Veterinary Surgery, 21, 4, 293-298, 1992

Evaluation of Canine Cortical Bone Graft Remodeling ANN L. JOHNSON, DVM, MS, Diplomate ACVS, JO ANN C. EURELL,

DVM. PhD,

and DAVID J. SCHAEFFER, PhD

A stable cortical bone fracture model was develoDed to evaluate the remodelina rate of cortical bone grafts. Samples of cortical bone were harvestedwith a trephine and press Et into predrill4 holes in the femoral diaphyses of four live dogs. The percentagesof new bone, unremodeled graft bone, porosity, forming bone surface area, and resorbing bone surface area were determined morphometrically and compared in cortical autografts, cortical allografts sterilized with 84% ethylene oxide (EO), and allografts sterilizedwith 12% EO. The host-graft interfaces healed without formation of fibrous tissue or cartilage, indicating a stable fracture surface. The amount of new bone formed in cortical autografts and allografts sterilized with 84% EO was significantly greater than the amount of new bone in allografts sterilized with 12% EO. There was no significant difference betweenthe amounts of new bone formed in the allografts sterilized with 84% EO and the cortical autografts. No significant differences were detected in percentages of porosity or bone surface areas.

are used clinically in the reC pair of severely comminuted diaphyseal fractures when the mechanical support of cortical bone is necessary ORTICAL BONE GRAFTS

for rigid stabilization of the fracture.'-' Methods of bone banking have been developed to ensure sterility and safe storage of a selection of bone Sterilization of cortical bone with ethylene oxide (EO) eliminates the need to follow aseptic harvesting procedure^.^,^ There are several protocols for ethylene oxide sterilization of cortical bone with differing percentages of ethylene oxide, environmental temperatures during treatment, lengths of treatment, and storage environment.' Varying these parameters alters the mechanical properties of an allograft.' The alteration in physical structure that results in mechanical changes after sterilization with EO could also cause changes in biologic properties and affect the remodeling of the allograft. Fracture healing with a cortical graft involves osteoconduction of new host bone along the trellis afforded by the graft.6*9-"Remodeling of the graft occurs by vascularization, resorption of the donor bone, and replacement by host b ~ n e . ~I ,The ~ - ' initial resorption weakens the graft, and timely replacement of the graft with host bone is necessary to maintain the integrity of the healing bone. Cortical bone grafts do not appear to remodel completely, with areas of inert donor bone surrounded by newly

formed host Measurement of the rate and completeness of host replacement of experimental cortical bone grafts yields important information about fracture stabilization in clinical patients. The model used to evaluate remodeling must approximate the clinical condition of immobilization of a cortical allograft in a cortical bone environment. The objectives of this study were to develop a stable cortical model and morphometric techniques for evaluating remodeling of cortical allografts and to gain preliminary data on the effects of two methods of ethylene oxide sterilization on allograft remodeling. Materials and Methods Two femora were harvested from a dog euthanatized after a surgical teaching laboratory. The soft tissues, epiphyses, and bone marrow were removed. One femur was sterilized with 84% ethylene oxide* at 22°C and atmospheric pressure, aerated for 72 hours at 22°C and atmospheric pressure, and stored at 0°C for 2 months.' The other femur was sterilized in 12%ethylene oxide? at 45°C and 7.59 X lo5 kilopascals for 2 hours, aerated at 54°C for 8 hours, and stored at 0°C for 2 months.' * Anproline, HW Anderson Product, Ltd., Osler Bay, New Jersey.

t Ethylene Oxide Freon Mixture, ACA Gas, Inc., Secaucus,New Jersey.

From the Departments of Veterinary Clinical Medicine (Johnson) and Veterinary Biosciences (Eurell, Schaeffer), University of Illinois, Urbana, Illinois. Supported by NIH, Biomedical Research Support Grant. Reprint requests: Ann L. Johnson, DVM, MS, University of Illinois 1008 Hazelwood Drive, Urbana, IL 61801.

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Four conditioned adult dogs weighing 20 to 30 kg were examined radiographically to document physeal closure and normal anatomy of the femora. The dogs were anesthetized, and both hind limbs were prepared for aseptic surgery. Ampicillin (25 mg/kg) was administered intravenously at induction of anesthesia and repeated orally at the appropriate dosage for 24 hours after surgery. A surgical approach was made to the craniolateral surface of each femur.” Six transverse holes, 1 cm apart, were drilled through the lateral cortex of each bone with a 13/ 64 inch (6 mm) drill bit inserted into a custom-made drill guide. A 6 mm scaphoid trephine$ fitted to a power drill was used to harvest allogeneic cortical bone samples from the treated femurs and autogenous cortical bone samples from the cranial aspect of the diaphysis of the femurs to be grafted. The cortical bone samples were assigned randomly and press fit into the drilled holes. Two dogs (four femora) received 12 allogeneic cortical bone samples sterilized with 84% EO and six autogenous samples, and six holes were left unfilled to serve as negative controls. Two dogs (four femora) received 12 allogeneic samples sterilized with 12% EO and six autogenous samples, and six holes were left unfilled. The soft tissues and skin were sutured. Radiographs of the femora were made immediately and at monthly intervals to document the location and appearance of the graft sites. All dogs were housed and treated in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and the Animal Welfare Acts. Analgesics were administered as needed. Fluorochrome labels were administered sequentially to allow retrospective histologic evaluation of the time sequence of bone formation. Chlortetracycline (10 mg/kg orally) was administered every 8 hours for 3 days at week 2; calcein greens (20 mg/kg subcutaneously [sc]) was administered at week 5 ; xylenol oranges (90 mg/kg sc) was administered at week 8; and alizarin complexones (30 mg/kg sc) was administered at week 1 1. The dogs were euthanatized with sodium pentobarbital administered intravenously at week 12. The femora were removed and cleaned of soft tissues, the epiphyses were removed, and the diaphyses were fixed in 10% neutral buffered formalin. The femora were transected proximal and distal to each bone graft. Each segment was dehydrated in a graded series of ethanol solutions and embedded in methylmetha~rylate.’~ The bone segments were sectioned serially with a diamond saw at 300 pm intervals until the center of each graft was located. The center was determined by measuring the width of the graft. The center section of each implant was ultramilled, examined under

$ Stryker Corporation, Kalamazoo, Michigan. 5 Sigma Chemical Co., St. Louis, Missouri.

incident fluorescent light, and photographed to record the spatial patterns of the fluorochrome labels. Surfaces of the same undecalcified sections were stained with toluidine blue and McNeal’s tetrachrome (TBMT). In test slides, the combined stains colored newly formed bone dark blue and mature bone light blue. To verify the color differentiation, a segment of bone sterilized with each of the ethylene oxide concentrations was stained with TBMT and observed to be light blue. In contrast, all bone formed during the 3-month study within the unfilled holes (negative controls) was stained dark blue by TBMT. Furthermore, all bone within the allografts and autografts that was stained dark blue was associated with a fluorochrome label indicating that it had been formed during the previous 3 months. With these supportive data, we were confident that bone that stained dark blue within the samples had been formed within the previous 3 months, and the amount of bone formed during this time could be determined histomorphometrically from the TBMT-stained sections. The stained sections were examined for morphology of healing of the host-graft interfaces. Using a microscope with a camera lucida, a digitizing tablet linked to a personal computer, and digitizing software, 11 histomorphometric measurements of the total tissue volume, volume of unremodeled graft bone (light blue), volume of new bone (dark blue), volume of resorbing cavities, forming surface area (surface of bone covered with osteoid), resorbing surface area (surface of bone covered with Howship’s lacunae), and total bone surface area were measured from three predetermined areas of the allografts and autografts (Fig. 1). The measured areas included three fields at the edges adjacent to the host-graft interface (periphery of the graft) and the center of the graft. The percentage of unremodeled graft bone (volume of unremodeled graft bone/total bone volume), percentage of new bone (volume of new bone/total bone volume), percentage of porosity (volume of resorbing cavities/total bone volume), percentage of forming surface (area of bone surface covered with osteoid/total bone surface area), and percentage of resorbing surface (area of bone surface covered with Howship’s lacunae/total bone surface area) were calculated from the measured data. The data were compared with a multivariate repeated measures analysis to determine if there were significant differences between the two groups of allografts and the group of autografts. In this analysis, the location of the measurement in the implant (edges and center) was the trials (repeated) factor, and treatment was the grouping factor. This permitted the use of post hoc linear contrast tests to compare treatments and location. Significance was set at p I .05. Because all I 3 3 l 4

~

~~~~

1) Sigma Scan. Jandel Scientific. Cone Madera, California.

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JOHNSON, EURELL, AND SCHAEFFER

Graft

Fig. 1. The nine fields (lined areas)of the graft frm which histomorphomettic data were obtained.

treatments were not replicated in all dogs, treatment could not be used as a trials (repeated measurement) factor. Results It was seen radiographically that two grafts migrated into the marrow canal by month 1. The rest of the samples remained stable during the study. Three samples were damaged during processing. Ten cortical autografts, 11 allografts sterilized in 12% EO, ten allografts sterilized in 84% EO, and 14 unfilled holes were available for morphometric analysis. The unfilled holes were incompletely filled with host bone by month 3 and were not evaluated morphometrically because they contained only newly formed bone. Fluorochrome-labeled sections of the autografts from all four dogs and the 84% EO treated allografts from two dogs appeared to have more calcein green label in the center of the grafts than the 12% EO treated allografts from two dogs, indicating that bone remodeling had proceeded more quickly in the first two types of grafts and was active during the second month. Xylenol orange and alizarin red were the predominant labels in the centers of the 12%EO-treated allografts, indicating that active bone remodeling was occumng during the third month. There was an increase in newly formed bone in the host and the graft along all host-graft interfaces. The fluorochrome-labeled regions of all specimens corresponded to the dark blue stained areas visible after surface staining of the section (Fig. 2). There was no evidence of fibrous tissue or cartilage at the host-graft interfaces (Fig. 2).

There was significantly more new bone in ,autografts and allografts treated with 84% EO than in allografts treated with 12% EO. There was no difference in amount of new bone between autografts and allografts treated with 84% EO. Conversely, there was significantly less unremodeled graft bone in autografts and allografts treated with 84% EO than in allografts treated with 12%EO. There was no significant difference in the amount of unremodeled graft bone between autografts and allografts treated with 84% EO. There were no differencesin the percentage of forming or resorbing surfaces within the autografts and allografts (Table 1). There was significantly more unremodeled bone at the centers and more newly formed bone at the periphery of the grafts of all three groups. There were no significant differences in porosity, percentage of forming surfaces, or percentage of resorbing surfaces between the centers and the periphery of the grafts (Table 1). Although there were no significant differences detected morphometrically in porosity of any of the samples, there was an impression that endosteal surfaces of allografts treated with 84% EO had been resorbed but not replaced with host bone (Fig. 3).

Discussion The cortical hole model developed in this study provided a stable environment for press-fit cortical bone grafts. The surface friction created when the samples were press fit into precision drilled holes was sufficient to immobilize 34 of 36 samples. The lack of cartilage or fibrous tissue formation at the healing sites confirmed that the

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BONE GRAFT REMODELING

Fig. 2. (A) Forming bone labeled with fluorochromes in the host bone [HI,interface[I], and allograft [GIin a specimen treated with 12% ethylene oxide. (B)The forming bone in the same section is dark blue (arrow) after staining with toluidine blue and McNeal’s tetrachrome (x160).

host-graft interfaces were immobilized sufficiently for pnmary bone healing to occur.15 The main objective of this project was to develop a technique for evaluating remodeling rates of cortical bone grafts. With the technique validated, it can be used to evaluate more than one treatment within the same dog, modulating the effect of biologic differences between animals. Although this preliminary project design did not allow placement of samples from each treatment group within one dog, such a design would be preferable. The advantages of the cortical hole model over a segmental cortical allograft stabilized with plate and screws include these: 1. Fewer animals are needed. 2. Expensive metal implants are unnecessary. 3. Remodeling is completed sooner in small bone samples. 4. The effect of biologic differences between dogs is minimized. segment This mode’ has an advantage Over the model,8,10too, in the stability achieved at the host-graft interface.

In the process of developing the technique, we obtained preliminary data comparing cortical allografts treated with two EO protocols to cortical autografts. The type of EO treatment appeared to have an effect on the rate of allograft

Fig. 3. An allograft treated with 84% EO, stained with toluidine blue and McNeal’s tetrachrome. There is extensive resorptionof the allograft and minimal new bone formation on the endosteal surface (X30).

297

JOHNSON, EURELL, AND SCHAEFFER TABLE 1. Data from the Perbherv and Center Fields Evaluated in Each Imdant or Graft Parameters Percent Unrernodeled Graft Bone Periphery Center Periphery Percent New Bone Periphery Center Periphery Percent Porosity Periphery Center Periphery Percent Forming Surface Periphery Center Periphery Percent Resorbing Surface Periphery Center Periphery

Autograft

Allograft 84% EO

Allograft 12% EO

17.09 f 11.47 44.89 k 22.59 21.09 f 16.22

9.48f 6.42 26.92f 17.98 10.23 f 13.44

45.64k 21.05 58.37 f 22.12 37.59 f 15.62

63.88f 10.18 36.69 f 17.52 63.99f 13.43

76.06f 7.98 53.12? 23.60 71.34f 18.60

43.87f 19.39 29.60f 17.09 49.92f 13.60

19.04 f 9.87 18.42 f 10.69 14.92 f 8.25

14.47 f 6.61 19.96 f 11.23 18.43 f 9.36

10.51 f 7.78 12.04f 9.54 12.49f 5.94

65.43f 9.74 66.89f 16.26 76.76 f 12.73

70.22k 14.37 82.64f 10.99 73.40+- 11.75

55.07 f 14.60 55.97 f 14.60 61.35f 12.62

9.74f 10.24 3.68f 4.71 6.18f 4.94

7.09 f 8.36 3.06 f 4.79 1.23 +- 1.57

6.41 f 9.38 4.05f 7.79 4.86 f 7.79

Mean and standard deviation. EO = Ethylene oxide.

remodeling. Cortical bone treated with 12% EO in an increased temperature and pressure environment was slower to remodel than the cortical autograft, but there were no differences in the remodeling rates of cortical bone treated with 84%EO at room temperature andatmospheric pressure and cortical autografts. Cortical bone treated with 12% EO in an increased temperature and pressure environment has been shown to have altered physical properties, becoming more brittle after treatment than untreated cortical bone and cortical bone treated with 84% EO at room temperature and atmospheric pressure.’ It is undetermined whether the changes in physical properties are caused by the EO or the altered temperature and pressure. Ethylene oxide and increased temperature can alter protein structure.l6,I7Alteration of bone morphogenetic protein could decrease the stimulus for remodeling of the allograft. Conflicting results have been obtained when bone morphogenetic protein was subjected to EO stenli ~ a t i o n . l ~ -Bone ~ ’ induction properties have been destroyed in a dose-dependent manner.” However, other workers routinely use EO to sterilize bone matrix before implantation. ”-” Further investigation is needed to identify factors associated with treatment of the implant that alter cortical allograft remodeling rates. Clinical use of implanted cortical allografts would be enhanced if complete remodeling of the allograft could be accelerated as in remodeling of cortical autografts. Although there was no statistical difference between the porosity of allografts treated with 84% EO and the other groups, our

impression of delayed endosteal bone formation in several of the samples should be investigated more thoroughly before this method of sterilization can be considered optimal treatment for cortical bone used clinically in fracture repair. In conclusion, the cortical hole model was successful in mimicking stable clinical conditions and allowed evaluation of two treatments within the same animal. Sterilization with EO appeared to alter the remodeling rate of cortical allografts. Further investigation is needed to identify optimal methods of treating cortical bone for use in fracture repair.

References I . Alexander JW. Use of a combination of cortical bone allografts and cancellous bone autografts to replace massive bone loss in fresh fractures and selected nonunions. J Am Anim Hosp Assoc 1983; 191671-678. 2. Henry WB, Wadsworth PL. Diaphyseal allografts in the repair of long bone fractures. J Am Anim Hosp Assoc 1981; 17525-534. 3. Johnson AL, Roe SC, Harari J. Ethylene oxide sterilization of cortical bone for bone banking: technique and results in three dogs and one cat. Vet Surg 1986; I5:49-53. 4. Sinabaldi K. Evaluation of full cortical allografts in 25 dogs. J Am Vet Med Assoc 1989; 1941570-1577. 5. LaRue SM, Withrow 9, Powers BE, et al. Limb sparing treatment for osteosarcoma in dogs. J Am Vet Med Assoc 1989; 195:1734-1744. 6. Johnson AL, Stein LE. Morphologic comparison ofhealing patterns in ethylene oxidesterilized cortical allografts and untreated cortical autografts in the dog. Am J Vet Res 1988;49:101-105.

BONE GRAFT REMODELING 7. Burchardt H, Jones H, Glowczewski Fetal. Freeze-dried allogeneic segmental cortical-bone grafts in dogs. J Bone Joint Surg Am 1978;60:1082-1090. 8. Johnson AL, Moutray M, Hoffmann WE. Effect of ethylene oxide sterilization and storage conditions on canine cortical bone harvested for banking. Vet Surg 1987; 16:418-422. 9. Goldberg VM, Stevenson S. Natural history of autografts and allografts. Clin Orthop Re1 Res 1987;225:7-16. 10. Basset CAL. Clinical implications of cell function in bone grafting. Clin Orthop Re1 Res 1972;87:49-59. I I . Enneking WF, Burchardt H, Puhl JJ, Piotrowski G. Physical and biological aspects of repair in dog cortical bone transplants. J Bone Joint Surg Am 1975;57:237-252. 12. Piermattei DL, Greeley RG. An Atlas ofSurgical Approaches to the Bones gf f h e Dog and Cut. 2nd ed. Philadelphia: WB Saunders, 1979:162-163. 13. Sterchi DL, Eurell JA. A new method for preparation of undecalcified bone sections. Stain Tech 1989;64201-205. 14. Schenk RK, Olah AJ, Herrrnann W. Preparation of calcified tissues for light microscopy. In: Dickson GR (ed). Methods o/Culcified Time Prepuruiion. New York: Elsevier, 1984: 1-56.

15. Rahn BA. Bone healing: histologic and physiologic concepts. In:

Summer Smith G (ed). Bone in Clinical Orthopedics.Philadelphia: WB Saunders, 1982:335-386. 16. Smith RM, Young JA. Simplified gas sterilization: a new answer for an old problem. Br J Anesth 1968;40:909-9 15. 17. Moore TM, Artal R, Arenas M, Gendler E. Influence of postmortem time and temperature on osteoinductive activity of demineralized microperforated ethylene oxide sterilized syngeneic bone implant in the rat. Clin Orthop Re1 Res 1990;259: 239-244. 18. Harakas NK. Demineralized bone-matrix-induced osteogenesis. Clin Orthop Re1 Res 1984: 188:239-25 I . 19. Unst MR. Surfacedecalcifiedallogeneic bone (SDAB) implants. Clin Orthop Re1 Res 1968;56:37-50. 20. Nilsson DS, Urist MR, Dawsod EG, et al. Bone repair induced by bone morphogenetic protein in ulnar defects in dogs. J Bone Joint Surg Br 1986;68:635-642. 2 1. Aspenberg P, Johnsson E, Thongren KG. Dose dependent reduction ofbone inductive properties by ethylene oxide. J Bone Joint Surg Br 1990:72: 1036-1 037.

Evaluation of canine cortical bone graft remodeling.

A stable cortical bone fracture model was developed to evaluate the remodeling rate of cortical bone grafts. Samples of cortical bone were harvested w...
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