Veterinary Surgery, 20, 4, 229-234, 1991
Healing of Canine Autogenous Cancellous Bone Graft Donor Sites ROGER
c. PENWICK, VMD. Diplomate ACVS. DEREK A. MOSIER, DVM. Diplomate ACVP, and DAVID M.CLARK,
DVM, Diplomate ACVS
Autogenous cancellous bone was procured from the proximal ends of one humerus and the contralateral tibia of eight adult mixed-breed dogs. Greater weights of bone were consistently harvested from the humerus than from the tibia. Restoration of cancellous bone was more rapid and complete in the humerus than in the tibia. The tibial harvest site filled primarily with fibrous tissue rather than with cancellous bone.
of "bone grafting" human surgery T was apparently recorded Since then. transferring techniques have become so commonplace HE FIRST USE:
in
in 188 I . I
bfaterials and klethods
bone
that in human surgery today bone is "transplanted" with greater frequency than any other organ except blood.'.' Veterinary surgeons have also shown considerable interest in experimental and clinical bone grafting techniques. 'The physiology of bone transplantation. including the concepts of osteopotency. osteoinduction. and osteoconduction. has been examined in detail.',''x and the appropriatc terminology has been claritied.' Although autogenous cancellous bone grafts provide little mechanical support. their osteogenic capacity makes their use attractive. Despite the extensive literature dealing with gmft-recipient site interactions and the physiology of osteogenesis. alterations within the donor sites ofcancellous bone have been given little attention. Failure o r delayed union of an arthrodesis or a nonunion fracture may require niorc than one bone grafting procedure. but it is not known whcthcr a site previously used for cancellous bone graft procurement can be used again for that purpose. Problems such as delayed soft tissue healing and incomplete fracture of the tibial cortex at the donor site have been documented." I t is important to examine the processes of hone and wound healing at a cancellous graft donor site before recommendations about the manner of harvesting cancellous grafts or the feasibility of obtaining a second grafl from a single site can be made. The purpose of this study is t o examine the process of bone healing at the collection site of cancellous bone in the canine humerus and tibia.
Four male and { b u r female adult, mixed-breed dogs weighing 12.3 to 23.h kg were sedated with acetylprornaLine maleate (0.I mg/kg intramuscularly) before induction of genenll anesthesia with sodium pen tothal ad in in tstcrcd intrabenousl? to efl'tct. ,Anesthesia was maintained via a semiclosed rebreathing system with halothane and oxygen. Aseptic technique was used for all surgical procedures. Cancellous bone graft material was procured from the proximal ends of one humerus and the contralateral tibia. All harvesting procedures were pertbi-med by the same surgeon (Penwick). N o efbrt was made to randomize which side was used. Alier- four-quadrant draping. approaches for hanesting cancellous bone were made to thc craniolateral aspect of the proximal end of the humerus and the medial aspect o f the proximal end ot'the tibia."' The cortex was penetrated with a 4.5 nim twist drill hit in a nitrogen powered drill, and a 5.0 mm curette was used to enlarge the portal slightly and harvest cancellous bone. Harvesting was continued at each site until no more cancellous bone could be obtained. Specimens ofcancellous bone were placed immediately into sterile, preweighed Petri dishes and weighed. Although the specitnens were not \hashed. etforts were made to remove gross amounts of blood or blood clots. A single 2.7 X 8 mm cortical bone screw was inserted a t the level of the graft donor site i n an adjacent region of the bone to facilitate later identification of the tlonor site. All wounds were closed with simple interrupted sutures of size 2-0 chromic
From the Departments of Medicine and Surgery (Penwick Clark) and Pathology (Mosier) College of Veterinary Medicine Oklahoma State University Stillwater Oklahoma Supported by Oklahoma State University, College of Veterinary Medicine, Seed Grant #5-1-50010 Reprint requests Roger C Penwick. VMD, Tucson Veterinary Surgical Service. PC 3801 E Ft Lowell Suite 101 Tucson, AZ 85716
229
230 TABLE 1
Doa No.
HEALING
OF BONE GRAFT DONOR SITES
Cancellous Bone Graft Material Obtained (Grams) Shoulder (Humerus)
Tibia
Difference’
2.26 1.63 3.04 2.82 2.75 2.41 2.06 2.10
1.39 1.83 1.51 1.07 0.99 0.88 1.01 1.47
+0.87 -0.20 +1.53 +1.75 +1.76 +1.53 +1.05 i 0.63
* Difference is expressed as a positive number where the weight of bone obtained from the humerus exceeded that obtained from the tibia, and as a negative number where the reverse applied.
gut in the subcutis and size 3-0 monofilament nylon in the skin. The periosteum and other deep structures were not sutured. Time-zero specimens were obtained by performing identical graft harvesting and processing procedures on both humeri and tibiae of a healthy dog cuthanatized for other purposes. Recovery from anesthesia was uneventful. All dogs were permitted to resume full activity in 1 X 2 meter runs and were housed and observed in compliance with NIH guidelines for the care and use of animals and the US Animal Welfare Acts. All dogs were monitored closely Ihr lameness. swelling, or inflammation at the operative sites. Body temperatures and other vital signs were recorded daily for 72 hours after surgery. Two dogs were euthanatized at each of the following time intervals: I week, 2 weeks, 4 weeks, and 8 weeks. Tibiae and humeri were removed immediately and cleansed of all soft tissues. The identifying cortical screw was located and removed. Serial transverse sections approximately 3 m m wide were cut with a band saw through the donor site and the corresponding region of the contralateral bone. Each transverse section was described, photographed, and fixed in 10%)neutral buffered formalin. Fixed sections were decalcified in 20% sodium citrate with 513% formic acid solution. Specimens were prepared and stained with hernatoxylin and eosin or trichrome for histologic evaluation.
’’
Fig. 1. Radiographs of time-zero tibia (left) and humerus (right) cancellous graft donor specimens. The location and extent of typical harvest site defects are visible at the proximal ends of the bones.
Transverse sections through the harvest sites of the humerus and tibia were characterized by central, depressed. dark red regions that replaced the harvested cancellous bone (Figs. 2a and 3a). The harvest site in the tibia occupied a relatively greater volume of cancellous bone than the corresponding site in the humerus. There was moderate fibrous periosteal thickening overlying cortical bone at the access portal. Histologically, the changes in the humerus and tibia were similar. The harvested cancellous bone was replaced by a dense coagulum of fibrin with numerous erythrocytes, a few leukocytes, and rare fibroblasts (Fig. 4). Adjacent to this coagulum was a dense zone of fibroblastic proliferation (Fig. 5). In deeper regions, the fibroblastic tissue
Results In all dogs, vital signs remained normal and wound healing proceeded without complications. There were no adverse sequelae to the 16 cancellous bone harvesting procedures. N o animal exhibited any signs that suggested a need for analgesics. Consistently greater amounts of cancellous bone were harvested from the humerus than from the tibia (Table 1). Typical defects created during the cancellous bone harvesting procedure are shown in Figure 1.
Fig. 2. Cross sections of tibia harvest sites at week 1 (A), week 2 (B), week 4 (C), and week 8 (D).
PENWICK, MOSIER, AND CLARK
Fig. 3 Cross sections of humerus harvest sites at week 1 (A), week 2 (B), week 4 (C). and week 8 (D)
contained small, irregular islands of ostecid co\ erect b y hypertrophic osteoblasts. The cortical access portal contained fibroblasts and fibrin that blcndcd with the thickened immature fibrous tissue of the overlying periostcuin. Periosteal neovascularization and granulation acconipanied by moderate t o large amounts of hemorrhage uere also present.
231
Fig 5. Humerus donor site (week 1) Higher magnification of fibroblastic zone Neovascularization and extensive fibroblastic proliferation are present (Hematoxylin and eosin 100 )
Transverse sections of the tibia contained fibrous tissue that filled a large amount of the harvest site and was separated from normal adjacent cancellous bone bq dark red tissue (Fig. 2b). The tibial periosteum was thickened Ii)cally, and a dense fibrous band extended through thc cortical defect into the harvest site. In thc humerus. dark red
tissue at the center o f ihe haintH site was bordered by a thin, irregular band of Ijbrous connective tissue (Fig. 3b). Changes i n the periosteum and cortical defect in the h u merus were similar to. hut much less prominent than. those in the tibia. l ' h e region of atfected cancellous bone was relativel? greater in the tibia than in the humerus. Histologicall). the tibia1 hanest site contained moderate fibrosis. heniorrhagt,. and w m c hemosiderin (Fig. 6 ) . There was no bone tbrmation Lcithin the connective tissue. Bone trabeculae adJacent to the fibrous tissue were thin. immature. and widely separated hy loose, stellate. fibrous tissue. Irrcgularl! arranged iibroblasts, surrounded by small amounts of' mature collagen and nests of hyaline cartilage. were prcsenl in the tibial periosteum. Small. irregular. immature hone trabeculae extended peripht,ral\>
Fig 4 Humerus donor site (week 1) A large coagulum of erythrocytes and fibrin replacing harvested bone IS separated from normal adjacent bone by a fibroblastic zone (Hematoxylin and eosin, 120 )
Fig 6 Tibia donor site (week 2) Fibroblastic zone is dense and cellular Extensive fibroblastic invasion of adjacent normal bone IS present (Hematoxylin and eosin 60 )
232
HEALING OF BONE GRAFT
DONOR SITES
Fig 7 Humerus donor site (week 2) Fibroblastic zone is thin and well defined Slight myxornatous fibroblastic invasion of adjacent normal bone is present (Hematoxylin and eosin, x40 )
Fig 8 Tibia donor site (week 4) There is dense periosteal fibrosis with extension of a fibrous band through the cortex into the medulla (Hematoxylin and eosin, x20 )
beyond the cortical bone into the periosteum and the cortical defect. In the humerus, fibrous tissue proliferation at the harvest site and in the cortical defect and periosteum was less prominent. Fibrosis within the harvest site consisted of narrow bands of dense fibrous connective tissue surrounding a central small cavitation (Fig. 7). Fibrosis penpheral to this region was granular to myxomatous. poorly cellular, and interspersed with fine. narrow, immature bone trabeculae with hyperplastic osteoblasts. Adjacent to the harvested region, bonc trabeculae were mature and normal bone marrow elements were present.
W2c.k 8 Transverse sections of the tibia at the harvest site contained denser, more irregular fibrosis than at week 4 (Fig. 2d). In the humerus, gross alterations were not seen in the periosteum, cortex. or harvest site (Fig. 3d). Histologically, focal dense connective tissue was surrounded by cancellous bone at the tibial harvest site. A thin band of fibrous connective tissue extended from this site through the cortical defect to the thickened, overlying periosteum. Rare small foci of dense connective tlssue, interspersed among cancellous bone, were present within the humeral harvest site. N o penosteal or cortical bone changes were present.
Hkck 4 Transverse sections of the tibia contained a nest of pale ;gray fibrous connective tissue that filled the harvest site (Fig. 2c). A fibrous band extended from the harvest site through the cortical defect to moderately thickened penosteum. In the humerus, penosteal thickening was not detected and only occasional small, multifocal areas of fibrosis, interspersed with normal cancellous bone. were present at the harvest site (Fig. 3c). Histologically, the tibial harvest site contained a dense core of mature fibrous connective tissue surrounded by cancellous bone that was widely separated by a less cellular rnyxomatous connective tissue. Dense fibrous connective tissue was responsible for the penosteal thickening and filled the connective tissue defect (Fig. 8). In the humerus, small nests of dense fibrous connective tissue were widely scattered throughout the harvest site (Fig. 9). Surrounding tissues were qualitatively normal, and no defects in the Cortex were present.
Humerus donor site (week 4). There is a focal area of medullarv fibrosis. (Hernatoxylin and eosin, x20.) Fiq. 9.
PENWICK, MOSIER, AND CLARK
Discussion A consistent pattern of healing followed procurement ofcancellous bone. A large coagulum of fibrin and crytlirocytes filled the cavity and was subsequently invaded from the periphery by vascular and fibrous tissucs. New bone formation accompanied by fibrosis followed rc\ascularization ofthe peripheral regions. progressing centrally to replace the harvested bone. The major difTerensc i n the healing process between tibia and humerus was the degree of regeneration of cancellous bone compared to the amount of replacement by connective tissue. By week 2, the response in the tibia remained primririli fibrosis. although significant amounts of new bone formation had begun at the periphery of the donor site. I n contrasl, the fibrous response in the humerus at week 2 was less productive, more niyxomatous. and interspersed with tine. immature bone trabeculae. At week 8. the tibia still contained prominent amounts of connective tissue within the harvest site, while only small amounts of fibrosis were present in the humerus. These results d o nut agrec with others in which, after harvest of cancellous bone from the canine tibia, endosteal callus formed by weck 8 and normal intramedullary architecture was restored h! week 12.” Reasons for these differences are not apparent. Results of this study suggested that cancellous hone was replaced much more rapidly and completely i n the humerus than in the tibia. The reason for this tlitlercnce is unclear. hut several factors may be postulated. Thc medullary cavity at the proximal end of the canine tibia appears to be relatively smaller than that ofthe humerus. Despite the fact that consistently lower weights of cancellous bone were obtained from the tibia. the harvested bone may have constituted a greater percentage ol‘ the cancellous bone at this site (Figs. 2,3). I t is possiblc that cancellous bone harvesting is more inJurious to thc tibia than to the humerus because of more conipletc removal of trabecular bone. hematopoietic or stromal elcments. and possibly endosteum. One author believes that osteoblasts are derived trom stromal elements of bone marrow, while osteoclasts arise from marrow hematopoietic elements. ” Thesc cell lines do not undergo transformation from one to thc other.“ The hypothesis could be advanced that the more aggressive removal of stromal elements of the marrow of the tibia inhibits regrowth of new cancellous bone. Induction of new osteoblasts from any source may be impeded by removal of bone morphogenetic protein. along with trabecular bone and bone marrow stroma. If. as has also been hypothesized, osteoclast stem cells exist in circulating blood, osteoclasia may be inhibited to a lesser degree than osteogensis. ’’ It has been stated that “marrow in repair may have a dichotomy i n differentiation: under optimal conditions
233
to form new bone and i n adversity to become phagocytic.”“’ I f true. this may panially explain why infection leads to delayed fracture union. and the hypothesis may be equally applicahlc to evcnts i n the tibial canccllous donor site. Histologicall>. there appeared to be cortical thinning during healing ofthe tibia. possibly attributable to the osteolytic role of marrow elements in a hypovascular state. In the tibia. conditions for regeneration of cancellous bone may be comparable to healing of a long bone fracture in which masive aniounts of bone marrow and endosteum are lost. If an extf;iosseous vascular supply is not present or an autogenous cancellous graft is not supplied. delayed healing may result.’- I‘ During healing of the proximal tibial donor site. hyaline cartilage associated with the bony defect may habe been indicative of a hypovascular state. resulting in IOU oxhgcn tension in the region.” In fracture healing~ostcogenic cells that ditferentiate in more superficial nonLascular areas of thc provisional call us d i ffe re n t i ;1t e i n to chon d ro blast s a nd c ho nd roc y t es. However, u.hen capillary ingrowth keeps pace with osteogenic cell growth. these pluripotential cells differentiate in a more vascular cnvironment ofgreater oxygen tension and thus form bone rather than cartilage.’x.”’It is possible that the damage t o the tibia was sufficient t o rccult in decreased vascular supply and an impcdinicnt to hcaling. In contrast. the humerus may have sustained a less severe injury and ovcrcome thc trauma to heal more rapidly and completely. It is likely that this was related to the relative difference in s i x s of the cavitics created, because the harvest sites in both hones were similarly remote from the nutrient artery. Previous reports of “stre\s fractures” in the tibia after harvesting cancellous bone reinforce the theory that injur? to the tibia may be severe.’” Although such fracturcs ma! be attributed to the stress riser effect of a cortical cfeli.ct. the size and configuration of the detects make this seem unlikely.” The delay in healing of a graft donor site. as demonstrated in the tibiae in this study, could possibly contribute to cortical fractures. Although the number of animals in this study was small. the histologic appearance and apparent progression of events were consistent in all specimens and at all time intervals studied. Thus, the proximal end of thc humerus appears to be superior to the tibia as a donor site tor cancellous bonc because it yields larger quantities of cancellous bone and undergoes more complete and rapid healing. While nearly all of thc humera1 harvest site was replaced by cancellous bonc b j weck 8, centrally located cancellous bone appeared thinner and more fragile than that at the periphery ofthe harvest site. Other investigators found that these thinner trabeculae persisted near the center of a humeral cancellous graft donor site 4 months after graft harvesting.” The current data suggest that by
HEALING OF BONE GRAFT DONOR SITES month 2 it may be possible for the humerus lo provide a second cancellous graft. Biologic contributions ofthe second graft would depend on the osteogenic capacity of the newly formed bone. Further studies are needed to determine the precise time when a functional graft could be 'obtained from a previous donor site. The pattern of healing in the tibia was far less favorable, making it unlikely [hat a second graft could be procured from it. Despite its shortcomings, the tibia remains a useful graft donor site i n clinical small animal surgery and is frequently employed by the authors. In our experience, clinical problems after harvesting cancellous bone from any site have been virtually nonexistent. The results of this study suggest, however, that when possible or convenient, ihe proximal end of the humerus should be the site of preference for cancellous bone harvesting.
References I . MacEwen W. Observations concerning transplantatton of bone: II-
2.
3. 4.
5
6.
7.
lustrated b> a case of inter-human osseous transplantation. whereby oker two-thirds of the shaft ofthe humerus was restored. Proc R Soc Lond [Biol] 1881: 2 3 3 2 - 2 4 7 . Prolo DJ. Rodrigo J J . Contemporary bonegraft physiology and surgery. Clin Orthop 1985; 200:322-342. Friedlander GE. Current concepts review of bone grafts. J Bone Joint Surg 1987; 69A:786-790. Lance EM. Some observations of bone graft technology. Clin Orthop 19x5: 200:l 14-124. Nilsson 0s. Urist MR. Schmalztriede TP. Fineman GA. Bone repair induced by bone morphogenetic protein in ulnar defects in dogs. J Bone Joint Surg 1986: 68B:635-642. Oikarinen J. Korhonen LK. The bone inductive capacity of various bone transplanting materials used for treatment of experimental bone defects. Clin Orthop 1979; 140:208-215. Ray RD. Sabet TY. Bone grafts: Cellular survival versus induction. An experimental study in mice. 3 Bone Joint Surg 1963: 45.4: 337-344.
8. Urist MR. Bone Cormation by autoinduction. Science 1965: 150: 893-809. 9. Johnson KA. Caneellous bone graft collection from the tibia i n dogs. Vet Surg 1986: l5:334-338. 10. Schena CJ. The procurement of cancellous bone for grafting in small animal orthopedic surgery: A review of instrumentation. technique, and pathophysiology. J Am Anim Hosp Assoc 1983: 19: 695-704. II . Guide for the Care and Use of Laboratory Animals. US Department of Health and Human Services Publication No. 86-23, Prepared by the Committee on Care and Use of Laborator) Animals of the Institute of Laboratory Animal Resources. Commission of ILifc Sciences. National Research Council. Public Health Service. National Institutes of Health. Bethesda. Md. Contract No. I -RR2-2 135: Animal Resources Program. 12. Johnson KA. Histologic features ofthe healing pattern of bone graft donor sites in dogs. Am J Vet Res 19x8; 49:XX5-888. 13. Owen M . The origin of hone cells in the postnatal organism. Arthritis Rheum 1980; 23:1079-1086. 14. Owen M. Bone growth at the cellular layer: A perspective. In: Dixon AD. Sartnat BG, eds. Fmtorc. mid Mdinni.sm\ It?//iwwitigBonc~ Growth New York: Alan R. Liss, 1982:19. 15. Bonucci E. New knowledge of the origin. function. and fate of osteoclasts. Clin Orthop 19x1; I58:252-261. 16. Burwell RG. The function of bone marrow in the incorporation of a hone graft. Clin Orthop 1985; 200:125-141. 17. Cruess RI. Dumont J . Conditions influencing fracture healing. In: Ncwton CD. Nunamaker DM. eds. 7c.i-rhooX o/'Stncr//. - I ! i i m d Or//wpcdilic~.~ Philadelphia: JB Lippincott. 1985:58. 18. Simmons DJ. Fracture healing perspectives. Clin Orthop 1985: 200: 100-1 13. 19. Bassett CA. Hermann I. Influence of oxygen concentration and mechanical factors on differentiation of connective tissue in virro. Nature 1961: 190:460-461. 10. Ham AW. Bone: The healing of a simple fracture of a long bone. In: Ham AW. I/r.c.to/ogj. 7th ed. Philadelphia: JB Lippincort. 1974:433. 21. Clark CR. Morgan C, Sonstegard DA. Matthews LS: The effect of biopsy hole shape and siLe on bone strength. J Bone Joint Surg. 1977: 59-A:213-217. 17 Howard PE, Wilson JW. Ribble GA. Effects of gelatin sponge ini--. plantation in cancellous bone defects in dogs. J Am Vet Med Assoc 1988: 192633-637.
Healing of Canine Autogenous Cancellous Bone Graft Donor Sites ROGER
c. PENWICK, VMD. Diplomate ACVS. DEREK A. MOSIER, DVM. Diplomate ACVP, and DAVID M.CLARK,
DVM, Diplomate ACVS
Autogenous cancellous bone was procured from the proximal ends of one humerus and the contralateral tibia of eight adult mixed-breed dogs. Greater weights of bone were consistently harvested from the humerus than from the tibia. Restoration of cancellous bone was more rapid and complete in the humerus than in the tibia. The tibial harvest site filled primarily with fibrous tissue rather than with cancellous bone.
of "bone grafting" human surgery T was apparently recorded Since then. transferring techniques have become so commonplace HE FIRST USE:
in
in 188 I . I
bfaterials and klethods
bone
that in human surgery today bone is "transplanted" with greater frequency than any other organ except blood.'.' Veterinary surgeons have also shown considerable interest in experimental and clinical bone grafting techniques. 'The physiology of bone transplantation. including the concepts of osteopotency. osteoinduction. and osteoconduction. has been examined in detail.',''x and the appropriatc terminology has been claritied.' Although autogenous cancellous bone grafts provide little mechanical support. their osteogenic capacity makes their use attractive. Despite the extensive literature dealing with gmft-recipient site interactions and the physiology of osteogenesis. alterations within the donor sites ofcancellous bone have been given little attention. Failure o r delayed union of an arthrodesis or a nonunion fracture may require niorc than one bone grafting procedure. but it is not known whcthcr a site previously used for cancellous bone graft procurement can be used again for that purpose. Problems such as delayed soft tissue healing and incomplete fracture of the tibial cortex at the donor site have been documented." I t is important to examine the processes of hone and wound healing at a cancellous graft donor site before recommendations about the manner of harvesting cancellous grafts or the feasibility of obtaining a second grafl from a single site can be made. The purpose of this study is t o examine the process of bone healing at the collection site of cancellous bone in the canine humerus and tibia.
Four male and { b u r female adult, mixed-breed dogs weighing 12.3 to 23.h kg were sedated with acetylprornaLine maleate (0.I mg/kg intramuscularly) before induction of genenll anesthesia with sodium pen tothal ad in in tstcrcd intrabenousl? to efl'tct. ,Anesthesia was maintained via a semiclosed rebreathing system with halothane and oxygen. Aseptic technique was used for all surgical procedures. Cancellous bone graft material was procured from the proximal ends of one humerus and the contralateral tibia. All harvesting procedures were pertbi-med by the same surgeon (Penwick). N o efbrt was made to randomize which side was used. Alier- four-quadrant draping. approaches for hanesting cancellous bone were made to thc craniolateral aspect of the proximal end of the humerus and the medial aspect o f the proximal end ot'the tibia."' The cortex was penetrated with a 4.5 nim twist drill hit in a nitrogen powered drill, and a 5.0 mm curette was used to enlarge the portal slightly and harvest cancellous bone. Harvesting was continued at each site until no more cancellous bone could be obtained. Specimens ofcancellous bone were placed immediately into sterile, preweighed Petri dishes and weighed. Although the specitnens were not \hashed. etforts were made to remove gross amounts of blood or blood clots. A single 2.7 X 8 mm cortical bone screw was inserted a t the level of the graft donor site i n an adjacent region of the bone to facilitate later identification of the tlonor site. All wounds were closed with simple interrupted sutures of size 2-0 chromic
From the Departments of Medicine and Surgery (Penwick Clark) and Pathology (Mosier) College of Veterinary Medicine Oklahoma State University Stillwater Oklahoma Supported by Oklahoma State University, College of Veterinary Medicine, Seed Grant #5-1-50010 Reprint requests Roger C Penwick. VMD, Tucson Veterinary Surgical Service. PC 3801 E Ft Lowell Suite 101 Tucson, AZ 85716
229
230 TABLE 1
Doa No.
HEALING
OF BONE GRAFT DONOR SITES
Cancellous Bone Graft Material Obtained (Grams) Shoulder (Humerus)
Tibia
Difference’
2.26 1.63 3.04 2.82 2.75 2.41 2.06 2.10
1.39 1.83 1.51 1.07 0.99 0.88 1.01 1.47
+0.87 -0.20 +1.53 +1.75 +1.76 +1.53 +1.05 i 0.63
* Difference is expressed as a positive number where the weight of bone obtained from the humerus exceeded that obtained from the tibia, and as a negative number where the reverse applied.
gut in the subcutis and size 3-0 monofilament nylon in the skin. The periosteum and other deep structures were not sutured. Time-zero specimens were obtained by performing identical graft harvesting and processing procedures on both humeri and tibiae of a healthy dog cuthanatized for other purposes. Recovery from anesthesia was uneventful. All dogs were permitted to resume full activity in 1 X 2 meter runs and were housed and observed in compliance with NIH guidelines for the care and use of animals and the US Animal Welfare Acts. All dogs were monitored closely Ihr lameness. swelling, or inflammation at the operative sites. Body temperatures and other vital signs were recorded daily for 72 hours after surgery. Two dogs were euthanatized at each of the following time intervals: I week, 2 weeks, 4 weeks, and 8 weeks. Tibiae and humeri were removed immediately and cleansed of all soft tissues. The identifying cortical screw was located and removed. Serial transverse sections approximately 3 m m wide were cut with a band saw through the donor site and the corresponding region of the contralateral bone. Each transverse section was described, photographed, and fixed in 10%)neutral buffered formalin. Fixed sections were decalcified in 20% sodium citrate with 513% formic acid solution. Specimens were prepared and stained with hernatoxylin and eosin or trichrome for histologic evaluation.
’’
Fig. 1. Radiographs of time-zero tibia (left) and humerus (right) cancellous graft donor specimens. The location and extent of typical harvest site defects are visible at the proximal ends of the bones.
Transverse sections through the harvest sites of the humerus and tibia were characterized by central, depressed. dark red regions that replaced the harvested cancellous bone (Figs. 2a and 3a). The harvest site in the tibia occupied a relatively greater volume of cancellous bone than the corresponding site in the humerus. There was moderate fibrous periosteal thickening overlying cortical bone at the access portal. Histologically, the changes in the humerus and tibia were similar. The harvested cancellous bone was replaced by a dense coagulum of fibrin with numerous erythrocytes, a few leukocytes, and rare fibroblasts (Fig. 4). Adjacent to this coagulum was a dense zone of fibroblastic proliferation (Fig. 5). In deeper regions, the fibroblastic tissue
Results In all dogs, vital signs remained normal and wound healing proceeded without complications. There were no adverse sequelae to the 16 cancellous bone harvesting procedures. N o animal exhibited any signs that suggested a need for analgesics. Consistently greater amounts of cancellous bone were harvested from the humerus than from the tibia (Table 1). Typical defects created during the cancellous bone harvesting procedure are shown in Figure 1.
Fig. 2. Cross sections of tibia harvest sites at week 1 (A), week 2 (B), week 4 (C), and week 8 (D).
PENWICK, MOSIER, AND CLARK
Fig. 3 Cross sections of humerus harvest sites at week 1 (A), week 2 (B), week 4 (C). and week 8 (D)
contained small, irregular islands of ostecid co\ erect b y hypertrophic osteoblasts. The cortical access portal contained fibroblasts and fibrin that blcndcd with the thickened immature fibrous tissue of the overlying periostcuin. Periosteal neovascularization and granulation acconipanied by moderate t o large amounts of hemorrhage uere also present.
231
Fig 5. Humerus donor site (week 1) Higher magnification of fibroblastic zone Neovascularization and extensive fibroblastic proliferation are present (Hematoxylin and eosin 100 )
Transverse sections of the tibia contained fibrous tissue that filled a large amount of the harvest site and was separated from normal adjacent cancellous bone bq dark red tissue (Fig. 2b). The tibial periosteum was thickened Ii)cally, and a dense fibrous band extended through thc cortical defect into the harvest site. In thc humerus. dark red
tissue at the center o f ihe haintH site was bordered by a thin, irregular band of Ijbrous connective tissue (Fig. 3b). Changes i n the periosteum and cortical defect in the h u merus were similar to. hut much less prominent than. those in the tibia. l ' h e region of atfected cancellous bone was relativel? greater in the tibia than in the humerus. Histologicall). the tibia1 hanest site contained moderate fibrosis. heniorrhagt,. and w m c hemosiderin (Fig. 6 ) . There was no bone tbrmation Lcithin the connective tissue. Bone trabeculae adJacent to the fibrous tissue were thin. immature. and widely separated hy loose, stellate. fibrous tissue. Irrcgularl! arranged iibroblasts, surrounded by small amounts of' mature collagen and nests of hyaline cartilage. were prcsenl in the tibial periosteum. Small. irregular. immature hone trabeculae extended peripht,ral\>
Fig 4 Humerus donor site (week 1) A large coagulum of erythrocytes and fibrin replacing harvested bone IS separated from normal adjacent bone by a fibroblastic zone (Hematoxylin and eosin, 120 )
Fig 6 Tibia donor site (week 2) Fibroblastic zone is dense and cellular Extensive fibroblastic invasion of adjacent normal bone IS present (Hematoxylin and eosin 60 )
232
HEALING OF BONE GRAFT
DONOR SITES
Fig 7 Humerus donor site (week 2) Fibroblastic zone is thin and well defined Slight myxornatous fibroblastic invasion of adjacent normal bone is present (Hematoxylin and eosin, x40 )
Fig 8 Tibia donor site (week 4) There is dense periosteal fibrosis with extension of a fibrous band through the cortex into the medulla (Hematoxylin and eosin, x20 )
beyond the cortical bone into the periosteum and the cortical defect. In the humerus, fibrous tissue proliferation at the harvest site and in the cortical defect and periosteum was less prominent. Fibrosis within the harvest site consisted of narrow bands of dense fibrous connective tissue surrounding a central small cavitation (Fig. 7). Fibrosis penpheral to this region was granular to myxomatous. poorly cellular, and interspersed with fine. narrow, immature bone trabeculae with hyperplastic osteoblasts. Adjacent to the harvested region, bonc trabeculae were mature and normal bone marrow elements were present.
W2c.k 8 Transverse sections of the tibia at the harvest site contained denser, more irregular fibrosis than at week 4 (Fig. 2d). In the humerus, gross alterations were not seen in the periosteum, cortex. or harvest site (Fig. 3d). Histologically, focal dense connective tissue was surrounded by cancellous bone at the tibial harvest site. A thin band of fibrous connective tissue extended from this site through the cortical defect to the thickened, overlying periosteum. Rare small foci of dense connective tlssue, interspersed among cancellous bone, were present within the humeral harvest site. N o penosteal or cortical bone changes were present.
Hkck 4 Transverse sections of the tibia contained a nest of pale ;gray fibrous connective tissue that filled the harvest site (Fig. 2c). A fibrous band extended from the harvest site through the cortical defect to moderately thickened penosteum. In the humerus, penosteal thickening was not detected and only occasional small, multifocal areas of fibrosis, interspersed with normal cancellous bone. were present at the harvest site (Fig. 3c). Histologically, the tibial harvest site contained a dense core of mature fibrous connective tissue surrounded by cancellous bone that was widely separated by a less cellular rnyxomatous connective tissue. Dense fibrous connective tissue was responsible for the penosteal thickening and filled the connective tissue defect (Fig. 8). In the humerus, small nests of dense fibrous connective tissue were widely scattered throughout the harvest site (Fig. 9). Surrounding tissues were qualitatively normal, and no defects in the Cortex were present.
Humerus donor site (week 4). There is a focal area of medullarv fibrosis. (Hernatoxylin and eosin, x20.) Fiq. 9.
PENWICK, MOSIER, AND CLARK
Discussion A consistent pattern of healing followed procurement ofcancellous bone. A large coagulum of fibrin and crytlirocytes filled the cavity and was subsequently invaded from the periphery by vascular and fibrous tissucs. New bone formation accompanied by fibrosis followed rc\ascularization ofthe peripheral regions. progressing centrally to replace the harvested bone. The major difTerensc i n the healing process between tibia and humerus was the degree of regeneration of cancellous bone compared to the amount of replacement by connective tissue. By week 2, the response in the tibia remained primririli fibrosis. although significant amounts of new bone formation had begun at the periphery of the donor site. I n contrasl, the fibrous response in the humerus at week 2 was less productive, more niyxomatous. and interspersed with tine. immature bone trabeculae. At week 8. the tibia still contained prominent amounts of connective tissue within the harvest site, while only small amounts of fibrosis were present in the humerus. These results d o nut agrec with others in which, after harvest of cancellous bone from the canine tibia, endosteal callus formed by weck 8 and normal intramedullary architecture was restored h! week 12.” Reasons for these differences are not apparent. Results of this study suggested that cancellous hone was replaced much more rapidly and completely i n the humerus than in the tibia. The reason for this tlitlercnce is unclear. hut several factors may be postulated. Thc medullary cavity at the proximal end of the canine tibia appears to be relatively smaller than that ofthe humerus. Despite the fact that consistently lower weights of cancellous bone were obtained from the tibia. the harvested bone may have constituted a greater percentage ol‘ the cancellous bone at this site (Figs. 2,3). I t is possiblc that cancellous bone harvesting is more inJurious to thc tibia than to the humerus because of more conipletc removal of trabecular bone. hematopoietic or stromal elcments. and possibly endosteum. One author believes that osteoblasts are derived trom stromal elements of bone marrow, while osteoclasts arise from marrow hematopoietic elements. ” Thesc cell lines do not undergo transformation from one to thc other.“ The hypothesis could be advanced that the more aggressive removal of stromal elements of the marrow of the tibia inhibits regrowth of new cancellous bone. Induction of new osteoblasts from any source may be impeded by removal of bone morphogenetic protein. along with trabecular bone and bone marrow stroma. If. as has also been hypothesized, osteoclast stem cells exist in circulating blood, osteoclasia may be inhibited to a lesser degree than osteogensis. ’’ It has been stated that “marrow in repair may have a dichotomy i n differentiation: under optimal conditions
233
to form new bone and i n adversity to become phagocytic.”“’ I f true. this may panially explain why infection leads to delayed fracture union. and the hypothesis may be equally applicahlc to evcnts i n the tibial canccllous donor site. Histologicall>. there appeared to be cortical thinning during healing ofthe tibia. possibly attributable to the osteolytic role of marrow elements in a hypovascular state. In the tibia. conditions for regeneration of cancellous bone may be comparable to healing of a long bone fracture in which masive aniounts of bone marrow and endosteum are lost. If an extf;iosseous vascular supply is not present or an autogenous cancellous graft is not supplied. delayed healing may result.’- I‘ During healing of the proximal tibial donor site. hyaline cartilage associated with the bony defect may habe been indicative of a hypovascular state. resulting in IOU oxhgcn tension in the region.” In fracture healing~ostcogenic cells that ditferentiate in more superficial nonLascular areas of thc provisional call us d i ffe re n t i ;1t e i n to chon d ro blast s a nd c ho nd roc y t es. However, u.hen capillary ingrowth keeps pace with osteogenic cell growth. these pluripotential cells differentiate in a more vascular cnvironment ofgreater oxygen tension and thus form bone rather than cartilage.’x.”’It is possible that the damage t o the tibia was sufficient t o rccult in decreased vascular supply and an impcdinicnt to hcaling. In contrast. the humerus may have sustained a less severe injury and ovcrcome thc trauma to heal more rapidly and completely. It is likely that this was related to the relative difference in s i x s of the cavitics created, because the harvest sites in both hones were similarly remote from the nutrient artery. Previous reports of “stre\s fractures” in the tibia after harvesting cancellous bone reinforce the theory that injur? to the tibia may be severe.’” Although such fracturcs ma! be attributed to the stress riser effect of a cortical cfeli.ct. the size and configuration of the detects make this seem unlikely.” The delay in healing of a graft donor site. as demonstrated in the tibiae in this study, could possibly contribute to cortical fractures. Although the number of animals in this study was small. the histologic appearance and apparent progression of events were consistent in all specimens and at all time intervals studied. Thus, the proximal end of thc humerus appears to be superior to the tibia as a donor site tor cancellous bonc because it yields larger quantities of cancellous bone and undergoes more complete and rapid healing. While nearly all of thc humera1 harvest site was replaced by cancellous bonc b j weck 8, centrally located cancellous bone appeared thinner and more fragile than that at the periphery ofthe harvest site. Other investigators found that these thinner trabeculae persisted near the center of a humeral cancellous graft donor site 4 months after graft harvesting.” The current data suggest that by
HEALING OF BONE GRAFT DONOR SITES month 2 it may be possible for the humerus lo provide a second cancellous graft. Biologic contributions ofthe second graft would depend on the osteogenic capacity of the newly formed bone. Further studies are needed to determine the precise time when a functional graft could be 'obtained from a previous donor site. The pattern of healing in the tibia was far less favorable, making it unlikely [hat a second graft could be procured from it. Despite its shortcomings, the tibia remains a useful graft donor site i n clinical small animal surgery and is frequently employed by the authors. In our experience, clinical problems after harvesting cancellous bone from any site have been virtually nonexistent. The results of this study suggest, however, that when possible or convenient, ihe proximal end of the humerus should be the site of preference for cancellous bone harvesting.
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