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Animal models of osteomyelitis
Animal Models of Osteomyelitis
JON T. MADER, M.D. Galveston,
Clinical research involving osteomyelitis is difficult because of the multiple variables found in the disease process. In order to evaluate osteomyelitis critically, a more precise staging system is needed, along with reproducible animal models for each of the clinical stages. The current animal models for osteomyelitis include the rabbit models of Norden and Andriole, the rat model of Zak, and the dog models of Fitzgerald and Deysine. Although each animal model of osteomyelitis has certain advantages and limitations, they have provided us with a clearer understanding of this disease process and what forms of treatment may prove satisfactory.
Texas
Clinical research into osteomyelitis is difficult because of the multiple variables involved in the disease process and the variability in the treatment protocols. There are four major factors influencing the treatment and prognosis of chronic osteomyelitis: degree of necrosis, condition of the host, site and extent of involvement, the disabling effects of the disease itself. The classifications of osteomyelitis offered by Waldvogel et al [l] and Cierny and associates [2,3] stage the disease by the way the infection is induced and by anatomic and physiologic features, respectively (Table I). Staging systems provide a framework for describing and developing models of osteomyelitis, determining appropriate medical and surgical therapy for each stage, determining prognosis, and comparing results of therapy from institution to institution. The Cierny-Mader stages are dynamic and may be altered by successful therapy, host alteration, or treatment failure. Regardless of the clinical staging classification utilized, there are many subject, organism, bone, and antibiotic variables found in osteomyelitis that cannot be easily controlled in human studies. To control the multiple variables necessary for osteomyelitis research, attempts have been made over the years to develop reliable animal models of osteomyelitis. Since Staphylococcus aureus is the major pathogen isolated from patients with osteomyelitis, most of the in vivo experimentation has used this organism. However, before appropriate animal models of osteomyelitis can be described or developed, an appropriate classification system of the disease must be used as the framework. The current osteomyelitis models will be described using both the Waldvogel and Cierny-Mader staging systems.
From the Marine Biomedical institute and Department of Internal Medicine, Division of lnfecticus Diseases, University of Texas Medical Branch, Galveston, Texas. Requests for reprints should be addressed to Dr. Jon T. Mader, Marine Biomedical Institute, University of Texas Medical Branch, 200 University Boulevard, Galveston, Texas 77550.
HISTORY OF THE OSTEOMYELITIS MODEL In 1884, Rodet [4] produced experimental bone abscesses in rabbits by intravenous injection of S. aureus. Lexer [5,8] produced bone abscesses in rabbits by the intravenous injection of small amounts of attenuated S.
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TABLE
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Ciassificaton
OF INFECTIOUS
DISEASES-MADER
Systems
Waldvogal system Hematogenous osteomyelitis Contiguous focus osteomyelitis Osteomyelitis associated with vascular Chronic osteomyelitis
disease
Ciemy-Mader system Anatomic Stage Stage Stage Stage Physiologic A host: B host:
Stage 1: medullary osteomyelitis 2: superficial osteomyelitis 3: localized osteomyelitis 4: diffuse osteomyelitis Stage normal host systemic compromise (BS) local compromise W C host: Treatment worse than the disease
aureus. Only by using this method could he keep the animals alive long enough for the subsequent development of pathologic bone changes. Lexer noted that larger doses of S. aureus or a more virulent S. aureus strain invariably caused early death in young animals or suppurative arthritis in older animals. Even though bone abscesses could be produced in this model, he stated it was impossible to produce a progressive infection by intravenous injections of S. aureus. Other investigators [7-91 produced suppurative bone lesions that were largely confined to the site of inoculum (direct needle stick) or to the point where the bacteria localized (intravenous injection) and were also not progressive. Scheman et al [lo], in similar studies, noted the development of small localized bone abscesses after direct injection of S. aureus. If infected intravenously, most of the animals died within a few days. Postmortem examination of these animals demonstrated abscesses of the liver, kidneys, or spleen along with occasional bone abscesses. CURRENT
RABBIT
MODELS
OF OSTEOMYELITIS
Currently, there are two rabbit models used for the study of osteomyelitis. The Andriole model of progressive osteomyelitis was developed in 1974 [l 1,121. In New Zealand white rabbits, a drill was used to place a hole in the proximal end of the tibia, and S. aureus was injected into the intramedullary canal. Following S. aureus inoculation, two different techniques were used to produce a progressive bone infection. in one group, a stainless steel pin was inserted into the intramedullary canal. This technique was identical to that in the other group except that, before inoculation and pinning, a fracture was placed through the middle third of the tibia. The model produced progressive chronic osteomyelitis with osteoiytic changes, sequestra, and new bone formation. Osteomyelitis was documented in 100 percent of the first group and 66 percent of the fracture group. it was difficult ta separate the role of the
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fracture and the intramedullary nail in the infection. The fact that the intrameduliary nail alone resulted in a progressive infection suggested that the fracture was not a major pathogenic factor in the development of the progressive osteomyelitis. Local bone trauma is necessary to produce progressive osteomyelitis. The intramedullary nail disrupts intrameduliary blood flow, which leads to localized necrosis. Initially, localized necrosis protects the organism from the host defense mechanisms and appears to be necessary in the pathogenesis of experimental osteomyelitis. As in other studies, the rabbit tibia was extremely resistant to intramedullary infection, requiring as many as 1O* organisms. Without bone trauma, there was no development of progressive osteomyelitis. A lo-fold increase in the intrameduilary S. aureus inocuium proved to be rapidly fatal to the animals. The Andriole model resembles osteomyelitis that develops as a complication of internal fixation devices. The model is classified as chronic contiguous focus osteomyelitis in the Waldvogel system and a 1A osteomyelitis in the Cierny-Mader system. The most commonly used osteomyelitis rabbit model was developed by Scheman et al [lo] in 1941, and refined by Norden and Kennedy (131 in 1970. This New Zealand white rabbit model involves the intramedullary injection of 5 percent sodium morrhuate, a mild sclerosing agent prepared from the sodium salt of fatty acids of cod liver oil, into the tibiai metaphysis. Sodium morrhuate is sequentially followed by injection of S. aureus and sterile saline. The current model does not have the extensive animal mortality of earlier studies and produces a progressive osteomyelitis with periosteal reaction, lytic bone lesions, sequestra, and involucrum. Crane et al [14] and Norden et al [15], in detailed histologic studies, described abscess formation at the injection site, which was surrounded by an exudate composed primarily of poiymorphonuciear leukocytes and an advancing zone of edema. The infection spread both proximally and distally to involve the entire tibia. Progressive bony destruction with sequestra formation was present 14 days after osteomyelitis induction. The current S. aureus model successfully produces a progressive bone infection. This experimental model of osteomyelitis has been expanded in the past several years to include Pseudomonas aeruginosa [16,17], a clinically important and increasingly isolated bone pathogen [16-201. The procedure for infection is essentially the same as with the S. aureus model, with intrameduliary injection of sodium morrhuate into the tibia1 metaphysis followed by P. aeruginosa and saline. The progressive nature of osteomyelitis produced by P. aeruginosa is documented by radiographic, histologic, and culture findings. The organism is still present in approximately 90 percent of the control animals sacrificed 70
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days after infection. In comparison to the experimental S. aureus model, this infection is more indolent, less destructive, and rarely leads to extraosseous extension. Radiographically, there is less bony destruction and more new bone formation. Fibrous and bony repair is seen in histologic sections of the tibia 28 days after infection induction, of the major histologic elements in osteomyelitis, healing with fibrous and bony repair predominates in the P. aeruginosa osteomyelitis model over a suppurative destructive necrosis found in the S. aureus osteomyelitis model. The Norden model of osteomyelitis is difficult to classify in the Waldvogel staging system. Infection is induced in the tibia1 metaphysis and is similar to the localization of early hematogenous osteomyelitis. The infection is progressive, with radiographic and histologic development of macronecrosis. The presence of macronecrosis is consistent with chronic osteomyelitis. The infection can also be considered a chronic contiguous focus osteomyelitis, since the organism did not reach the bone by a hematogenous route, but by a direct contiguous inoculation. In the Cierny-Mader staging system, the model is a diffuse or 4A osteomyelitis. The osteomyelitis rabbit model has limitations. Either extensive trauma or sodium morrhuate is necessary to produce progressive infection. Sodium morrhuate produces aseptic necrosis of varying degrees. This drug acts directly on the marrow elements (marrow sclerosis) and the bone trabeculae (trabecular degeneration), rather than causing vascular occlusion [14]. Scheman et al [lo] found that bone repair was well advanced three weeks after the injection of sodium morrhuate. Norden and Kennedy [13] lessened the chemical effect by decreasing the amount of injected sodium morrhuate threefold. Histologic evaluation revealed only a small focus of fibrosis at the inoculation site seven days after the sodium morrhuate injection. The second limitation is that a high inoculum of bacterial organisms is required to produce the infection in rabbits (at least 1O6 S. aureus and lo* P. aeruginosa). Human infection is believed to be initiated by a relatively small inoculum of bacteria. Finally, the rabbit is relatively susceptible to the toxic effects of antibiotic therapy. Highdose long-term therapy with some antibiotics has been associated with significant rabbit mortality, probably related to idiopathic diarrhea or pseudomembranous colitis [21]. Despite these limitations, the rabbit model of osteomyelitis has given support to many clinical observations and has been used to assess pathophysiologic and therapeutic variables. RAT MODEL
DOG MODELS
DISEASES-MADER
OF OSTEOMYELITIS
Currently, there are two major dog models for the study of osteomyelitis. Adult mongrel dogs were used in the osteomyelitis model developed by Deysine et al [23] in 1976. The animals were anesthetized, and both tibia1 nutrient arteries were isolated utilizing a dissecting microscope. Then 0.1 ml of 20 percent barium sulfate was injected into both nutrient arteries. S. aureus (1 O5organisms in 0.1 ml) was injected only into the left nutrient artery. The soft tissue wounds were closed and the animals were returned to their cages. The infection was relentlessly progressive, resulting in death of the animals in four to 16 weeks. Radiography shortly after the operation demonstrated barium in the intramedullary arterial system of both tibias. In the infected tibias, serial radiography revealed loss of bone in the medullary canal followed by progressive periosteal new bone formation. Fractures at the epiphyseal-metaphyseal line were seen in some animals. In the noninfected tibias, no progressive radiographic changes were demonstrated. Microscopically, the bone specimens revealed typical osteomyelitic changes, characterized by necrosis and new bone formation. The noninfected tibias showed no histologic changes. Culture of specimens of the medullary canals from infected tibias yielded S. aureus that was identical to the infecting strain. Because of
Recently, a Wistar rat model of progressive osteomyelitis was described by Zak et al [22]. The model involves intramedullaty injection of 5 percent sodium morrhuate and S. aureus. Histopathologic examination 14 days after in-
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fection induction reveals severe purulent osteomyelitis with abscesses surrounded by granulation tissue in the cancellous bone and in the intramedullary canal. Sequestra and reactive bone formation were occasionally found. In rats sacrificed one, three, and four months after osteomyelitis induction, chronic bone infection was found. There were areas of microabscesses, sequestra, reactive new bone formation, and chronic inflammation with foamy macrophages. The model is produced by exposing the tibia surgically and boring a hole with a dental drill into the medullary cavity of the proximal tibia. Sequential injections of 0.05 ml of 5 percent sodium morrhuate and 0.05 ml S. aureus (2 x 10 colony-forming units in 0.05 ml) are made. The tibia1 hole is then plugged with dental gypsum. Experimental S. aureus osteomyelitis in rats is progressive. The model is reliable, inexpensive, and histopathologically similar to chronic human post-traumatic osteomyelitis. Rats reportedly are more resistant to the side effects of high-dose, long-term antibiotic therapy. Rat tibias are small and amenable to pulverization in an inexpensive bone mill. Thus, quantitative bacterial counts can be easily obtained with this model. The rat is a valuable model for osteomyelitis research. The Zak rat osteomyelitis model is consistent with chronic contiguous focus infection in the Waldvogel classification. In the Cierny-Mader classification, the model is a diffuse or 4A osteomyelitis.
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minimal sequestra formation, the Deysine model represents a subacute hematogenous osteomyelitis in the Waldvogel classification and a 1A osteomyelitis in the Cierny-Mader classification system. The Fitzgerald model of osteomyelitis [24] utilizes adult dogs. A cortical window measuring 1 cm2 is cut into the tibia1 metaphysis, and the underlying cancellous bone is removed. S. aureus (10’ organisms per 1 .Oml) is inserted into the newly created metaphyseal defect. The defect is then filled with polymethylmethacrylate. The model produces localized progressive osteomyelitis, as demonstrated radiographically and histologically, and confirmed by culture. Radiographically, at one week, an irregular lucent zone representing necrotic bone was seen around the acrylic implant. By the second week, lacy periosteal new bone was consistently observed, and early sclerotic bone around the implant was found by the sixth week. Dense sclerotic bone, which appeared to isolate the infectious process from normal bone was present by the 12th week. Histologically, a rich polymorphonuclear leukocyte and lymphocyte cellular response was seen by two weeks. Osteoclastic reabsorption of necrotic bone spicules occurred at four weeks, and involucrum formation was seen at six weeks. Dense sclerotic bone was present at week 12. Deep bacterial culture specimens taken two to five months after osteomyelitis induction grew S. aureus in 93 percent of the samples. Since there is minimal dead bone, the model represents a subacute contiguous focus osteomyelitis in the WaldVDgt?l staging system and a 3A osteomyelitis in the Ciemy-Mader staging system. The canine model of osteomyelitis has many potential benefits. Dogs are of sufficient size to allow evaluation of multiple surgical procedures, and are relatively resistant to the toxic effect of broad-spectrum antibiotics. The model produced by Deysine is technically difficult to produce, ultimately leads to death of the animal, and is much different from human osteomyelitis, in which the overall mortality from the infection is low but the morbidity is high. The Fitzgerald model is the best dog model of osteomyelitis. The osteomyelitis is relatively easy to produce and is localized. The model is amenable to antibiotic therapy and objective evaluation of different surgical procedures including saucerization, local muscle flaps, and free flaps. The major disadvantages of the model involves the initial cost of purchase and animal care expenses. It is also difficult and expensive to obtain inbred dogs.
It is sssential that an adequate classification system be in place for the categorization of the current animal models of osteomyelitis and for the development of new models. However, there has been little new development of osteomyelitis classification systems over the past century. The current classification scheme was expanded from other
systems by Waldvogel in 1970 [l]. The Waldvogel classification is an etiologic system that does not possess an adequate framework for the formulation of medical or surgical treatment of osteomyelitis or for the prediction of disease prognosis. The classification system developed by Cierny and Mader [2,3] evaluates the location, extent, and stability of the bone infection and also takes into account the status of the host. The current animal models have been described and categorized in both the Waldvogel and the Cierny-Mader classification systems. The models that are most commonly used or show the most promise are the rabbit model of Norden et al [13], rat model of Zak et al (221, and the dog model of Fitzgerald [24]. of all the animal models, the rabbit model developed by Norden has been the most extensively used in osteomyelitis research. Diffuse osteomyelitis develops in the majority of these animals. Antibiotic trials [16,17,25-331, determination of antibiotic tissue concentrations [16,17,25-331, histologic studies [14,15], hyperbaric oxygen evaluation [31], determination of tissue oxygen tensions (311, and blood flow studies [34] have been performed using the model. The model is reliable, inexpensive, and large enough to allow some surgical manipulation, including blood flow studies. However, the diffuse nature of the osteomyelitis and the small size of the animal preclude the evaluation of surgical procedures such as saucerization, local muscle flaps, or free flaps. A sclerosing agent and a large inoculum of organisms are necessary to produce the infection; Moreover, the bone infection may clear with time, especially when osteomyelitis is induced by P. aeruginosa. The rabbit tibia is too large for easy pulverization, and the bone culture samples are obtained by swabs and intramedullary washings. Finally, rabbits do not uniformly tolerate broad-spectrum antibiotic therapy, with fatal pseudomembranous colitis or idiopathic diarrhea developing in many animals [21]. The intestinal problems can be lessened with the concurrent administration of oral vancomycin [33], which is not absorbed from the gastrointestinal tract and does not appear to influence antibiotic studies. The rat model of Zak has been used for antibiotic trials, determination of antibiotic tissue concentrations, and histologic studies [22]. Rats are inexpensive, and the small size of the tibia allows for easy pulverization of the bone with quantitative counts of the infecting organism being obtained. Rats tolerate broad-spectrum antibiotic therapy with a minimum of side effects. However, the osteomyelitis produced is diffuse, and, with the small size of the animal, evaluation of surgical procedures is impossible. The dog model of Fitzgerald has been used for local antibiotic research using acrylic beads [24]. The model is also amenable to evaluation of systemic antibiotic therapy and tissue antibiotic concentrations. The infection pro-
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duced is a localized osteomyelitis, and given the large size of the animal, evaluation of surgical procedures is feasible. The major advantage of this model is its capacity for evaluation of surgical procedures, and the major disadvantage is its expense. Thus, the various animal models of osteomyelitis offer different advantages to researchers. The models simulate osteomyelitis clinically and histologically and permit control of multiple variables in the disease process. The ideal model of osteomyelitis would be one in which all stages of osteomyelitis could be produced, of sufficient size for
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evaluation of surgical procedures, tolerant to the toxic effects of antibiotics, and inexpensive. It is unlikely that there will ever be a perfect animal model for osteomyelitis. However, the current models have given support to many clinical observations and have been used successfully to assess pathophysiologic and therapeutic manipulations. ACKNOWLEDGEMENT I wish to thank Aurora Galvan and Joan Mader for manuscript research and preparation. This study was supported by a grant from Smith Kline and French.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 6. 9.
10. 11.
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15.
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17. 16.
19. 20.
Waldvogel FA, Medoff G, Swartz MN: Osteomyefiiis: a review of clinical features, therapeutic considerations and unusual aspects. N Engl J Med 1970; 262: 196-206, 260-266, 316322. Ciemy G, Mader JT: Adult chronic osteomyelitis. Drtho 1964; 7: 1557-l 564. Cierny G, Mader JT, Penninck JJ: A clinical staging system of adult osteomyelitis. Contemp Drthop 1986; 10: 17-37. Rodet A: Physfologie pathol&ique-etude experimentale sur I osteomyefffe infectieuse. CR Acad Sci 1686; 99: 569-571. Lexer E: Zur experimentellen erzeugung osteomyeliiischer herde. Arch Kfin Chir 1694; 48: lSl-200. Lexer E: Experfmente uber osteomyelitis. Arch Klin Chir 1696; 53: 266-277. Starr CL: Acute hematogenous osteomyelitis, Arch Surg 1922; 4: 567-567. Haldeman KO: Acute osteomyeliiis. A clinical and experimental study. Surg Gynecol Dbstet 1934; 59: 25-31. Thompson RHS, Dubos RJ: Production of experimental osteomyelitis in rabbii by intravenous injection of Staphylococcus aureus. J Exp Med 1936: 66: 191-206. Scheman L, Janota M, Lewin P: The production of experimental osteomyelitis. JAMA 1941; 117: 1525-l 529. Andriole VT, Nagel DA, Southwick WO: A paradigm for human chronic osteomyelitis. J Bone Joint Surg 1973; 55A: 151 l1515. Andriole VT, Nagel DA, Southwick WO: Chronic staphylococcal osteomyelitis. Norden CW, Kennedy E: Experimental osteomyelitis I. A description of the model. J Infect Dis 1970; 122: 410-416. Crane LR, Kapdi CC, Wolfe JN, Silberberg BK, Lemer AM: Xeroradiographic, bacteriologic, and pathologic studies in experimental staphylococcal osteomyelftis. Proc Sot Exp Biol Med 1977; 156: 303-314. Norden CW, Myerowitz RL, Keleti E: Experimental osteomyelitis due to Staphylococcus aureus or Pseudomonas aeruginosa: a radiographic-pathological correlative analysis. Br J Exp Pa9101 1980; 61: 451-460. Van Wingerden GI, Lolans V, Jackson GG: Experimental pseudomonas osteomyelitis. Treatment with sisomicin and carbenicillin. J Bone Joint Surg 1974; 56A: 1452-1458. Norden CW, Keleti E: Experimental osteomyelitis caused by Pseudomonas aeruginosa. J Infect Dis 1960; 141: 71-75. Lee Y-H, Kerstein MD: Osteomyelitis and septic arthritis. A complication of subclavlan venous catherization. N Engl J Med 1971; 285: 1179-1160. Leonard A, Comty, CM, Shapiro FL, Raij L: Osteomyelitis in hemodialysis patients. Ann Intern Med 1973; 78: 651-656. Holzman RS, Bishko F: Osteomyelitis in heroin addicts. Ann Intern bled 1971; 75: 693-696.
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LaMont JT, Sonnenblick EB, Rothman S: Role of clostridial toxin in the pathogenesis of clindamycin colitis in rabbis. Gastroenterology 1979; 70: 356-361. Zak 0, ak F, Rich R, Tosch W, Kradolfer F, Scheld WM: Experimental staphylococcal osteomyelitis in rats: therapy with rtfampin and cloxacillin alone or in combination. In: Periti P, Grassi GG, eds. Current Chemotherapy and Immunotherapy. Washington: American Society for Microbiology, 1962: 973974. Desyine M, Rosano E, Isenberg, HD: Acute hematogenous osteomyelitis: an experimental model. Surgery 1976; 79: 97-99. Fitzgerald RH: Experimental osteomyelitis: description of a canine model and the role of depot administration of antibiotics in the prevention and treatment of sepsis. J Bone Joint Surg 1983; 65A: 371-360. Norden CW, Kennedy E: Experimental osteomyelitis. II. Therapeutic trials and measurement of antibiotic levels in bone. J Infect Dis 1971; 124: 565-571. Norden CW, Dickens DR: Experimental osteomyelitis. Ill. Treatment with cephaloridine. J Infect Dis 1973; 127: 525-526. Norden CW: Experimental osteomyelitis. IV. Therapeutic trials with rifampin alone and In combination with gentamicin, sisomicin, and cephafothin. J Infect Dis 1975; 132: 493-499. Norden CW: Experimental osteomyelkis. V. Therapeutic trials wfth oxacilfin and slsomicin alone and in combination. J Infect Dis 1976; 137: 155-160. Norden CW, Keleti E: Treatment of experimental staphylococcal osteomyelitis with rffampin and trfmethoprim, alone and in combination. Antimicrob Agents Chemother 1980; 17: 591594. Norden CW, Shaffer M: Treatment of experimental chronic osteomyelitis due to Staphylococcus aureus wfth vancomycin and rifampin. J Infect Dis 1983; 147: 352-357. Mader JT, Guckian JC, Glass DL, Reinarz JA: Therapy with hyperbaric oxygen for experimental osteomyelitis due to Staphylococcus aureus in rabbis. J Infect Dis 1978; 138: 312-316. Mader JT, Wilson KJ: Comparative evaluation of cefamandole and cephalothin in the treatment of experimental Staphylococcus aureus osteomyelitis in rabbits. J Bone Joint Surg 1983; 66A: 507-513. Mader JT, Wilson KJ: Comparative evaluation of moxalactam and clindamycin in the treatment of experimental Staphylococcus aureus osteomyelitis (abstr 105). Presented at the 22nd Interscience Conference on Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Miami, October 1962. Mader JT, Brown GL, Guckian JC, Wells CH, Reinarz JA: A mechanism for the amelioration by hyperbaric oxygen of experimental staphylococcal osteomyelitis in rabbits. J Infect Dis 1960; 142: 915-922.
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JON T. MADER, M.D. Galveston,
Clinical research involving osteomyelitis is difficult because of the multiple variables found in the disease process. In order to evaluate osteomyelitis critically, a more precise staging system is needed, along with reproducible animal models for each of the clinical stages. The current animal models for osteomyelitis include the rabbit models of Norden and Andriole, the rat model of Zak, and the dog models of Fitzgerald and Deysine. Although each animal model of osteomyelitis has certain advantages and limitations, they have provided us with a clearer understanding of this disease process and what forms of treatment may prove satisfactory.
Texas
Clinical research into osteomyelitis is difficult because of the multiple variables involved in the disease process and the variability in the treatment protocols. There are four major factors influencing the treatment and prognosis of chronic osteomyelitis: degree of necrosis, condition of the host, site and extent of involvement, the disabling effects of the disease itself. The classifications of osteomyelitis offered by Waldvogel et al [l] and Cierny and associates [2,3] stage the disease by the way the infection is induced and by anatomic and physiologic features, respectively (Table I). Staging systems provide a framework for describing and developing models of osteomyelitis, determining appropriate medical and surgical therapy for each stage, determining prognosis, and comparing results of therapy from institution to institution. The Cierny-Mader stages are dynamic and may be altered by successful therapy, host alteration, or treatment failure. Regardless of the clinical staging classification utilized, there are many subject, organism, bone, and antibiotic variables found in osteomyelitis that cannot be easily controlled in human studies. To control the multiple variables necessary for osteomyelitis research, attempts have been made over the years to develop reliable animal models of osteomyelitis. Since Staphylococcus aureus is the major pathogen isolated from patients with osteomyelitis, most of the in vivo experimentation has used this organism. However, before appropriate animal models of osteomyelitis can be described or developed, an appropriate classification system of the disease must be used as the framework. The current osteomyelitis models will be described using both the Waldvogel and Cierny-Mader staging systems.
From the Marine Biomedical institute and Department of Internal Medicine, Division of lnfecticus Diseases, University of Texas Medical Branch, Galveston, Texas. Requests for reprints should be addressed to Dr. Jon T. Mader, Marine Biomedical Institute, University of Texas Medical Branch, 200 University Boulevard, Galveston, Texas 77550.
HISTORY OF THE OSTEOMYELITIS MODEL In 1884, Rodet [4] produced experimental bone abscesses in rabbits by intravenous injection of S. aureus. Lexer [5,8] produced bone abscesses in rabbits by the intravenous injection of small amounts of attenuated S.
June
28,198!5
The American
Journal
of Medicine
Volume
78 (auppl
86)
213
MANAGEMENT
TABLE
I
AND PREVENTION
Ciassificaton
OF INFECTIOUS
DISEASES-MADER
Systems
Waldvogal system Hematogenous osteomyelitis Contiguous focus osteomyelitis Osteomyelitis associated with vascular Chronic osteomyelitis
disease
Ciemy-Mader system Anatomic Stage Stage Stage Stage Physiologic A host: B host:
Stage 1: medullary osteomyelitis 2: superficial osteomyelitis 3: localized osteomyelitis 4: diffuse osteomyelitis Stage normal host systemic compromise (BS) local compromise W C host: Treatment worse than the disease
aureus. Only by using this method could he keep the animals alive long enough for the subsequent development of pathologic bone changes. Lexer noted that larger doses of S. aureus or a more virulent S. aureus strain invariably caused early death in young animals or suppurative arthritis in older animals. Even though bone abscesses could be produced in this model, he stated it was impossible to produce a progressive infection by intravenous injections of S. aureus. Other investigators [7-91 produced suppurative bone lesions that were largely confined to the site of inoculum (direct needle stick) or to the point where the bacteria localized (intravenous injection) and were also not progressive. Scheman et al [lo], in similar studies, noted the development of small localized bone abscesses after direct injection of S. aureus. If infected intravenously, most of the animals died within a few days. Postmortem examination of these animals demonstrated abscesses of the liver, kidneys, or spleen along with occasional bone abscesses. CURRENT
RABBIT
MODELS
OF OSTEOMYELITIS
Currently, there are two rabbit models used for the study of osteomyelitis. The Andriole model of progressive osteomyelitis was developed in 1974 [l 1,121. In New Zealand white rabbits, a drill was used to place a hole in the proximal end of the tibia, and S. aureus was injected into the intramedullary canal. Following S. aureus inoculation, two different techniques were used to produce a progressive bone infection. in one group, a stainless steel pin was inserted into the intramedullary canal. This technique was identical to that in the other group except that, before inoculation and pinning, a fracture was placed through the middle third of the tibia. The model produced progressive chronic osteomyelitis with osteoiytic changes, sequestra, and new bone formation. Osteomyelitis was documented in 100 percent of the first group and 66 percent of the fracture group. it was difficult ta separate the role of the
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fracture and the intramedullary nail in the infection. The fact that the intrameduliary nail alone resulted in a progressive infection suggested that the fracture was not a major pathogenic factor in the development of the progressive osteomyelitis. Local bone trauma is necessary to produce progressive osteomyelitis. The intramedullary nail disrupts intrameduliary blood flow, which leads to localized necrosis. Initially, localized necrosis protects the organism from the host defense mechanisms and appears to be necessary in the pathogenesis of experimental osteomyelitis. As in other studies, the rabbit tibia was extremely resistant to intramedullary infection, requiring as many as 1O* organisms. Without bone trauma, there was no development of progressive osteomyelitis. A lo-fold increase in the intrameduilary S. aureus inocuium proved to be rapidly fatal to the animals. The Andriole model resembles osteomyelitis that develops as a complication of internal fixation devices. The model is classified as chronic contiguous focus osteomyelitis in the Waldvogel system and a 1A osteomyelitis in the Cierny-Mader system. The most commonly used osteomyelitis rabbit model was developed by Scheman et al [lo] in 1941, and refined by Norden and Kennedy (131 in 1970. This New Zealand white rabbit model involves the intramedullary injection of 5 percent sodium morrhuate, a mild sclerosing agent prepared from the sodium salt of fatty acids of cod liver oil, into the tibiai metaphysis. Sodium morrhuate is sequentially followed by injection of S. aureus and sterile saline. The current model does not have the extensive animal mortality of earlier studies and produces a progressive osteomyelitis with periosteal reaction, lytic bone lesions, sequestra, and involucrum. Crane et al [14] and Norden et al [15], in detailed histologic studies, described abscess formation at the injection site, which was surrounded by an exudate composed primarily of poiymorphonuciear leukocytes and an advancing zone of edema. The infection spread both proximally and distally to involve the entire tibia. Progressive bony destruction with sequestra formation was present 14 days after osteomyelitis induction. The current S. aureus model successfully produces a progressive bone infection. This experimental model of osteomyelitis has been expanded in the past several years to include Pseudomonas aeruginosa [16,17], a clinically important and increasingly isolated bone pathogen [16-201. The procedure for infection is essentially the same as with the S. aureus model, with intrameduliary injection of sodium morrhuate into the tibia1 metaphysis followed by P. aeruginosa and saline. The progressive nature of osteomyelitis produced by P. aeruginosa is documented by radiographic, histologic, and culture findings. The organism is still present in approximately 90 percent of the control animals sacrificed 70
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days after infection. In comparison to the experimental S. aureus model, this infection is more indolent, less destructive, and rarely leads to extraosseous extension. Radiographically, there is less bony destruction and more new bone formation. Fibrous and bony repair is seen in histologic sections of the tibia 28 days after infection induction, of the major histologic elements in osteomyelitis, healing with fibrous and bony repair predominates in the P. aeruginosa osteomyelitis model over a suppurative destructive necrosis found in the S. aureus osteomyelitis model. The Norden model of osteomyelitis is difficult to classify in the Waldvogel staging system. Infection is induced in the tibia1 metaphysis and is similar to the localization of early hematogenous osteomyelitis. The infection is progressive, with radiographic and histologic development of macronecrosis. The presence of macronecrosis is consistent with chronic osteomyelitis. The infection can also be considered a chronic contiguous focus osteomyelitis, since the organism did not reach the bone by a hematogenous route, but by a direct contiguous inoculation. In the Cierny-Mader staging system, the model is a diffuse or 4A osteomyelitis. The osteomyelitis rabbit model has limitations. Either extensive trauma or sodium morrhuate is necessary to produce progressive infection. Sodium morrhuate produces aseptic necrosis of varying degrees. This drug acts directly on the marrow elements (marrow sclerosis) and the bone trabeculae (trabecular degeneration), rather than causing vascular occlusion [14]. Scheman et al [lo] found that bone repair was well advanced three weeks after the injection of sodium morrhuate. Norden and Kennedy [13] lessened the chemical effect by decreasing the amount of injected sodium morrhuate threefold. Histologic evaluation revealed only a small focus of fibrosis at the inoculation site seven days after the sodium morrhuate injection. The second limitation is that a high inoculum of bacterial organisms is required to produce the infection in rabbits (at least 1O6 S. aureus and lo* P. aeruginosa). Human infection is believed to be initiated by a relatively small inoculum of bacteria. Finally, the rabbit is relatively susceptible to the toxic effects of antibiotic therapy. Highdose long-term therapy with some antibiotics has been associated with significant rabbit mortality, probably related to idiopathic diarrhea or pseudomembranous colitis [21]. Despite these limitations, the rabbit model of osteomyelitis has given support to many clinical observations and has been used to assess pathophysiologic and therapeutic variables. RAT MODEL
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Currently, there are two major dog models for the study of osteomyelitis. Adult mongrel dogs were used in the osteomyelitis model developed by Deysine et al [23] in 1976. The animals were anesthetized, and both tibia1 nutrient arteries were isolated utilizing a dissecting microscope. Then 0.1 ml of 20 percent barium sulfate was injected into both nutrient arteries. S. aureus (1 O5organisms in 0.1 ml) was injected only into the left nutrient artery. The soft tissue wounds were closed and the animals were returned to their cages. The infection was relentlessly progressive, resulting in death of the animals in four to 16 weeks. Radiography shortly after the operation demonstrated barium in the intramedullary arterial system of both tibias. In the infected tibias, serial radiography revealed loss of bone in the medullary canal followed by progressive periosteal new bone formation. Fractures at the epiphyseal-metaphyseal line were seen in some animals. In the noninfected tibias, no progressive radiographic changes were demonstrated. Microscopically, the bone specimens revealed typical osteomyelitic changes, characterized by necrosis and new bone formation. The noninfected tibias showed no histologic changes. Culture of specimens of the medullary canals from infected tibias yielded S. aureus that was identical to the infecting strain. Because of
Recently, a Wistar rat model of progressive osteomyelitis was described by Zak et al [22]. The model involves intramedullaty injection of 5 percent sodium morrhuate and S. aureus. Histopathologic examination 14 days after in-
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fection induction reveals severe purulent osteomyelitis with abscesses surrounded by granulation tissue in the cancellous bone and in the intramedullary canal. Sequestra and reactive bone formation were occasionally found. In rats sacrificed one, three, and four months after osteomyelitis induction, chronic bone infection was found. There were areas of microabscesses, sequestra, reactive new bone formation, and chronic inflammation with foamy macrophages. The model is produced by exposing the tibia surgically and boring a hole with a dental drill into the medullary cavity of the proximal tibia. Sequential injections of 0.05 ml of 5 percent sodium morrhuate and 0.05 ml S. aureus (2 x 10 colony-forming units in 0.05 ml) are made. The tibia1 hole is then plugged with dental gypsum. Experimental S. aureus osteomyelitis in rats is progressive. The model is reliable, inexpensive, and histopathologically similar to chronic human post-traumatic osteomyelitis. Rats reportedly are more resistant to the side effects of high-dose, long-term antibiotic therapy. Rat tibias are small and amenable to pulverization in an inexpensive bone mill. Thus, quantitative bacterial counts can be easily obtained with this model. The rat is a valuable model for osteomyelitis research. The Zak rat osteomyelitis model is consistent with chronic contiguous focus infection in the Waldvogel classification. In the Cierny-Mader classification, the model is a diffuse or 4A osteomyelitis.
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minimal sequestra formation, the Deysine model represents a subacute hematogenous osteomyelitis in the Waldvogel classification and a 1A osteomyelitis in the Cierny-Mader classification system. The Fitzgerald model of osteomyelitis [24] utilizes adult dogs. A cortical window measuring 1 cm2 is cut into the tibia1 metaphysis, and the underlying cancellous bone is removed. S. aureus (10’ organisms per 1 .Oml) is inserted into the newly created metaphyseal defect. The defect is then filled with polymethylmethacrylate. The model produces localized progressive osteomyelitis, as demonstrated radiographically and histologically, and confirmed by culture. Radiographically, at one week, an irregular lucent zone representing necrotic bone was seen around the acrylic implant. By the second week, lacy periosteal new bone was consistently observed, and early sclerotic bone around the implant was found by the sixth week. Dense sclerotic bone, which appeared to isolate the infectious process from normal bone was present by the 12th week. Histologically, a rich polymorphonuclear leukocyte and lymphocyte cellular response was seen by two weeks. Osteoclastic reabsorption of necrotic bone spicules occurred at four weeks, and involucrum formation was seen at six weeks. Dense sclerotic bone was present at week 12. Deep bacterial culture specimens taken two to five months after osteomyelitis induction grew S. aureus in 93 percent of the samples. Since there is minimal dead bone, the model represents a subacute contiguous focus osteomyelitis in the WaldVDgt?l staging system and a 3A osteomyelitis in the Ciemy-Mader staging system. The canine model of osteomyelitis has many potential benefits. Dogs are of sufficient size to allow evaluation of multiple surgical procedures, and are relatively resistant to the toxic effect of broad-spectrum antibiotics. The model produced by Deysine is technically difficult to produce, ultimately leads to death of the animal, and is much different from human osteomyelitis, in which the overall mortality from the infection is low but the morbidity is high. The Fitzgerald model is the best dog model of osteomyelitis. The osteomyelitis is relatively easy to produce and is localized. The model is amenable to antibiotic therapy and objective evaluation of different surgical procedures including saucerization, local muscle flaps, and free flaps. The major disadvantages of the model involves the initial cost of purchase and animal care expenses. It is also difficult and expensive to obtain inbred dogs.
It is sssential that an adequate classification system be in place for the categorization of the current animal models of osteomyelitis and for the development of new models. However, there has been little new development of osteomyelitis classification systems over the past century. The current classification scheme was expanded from other
systems by Waldvogel in 1970 [l]. The Waldvogel classification is an etiologic system that does not possess an adequate framework for the formulation of medical or surgical treatment of osteomyelitis or for the prediction of disease prognosis. The classification system developed by Cierny and Mader [2,3] evaluates the location, extent, and stability of the bone infection and also takes into account the status of the host. The current animal models have been described and categorized in both the Waldvogel and the Cierny-Mader classification systems. The models that are most commonly used or show the most promise are the rabbit model of Norden et al [13], rat model of Zak et al (221, and the dog model of Fitzgerald [24]. of all the animal models, the rabbit model developed by Norden has been the most extensively used in osteomyelitis research. Diffuse osteomyelitis develops in the majority of these animals. Antibiotic trials [16,17,25-331, determination of antibiotic tissue concentrations [16,17,25-331, histologic studies [14,15], hyperbaric oxygen evaluation [31], determination of tissue oxygen tensions (311, and blood flow studies [34] have been performed using the model. The model is reliable, inexpensive, and large enough to allow some surgical manipulation, including blood flow studies. However, the diffuse nature of the osteomyelitis and the small size of the animal preclude the evaluation of surgical procedures such as saucerization, local muscle flaps, or free flaps. A sclerosing agent and a large inoculum of organisms are necessary to produce the infection; Moreover, the bone infection may clear with time, especially when osteomyelitis is induced by P. aeruginosa. The rabbit tibia is too large for easy pulverization, and the bone culture samples are obtained by swabs and intramedullary washings. Finally, rabbits do not uniformly tolerate broad-spectrum antibiotic therapy, with fatal pseudomembranous colitis or idiopathic diarrhea developing in many animals [21]. The intestinal problems can be lessened with the concurrent administration of oral vancomycin [33], which is not absorbed from the gastrointestinal tract and does not appear to influence antibiotic studies. The rat model of Zak has been used for antibiotic trials, determination of antibiotic tissue concentrations, and histologic studies [22]. Rats are inexpensive, and the small size of the tibia allows for easy pulverization of the bone with quantitative counts of the infecting organism being obtained. Rats tolerate broad-spectrum antibiotic therapy with a minimum of side effects. However, the osteomyelitis produced is diffuse, and, with the small size of the animal, evaluation of surgical procedures is impossible. The dog model of Fitzgerald has been used for local antibiotic research using acrylic beads [24]. The model is also amenable to evaluation of systemic antibiotic therapy and tissue antibiotic concentrations. The infection pro-
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duced is a localized osteomyelitis, and given the large size of the animal, evaluation of surgical procedures is feasible. The major advantage of this model is its capacity for evaluation of surgical procedures, and the major disadvantage is its expense. Thus, the various animal models of osteomyelitis offer different advantages to researchers. The models simulate osteomyelitis clinically and histologically and permit control of multiple variables in the disease process. The ideal model of osteomyelitis would be one in which all stages of osteomyelitis could be produced, of sufficient size for
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evaluation of surgical procedures, tolerant to the toxic effects of antibiotics, and inexpensive. It is unlikely that there will ever be a perfect animal model for osteomyelitis. However, the current models have given support to many clinical observations and have been used successfully to assess pathophysiologic and therapeutic manipulations. ACKNOWLEDGEMENT I wish to thank Aurora Galvan and Joan Mader for manuscript research and preparation. This study was supported by a grant from Smith Kline and French.
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