2.9 grade (about 25% of host bone size) and linearly declined to its lowest value (1.8) at 12 weeks (about 12% of host bone size) (Fig. 6). The mean clinical grade score in the postgrafting irradiation group also indicated early retardation of bone formation at two and three weeks. It then sharply improved at four weeks (3.9, or about .SO%of host bone size), reaching near control levels of bone formation by week five and 100% of control levels by week 12 (Figs. 5, 7). F’HYSICAL GRAFT CONSOLIDATION
A summary of the physical consolidation data is shown in Figure 8. The results indicate an inhibition of early bone production in the pregrafting irradiation group. They also strongly suggest a failure of the bone to mature. The graft reached a maximum resistance modulus of only 8.5% of controls at four weeks and lost further force resistance as each week progressed. In addition, the results show less inhibition of bone formation in grafts irradiated after placement. As indicated by the clinical grading scores. the fourth week seemed to be a critical time after which irradiation had less impact. The yield point and modulus of resistance each indicated an early production of osteoid that matured and consolidated to a resistance strength equal to 40-45% of ungrafted bone by five weeks and continued to at least 12 weeks. In contrast, the pregraft irradiation group also seemed to develop early osteoid. but the graft did not consolidate to develop any resistance strength, and it deteriorated even further with time. HISTOLOGIC
ASSESSMENT
The pregrafting irradiation group showed cellular loss in much of the transplanted bone. Osteocytes were absent in many lacunae and others showed nuclear changes consistent with cell death. Endosteal osteoblasts were also reduced in number. The stroma was fibrotic and poorly vascular. In the two-
FIGURE 6 (top). Pregrafting irradiation gross specimen (12 weeks) (above) and ungrafted irradiated mandible (below). Graft shows minimal new bone formation. as well as little structural integrity. FIGURE 7 (bottom). Postgrafting irradiation six-week specimen (above) and ungrafted irradiated mandible (below). Excellent bone formation and structural integrity values comparable to the ungrafted irradiated mandible is shown.
and three-week specimens osteoid production was sparse. In the four- and five-week specimens cellular osteoid production was evident but in a reduced quantity (Fig. 9). The later specimens, including the one at 12 weeks, showed osteoclastic activity and resorption of early osteoid without new bone replacement. In several areas the bone graft was replaced by a chondroid matrix (Fig. 10). The postgraft irradiation group also showed degenerative changes in the osteocytes. However, the endosteal osteoblasts were numerous, without evidence of nuclear changes. Osteoid production was sparse in the two- and three-week specimens but much more exuberant in the four- and five-week specimens (Fig. 11). In the later specimens, including the 12-week specimen, new bone formation could be observed arising from the host elements and the grafted elements. The osteoid tissue was
38
EFFECT
(IF
IKKAI>IAl‘ION
900
700 600
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GRAFT
HEALING
l
800
STRENGTH: mogpJs
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I
l
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RESISTANCE MFbP2/2E
X. rt’\i\t;tnce
time.
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graph bone
cover\
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of
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ungrafted
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which
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rt’-
and
a
PRE GRAFT IRR.
linear
r)
POST GRAFT IRR.
tegrity
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CONTROL
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group
that
wa\
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the
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I?-week
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It
further
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of
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100
postgrafting
point.
demon\trate5 the
in-
structural
or gain integ-
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01234567
8
9
10
11
12
TIME, WEEKS
becoming more organized and less cellular, and was forming a larger mineralized ossicle (Fig. 12). In some specimens a periosteum could be identified surrounding the grafted bone. Discussion
Bone graft healing, as proposed by Axhausen9 and documented by Gray et al.,rO can be regarded as cellular bone regeneration occurring in two phases. In the first (cellular phase), bone forms from the transplanted cellular elements, especially those of endosteal origin. In the second (replacement-remodeling phase), the transplanted bone is physiologically resorbed and replaced by a more mature bone of host cell origin. Pertinent to this study is the probable inhibition of both phases of bone regeneration in the pregrafting irradiation group. The poorly vascular host tissue both reduces survival of transplanted cellular elements and inhibits the metabolic activity required actively to produce osteoid. In addition, the fibrotic stroma lacks the cellular elements capable of producing the host-derived second phase bone and hence the observed reduction in clinical grade score and modulus of force resistance after four weeks. In fact, the ongoing reduction in modulus of force resistance and observed absence of bone can be regarded as the result of the expected resorption of whatever phase-one bone was formed without its replacement by phase-two bone. The presence of chondroid matrix supports the supposition that the irradiation of the tissue bed is mostly responsible for
the lack of new bone formation or its subsequent disappearance. Marx has shown irradiated jaws to be hypovascular-hypocellular and measurably et al. have, in turn. shown bone hypoxic. ‘I Basset ~ precursor cells to undergo chondroblastic differention in hypoxic environments up to the point of forming mature cartilage.” It seems reasonable to view the pregrafting irradiated tissue bed as hypoxic and incapable of adequately supporting either cellular survival or host cell bone differentiation. The response of bone graft elements to irradiation in the postgrafting irradiation group can also be interpreted in light of the two phase concept of bone regeneration. The initial phase of cellular proliferation and osteoid production may have been slowed by the cellular killing and damage of irradiation. However, our observations and those in the studies by Hulth and Wesherborn’3 and by Melonotte and Follis.r4 indicate a relative radiation resistance to endosteal osteoblasts. These cells appear more sensitive to irradiation in the first three weeks after transplantation, but are capable of some survival and subsequent bone formation. When the graft is irradiated at four weeks or later. bone formation is minimally affected. This finding can be interpreted to mean that by four weeks the cellular phase-one bone has mostly formed and that its replacement by host-derived phase-two bone is not greatly affected by irradiation. The observation that even the postgrafting irradiation group showed a modulus of force resistance only 42% of controls by 12 weeks can be explained by either of two possible mechanisms. The first is
MORALES
ET AL.
FIGURE 9 crop). Photomicrograph of a four-week inflammation, and fibrous tissue ingrowth replacing FIGURE formation
pregrafting irradiation the transplanted bone.
10 (bottom). Chondroid matrix within an area of cellular may be secondary to postirradiation hypoxia. (Hematoxylin
specimen showing minimal new osteoid (Hematoxylin and eosin. Magnification.
bone in a I?-week pregrafting irradiation and eosin. Magnification, x IO.)
formation, x 10.)
specimen.
sevc :re
Chondroid
40
EFFECT
‘.
*.
OF IRRADIATION
ON BONE
GRAFT
HEALING
“_
FIGURE 11 (top). Exuberar __ .Magmhcation, X 10.)
It cellular
osteoid
formation
FIGURE 12 (bottom). A I?-week postgrafting irradiation tive tissue stroma. (Hematoxylin and eosin. Magnification.
in a four-week specimen ~4.)
postgrafting
demonstrating
irradiation a mature
specimen.
mineralized
(Hematoxylin
ossicle
and eosin.
and minimal
connec-
41
MORALES E:T AL.
that after four or five weeks bone grafts are somewhat resistant to irradiation damage or at least no more vulnerable than ungrafted host bone. The fact that force resistance was less than one-half that of host bone may merely be due to a greater than 12week requirement for the grafts to develop similar strength characteristics. Phase-two remodeling may continue at a slow pace and add structural integrity to a graft over many years. The slope of the control data in Figure 8 suggests this mechanism. A reduction in structural integrity of even ungrafted bone was seen in early irradiated mandibles. Although there was some individual variation, probably due to size differences between jaws, a redevelopment of measured structural integrity was seen. This observation implies that ungrafted bone is also weakened by irradiation and recovers slowly and incompletely. The postgrafting irradiated grafts were probably still in a recovery phase, although their clinical appearance was similar to ungrafted bone at 12:weeks. The second possible explanation for a reduced modulus of force resistance is that irradiation of a bone graft at four weeks or later may have a small or negligible effect on the graft itself but that it does lead to soft tissue changes of a hypovascular-hypocellular-hypoxic nature similar to those created in the pregrafting irradiated group, but of a lesser degree. Such an effect on the soft tissue envelope can inhibit full development of the graft by affecting later phase-two remodeling. Although much of the phase-two replacement and remodeling has occurred by the time the chronic hypoxic state develops from irradiation damage, late phase-two bone maintenance may be altered to create a weaker bone. Further studies with long-term follow-up are required to elucidate the correct explanation. Data obtained from animal models should be interpreted with caution. Bone physiology, structure, and healing rates vary between species and among members of the same species. The results obtained in this study, however, suggest a more detrimental effect on attempted bone formation in tissue previously altered by irradiation than to bone irradiated during its formative period. Bone grafts placed into irradiated tissue beds suffer loss in both phases of expected bone regeneration. The wellknown low success rates of this type of clinical reconstruction is attested to by the findings of this study. The hypovascular-hypoxic-hypocellular tissue bed seems to be the critical tissue effect. The current use of graft systems that circumvent this ir-
radiation effect, such as microvascular bone-periosteal grafts, osteomyocutaneous grafts. and hyperbaric oxygen-supported grafts, is supported by these findings. Bone grafts which are irradiated are, no doubt, affected by the cellular damage of high energy transfer. However, particulate bone and cancellous marrow grafts were found to be surprisingly tolerant of irradiation. A four-week period of phaseone bone initiation prior to irradiation appears to be the critical time for bone to develop in a near normal fashion. Such evidence establishes a scientific basis for the consideration of early jaw reconstruction in cancer patients who may require irradiation after grafting. It would seem that if irradiation were deferred for four weeks after jaw reconstruction, the clinical results of current bone graft systems could be as successful as nonirradiated grafts and certainly more successful than a graft placed into an unimproved irradiated tissue bed.
References 1. Perez CA, Lee FA, Ackerman LV, et al: Nonrandomized comparison of preoperative irradiation and surgery (vs) irradiation alone in the management of carcinoma of the tonsil. Am J Roentgen 126:248, 1976 2. Marx RE: A new concept in the treatment of osteoradionecrosis. J Oral Maxillofac Sum. 41:351. 1983 3. Marx RE, Ames JR: The use OFhyperbaric oxygen therapy in bone reconstruction of the irradiated and tissue-deficient patient. J Oral Maxillofac Surg 40:412, 1982 4. McGraw JB, Dibbell DC, Cart-away JH: Clinical definition of independent myocutaneous vascular territories. Plast Reconstr Surg 60:341, 1977 5. Donoff RB, May JW: Microvascular mandibular reconstruction. J Maxillofac Surg 40: 122, 1982 6. Fayas JV, Lampe I: Radiotherapy of squamous cell carcinoma of the oral portion of the tongue. Arch Surg 94:316, 1967 7. Bear SE, Green RK, Wentz WW: Stainless steel wire mesh -an aid in difficult oral surgery problems. J Oral Surg 29:27, 1971 8. Marx RE: Current Principles and Techniques in Major Jaw Reconstruction. Mini Lecture Series “A.” 66th Annual AAOMS Meeting, 14 September 1984. New York 9. Axhausen W: The osteogenetic phases of regeneration of bone. J Bone Joint Surg 38A:593, 1956 10. Gray J, Phil M, Elves M: Donor cells contribution to osteogenesis in experimental cancellous bone grafts. Clin Orthop 163:261, 1982 Il. Marx RE: Osteoradionecrosis: a new concept of its pathophysiology. J Oral Maxillofac Surg 41:283. 1983 12. Bassett CAL, et al: Clinical implications of cell function in bone grafting. Clin Orthop 87:49, 1972 13. Hulth A, Westerbom 0: Early changes of the growth zone in rabbit following roentgen irradiation. Acta Orthop Stand 30:155, 1960 14. Melanotte PL, Follis RH: Early effects of x-irradiation on cartilage and bone. Am J Pathol 39: 1, 1965