Antibiotics in Acrylic Bone Cement. In Vitro Studies ARDYCE B. WELCH, Section of Microbiology, School of L i f e Sciences, University of Nebraska, Lincoln, Nebraska 68588
Summary The effect of combining gentamicin and cephalothin with Surgical Simplex bone cement was tested in uitro. Experiments were designed to determine (1) quantitative elution rates and period of time the antibiotics were eluted and (2) effective bactericidal qualities. Both antibiotics were eluted from polymerized acrylic cement in large quantities within 24-48 hr after coming in contact with fluid and continued to be released in smaller amounts for 21 days to more than 175 days. This varied with the antibiotic, concentration of antibiotic in the cement, surface area of cement, and volume of elution fluid. Quantitative determinations of numbers of bacteria in broth cultures demonstrated that sufficient quantities of antibiotics were released to be bactericidal to microorganisms within 45 min to 20 hr. This was dependent on the type of antibiotic, type of bacteria, concentration of antibiotic in the cement, and time of contact.
INTRODUCTION Arthroplastic joint replacement is an accepted procedure for surgical correction of various orthopedic diseases and congenital malformations. In the majority of total hip and knee replacements poly(methy1methacrylate) (PMMA) is used as a filler to provide mechanical fixation of the prosthetic components to bone. Infection is one of the most serious complications following arthroplastic joint replacement, with reported rates varying from 11to less than 1 % . l y 2 Various techniques have been advocated to reduce infection rates, including use of clean-air surgical rooms, impermeable drapes and gowns, prophylactic systemic antibiotics pre- and postoperative, and intraoperative irrigation of the wound with antibiotics. However, infections may occur, in spite of the most rigid precautions, from exogenous sources or from undetected foci of infection. The observation that a PMMA bone cement, Palacos R (manu-
Journal of Biomedical Materials Research, Vol. 12,679-700 (1978) 0 1978John Wiley & Sons, Inc. 0021-9304/78/0012-0679$01.00
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factured by Kulzer, Bad Homburg), released residual monomer over an extended period of time gave Buchholz and Engelbrecht3 the idea of mixing antibiotics with the cement. The reasoning was that antibiotics should be released from the cement for a prolonged period after surgery, which should reduce the possibility of infection a t the implant-bone boundary. They reported a decrease in deep infection in over 1000 operations by using antibiotic additives. Antibiotics introduced a t the site where infections may develop have the advantage of being more effective than parenteral or oral therapy. It may be possible that a lower dose of antibiotics would be effective, thereby eliminating some of the undesirable side effects of large doses administered over long periods of time. This technique could also eliminate the possibility of altering normal flora of the body by prolonged systemic treatment. Until recently the only PMMA bone cement licensed for use in the United States was Surgical Simplex, which has a slightly different composition from other PMMA bone cements, The majority of reports in the literature regarding release of antibiotics from acrylic cements in uitro are based on use of Palacos R, Palacos E, or CMW.3-9 Wahlig et al.,5using Palacos R, Palacos E, and two other PMMA cements, demonstrated that release of antibiotics varies with the composition of the PMMA. Similar results were reported by Elson et al.9 Wahlig et al.5 stated that usability of an antibiotic-bone cement mixture must be demonstrated by detailed experimental tests, not only with respect to suitability of the antibiotic, but also with respect to the properties of the cement. Results obtained with one antibiotic and one bone cement cannot be transferred to other antibiotics or bone cements. There is a need for a better understanding of the effects of adding antibiotics to Surgical Simplex, since there has been a limited amount of research in this The present study was undertaken to determine long-term release rates of gentamicin and cephalothin quantitatively from Surgical Simplex cement under carefully controlled laboratory conditions. These antibiotics were selected since they have many properties that would be desirable in antibiotic-bone cement combination and they are frequently used in orthopedic surgery. Also, bone cement combined with antibiotics was tested quantitatively for effective inhibition of microorganisms commonly reported to be the cause of infections following orthopedic surgery.
ANTIBIOTICS IN ACRYLIC BONE CEMENT
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MATERIALS AND METHODS Antibiotics Gentamicin sulfate powder, potency 580 ,ug/mg, was supplied by Schering Corp., Bloomfield, N.J. Concentrations reported in this article are distinguished as being based on weight of the gentamicin sulfate powder and on potency of the gentamicin. Sterile sodium cephalothin was supplied by Eli Lilly and Co., Indianapolis, Ind.
Preparation of Cylinders and Disks
A teflon mold, 55 X 65 mm, was constructed containing 20 cylindrical holes 6 mm in diameter and 12 mm deep. Commercially available PMMA bone cement, Surgical Simplex P Radiopaque (manufactured by North Hill Plastics, Ltd., 49 Grayling, London N16, England), supplied by Howmedica, Inc., Medical Division, Rutherford, 'N.J., was prepared in accordance with the manufacturer's directions at a ratio of 40 g powder and 20 ml liquid monomer. Varying amounts of gentamicin sulfate and cephalothin, either separately or in combination, were thoroughly mixed with the PMMA powder in a ceramic dish. Liquid monomer was added to the dry mixture and stirred gently with a teflon-coated spatula for approximately 11/2 to 2 min until just prior to the dough stage. The viscous mixture was placed in the holes of the teflon mold, being careful to have each hole well packed so that no air bubbles were formed inside the cylinders. The sides of the mold were scraped with the spatula to remove any excess PMMA in order to form even ends of cylinders. The mold was allowed to stand at room temperature until the mixture had polymerized, and then cylinders were punched out of the mold. Disks were prepared by rolling the material in the dough stage to a sheet 2 mm thick and cutting circular disks 15 mm in diameter. All procedures were carried out using sterile equipment and aseptic technique. Calculation of Theoretical Amount of Antibiotic in Cylinders A large number of samples of PMMA powder and liquid monomer were weighed, and it was determined that 1ml of liquid monomer weighed 0.93 g. The liquid was mixed with PMMA powder at the prescribed ratio of 1 ml to 2 g powder. Weights were determined
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immediately after polymerization and at intervals up to 18 hr at 235°C and relative humidity of 44%. By determining the loss of weight in the polymerized bone cement due to volatilization of the monomer, the theoretical amount of antibiotic could be calculated. Accordingly, each cylinder was weighed prior to using the calculated amount of antibiotic in each cylinder determined.
Antibiotic Standards and Quantitative Procedures Stock solutions of antibiotics at concentrations of 1000 pg/ml were prepared in sterile distilled water, dispensed into small portions, and frozen at -20°C. Working standards, ranging from 1.0 to 20 pg/ml were prepared by diluting the stock solutions in 0.1M phosphate buffer plus 0.85% NaCl (PBS). Gentamicin sulfate was diluted in PBS pH 7.4, since this pH approximates that found in normal mammalian blood. Cephalothin was diluted in PSB pH 7.2, because it is the sodium salt and is not ionized between pH 6.8 and 7.2. Preliminary tests using phosphate buffer, PBS, phosphate buffer plus 5%normal human serum, and normal human serum as diluents produced almost identical results. Working solutions of gentamicin sulfate were stored at 4°C and used within 4 days, whereas working solutions of cephalothin were prepared fresh daily. Antibiotics were measured quantitatively by an agar diffusion method similar to that described by Sabath and Matsen.13 Antibiotic medium agar # 5 (Difco Laboratories, Detroit, Mich.) was dispensed in 25 ml amounts into 150 X 15 mm plastic petri dishes in a laminar flow hood with a level bench and allowed to set until the surface of the agar was dry (approximately '/z hr). An overnight broth culture of Staphylococcus epidermidis ATCC 12228 was diluted to approximately 1X lo6 organisms/ml and 0.1 ml spread evenly on the surface of the agar. Filter-paper disks, 6.35 mm diameter (#740-E, Schleicher & Schuell, Inc., Keene, N.J.), were placed in sterile plastic pertri dishes. For each assay, 0.02-ml portions of standard antibiotic or unknown test solutions were pipetted with a MLA pipette (Medical Laboratories Automation, Inc., Mt. Vernon, N.Y.) fitted with sterile disposable tips onto each of two filter-paper disks. Four standard antibiotic concentrations, ranging from 1or 2.5 to 20 pg/ml, were used on each plate. The disks were then placed in pairs on the agar surface, with members of the same pair placed directly opposite each other. After overnight incubation at 35"C, zone diameters were measured
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with a vernier calipers and recorded to the nearest 0.1 mm. Zone diameters of each antibiotic standard were averaged, and a dose response curve was plotted on semilog graph paper using point-to-point connection relating the concentration of antibiotic in the standard (log scale) to the diameter (in mm) of the zone of inhibition produced. A separate graph was prepared for each of the plates. Diameters of the zones of the test samples were measured, determined individually from the graph, and then averaged. Quantitative Measurement of Elution of Antibiotics
Cylinders prepared with 0.5, 1.0, and 2.0 g gentamicin sulfate or cephalothin per 10 g PMMA powder were placed individually in sterile screw-cap tubes. Five cylinders of each concentration were used. Five ml PBS was added to each tube as eluting fluid, and the tubes were incubated at 37OC. At intervals cylinders were removed, dried on filter paper, and placed in fresh solutions. The elution fluid was quantitatively tested for amount of antibiotic by the assay procedure. Standard working solutions of cephalothin were also incubated at the same time and tested at intervals to determine stability of this antibiotic under the same conditions. In order to test the influence of volume of eluting fluid on amount of antibiotic eluted, cylinders were prepared with 0.5 g gentamicin sulfate per 10 g PMMA powder. Cylinders that differed no more than 0.00048 g in weight were used. Two ml PBS were added to each cylinder in one set and 10 ml PBS to each cylinder in the other set. The amount of gentamicin eluted at 2 days and a t 7 days was determined. The effect of the surface area of the bone cement used was determined by preparing disks, 15.0 mm in diameter and 2.0 mm thick, with 0.5 g gentamicin sulfate per 10 g PMMA powder. Two ml PBS were added to each tube containing individual disks, and the amount of gentamicin eluted was determined at intervals for a total period of 65 days. Microorganisms Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and a clinical isolate of Pseudomonas aeruginosa (DEW) were used to test efficacy of anti-
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biotics eluted from PMMA cylinders. These microorganisms were selected as representative strains of bacteria most frequently reported to be isolated from orthopedic infections. Each organism was tested for susceptibility to gentamicin and cephalothin by the standard disk diffusion meth0d.1~ Minimal inhibitory concentrations (MIC) were determined by the tube dilution method,15using Antibiotic medium # 3 (Difco Laboratories, Detroit, Mich.). After 18 hr incubation at 37°C the lowest concentration of antibiotics resulting in complete inhibition of visible growth was considered to be the MIC. All tubes not showing visible growth were subcultured on nutrient agar plates to determine minimal lethal concentrations (MLC).16 The lowest concentration of antibiotic resulting in 99.9% reduction in total bacterial count after 18 hr incubation of the broth dilution series was considered to be the MLC. In addition, the MIC and MLC resulting from a combination of the two antibiotics were determined by the method of Barry and Sabath.16
Agar Plates Seeded with Microorganisms Cylinders were prepared using a ratio of 0.005,0.05, and 0.5 g gentamicin sulfate and 0.5 g Keflin to 10 g PMMA powder. Cylinders were kept a t room temperature for 2 hr before using. Forty ml of Mueller-Hinton agar was cooled to 50°C, 0.1 ml of the individual microorganisms a t a concentration of approximately 1X lo7 added, mixed, and poured into a 100 X 15 mm petri dish. One cylinder was placed in each plate, the agar allowed to solidify, and incubated a t 37°C. Triplicate sets were prepared and results averaged. Plates were observed for 7 weeks.
Efficacy Testing with Broth Cultures of Microorganisms Cylinders were prepared using a ratio of 0.05, 0.5, 1.0, and 2.0 g gentamicin sulfate or cephalothin to 10 g PMMA powder 2 hr prior to using. Broth cultures of the microorganisms were diluted to approximately 1X lo5 to 1 X lo6 microorganisms per ml in Trypticase soy broth (TSB) and incubated approximately 20-30 min in order to obtain organisms in the exponential growth phase. Individual cylinders were placed in 5 ml of the broth cultures and incubated a t 37°C. At intervals numbers of viable organisms were determined by plating the cultures on Trypticase soy agar (TSA).
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In another set of experiments cylinders were prepared using a combination of 0.025,0.25,0.5, or 1.0 g each of gentamicin sulfate and cephalothin per 10 g PMMA powder. These were tested with broth cultures of P. aeruginosa, a combination of P. aeruginosa and S. epidermidis, and a combination of P. aeruginosa and S. aureus.
RESULTS Cylinders and Disks Dough and set time of the bone cement varied slightly, depending on the temperature and relative humidity of the room. Usually the mixture reached the dough stage approximately 3 min after addition of the monomer and was polymerized a t approximately 13 min. There was no extreme variation due to addition of the antibiotics in the concentrations used. Average measurements of cylinders were weight 382.356 mg f 15.937, diameter 6.0071 mm f 0.03048, length 12.0777 mm f 0.237, volume 339.3 mm3, and surface area 282.7 mm2. Measurements for disks were weight 254.2 mg f 34.5, diameter 15.0 mm, thickness 2.0 mm, volume 353.4 mm3, and surface area 447.6 mm2. The greatest variation among individual samples was in weight, but this was not correlated with amount of antibiotic used. The greatest weight loss of the combined PMMA powder and monomer occurred immediately after or during polymerization and was 10.35%. At 18 hr the total weight loss was calculated to be 12.45%.
Antibiotic Susceptibility of Microorganisms Susceptibility of the microorganisms to gentamicin and cephalothin is shown in Table I. All are within the range considered to be sensitive, except P. aeruginosa, which was resistant to cephalothin. There were no marked synergistic or antagonistic effects when organisms were tested with a combination of the two antibiotics.
Elution of Antibiotics Results of the quantitative elution of antibiotics from cylinders are presented in Figure 1 (cephalothin) and Figure 2 (gentamicin). The graphs have been plotted to show additive values of the percentage
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WELCH TABLE I Susceptibility of Microorganisms to Antibiotics
Microorganism
S. epidermidis S. aureus E. coli P. aeruginosa
Zone Sizea MICb MLC' Genta- Cephalo- Genta- Cephalo- Genta- Cephalomicin thin micind thin micind thin 29.0 20.7 20.7 22.7
34.6 31.3 19.3 None
0.10 0.15 3.5 1.5
0.25 0.25 20.0 >50.0
0.3 1.5 5.0 3.0
2.0 2.0 50.0 >50.0
a Inhibition zone diameter in millimeters by the disk diffusion method using standard GM lO-Kg disks of gentamicin and CF 30-unit disks of cephalothin (BBL, Division of Becton, Dickinson & Co.). Minimal inhibitory concentration in gg/ml. Minimal lethal concentration in pg/ml. Based on potency of gentamicin.
eluted. The amount of antibiotic per ml was determined by the assay procedure, multiplied by ml of elution fluid, and divided by the calculated amount of antibiotic in the cylinder. Each point represents an average of five individual cylinders. The largest amount of antibiotic was eluted within the first 24-48 hr after placing cylinders in the fluid, and proportionally smaller amounts were eluted with time. The assays were terminated a t 175 days or when the amount of antibiotic in solution measured less than 2 pg/ml. Amounts of gentamicin greater than 2 pg/ml were eluted after 175 days from cylinders prepared with 1and 2 g per 10 ml PMMA powder. In the preceding determinations the volume of PBS used for each cylinder was 5 ml. The effect of volume of fluid in which cylinders were placed is shown in Table 11. The smaller amount of eluting fluid contained more pg/ml of antibiotic, but a larger total amount was eluted into the larger volume. The 2 ml volume of fluid contained 105 pg gentamicin per ml, whereas the 10 ml volume contained only 49 pg/ml after 2 days incubation. If the total volumes are considered, the 10 ml volume contained only 2.3 times as much gentamicin as the 2 ml volume. A comparison of results obtained using the disks and cylinders is shown in Figure 3. The volume of eluting fluid was 5 ml with the cylinders and 2 ml with the disks. The total percentage of gentamicin eluted at 40 days was the same, although a slightly greater percentage was eluted from the cylinders from the second to the thirty-fifth day. A larger percentage was eluted from the disks within the first 24 hr.
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Elution of Keflin Additive Values
I : : :
: :
8h I 2 3 4
67
I1 1213 15
18
21
25 27 29
DAYS
Fig. 1. Elution of Keflin from Surgical Simplex cement. Additive values of the percentage eluted from cylinders. Cylinders prepared with 10 g PMMA powder and ( 0 )0.5 g Keflin (13.9 mg Keflin per cylinder), (A)1g (28.1 mg per cylinder), and (m) 2 g (56.4 mg per cylinder).
After 40 days the amount of gentamicin per ml was less than 2 pglml with the cylinders, but larger amounts were eluted from the disks until the assays were terminated at 65 days.
Agar Plates Seeded with Microorganisms Results shown in Table I11 demonstrated that gentamicin was eluted from all the cylinders in sufficient quantities to inhibit growth
WELCH
688
7
28.. 27.-
26.25. 24-
23-
/
22-
21
.-
20-
6.-
/
f
5.-
gentamicin per cylinder).
of S. epidermidis and S. aureus. However, cylinders prepared with 0.5 g gentamicin sulfate per 10 g PMMA powder were required to produce a zone of inhibition against E. coli that remained without being overgrown by the bacteria after 5 days. The same concentration inhibited growth of P. aeruginosa, but this organism grew into the zone of inhibition after 5 days.
ANTIBIOTICS IN ACRYLIC BONE CEMENT
689
TABLE I1 Effect of Volume of Fluid on Elution of Gentamicin Calculated Amount Gentamicin Gentamicin Gentamicin in Total Weight of per Calculated Cylinder Recovery Volume Cylinder per ml Volume of PBS (ml) PBS (Fg) PBS (pg) (mg) (ms) (%) 2 days 7 days
2.0 10.0 1.5 9.5
105 49 370 117
210 490 555 1111.5
391.99 392.47 391.99 392.47
8.57 8.58 8.57 8.58
2.45 5.71 6.48 12.95
Efficacy in Broth Cultures The effect of cylinders containing cephalothin on broth cultures of S. aureus and E. coli is shown in Figure 4. Control cultures without antibiotics multiplied in a normal exponential fashion. There was a marked reduction in the total number of S. aureus after addition of a cylinder containing 1.32 mg cephalothin which was even more pronounced a t 20 hr. Cylinders containing 12.6 mg cephalothin or greater were bactericidal within 20 hr. Results obtained using S. epidermidis are not shown, since they so closely paralleled those obtained with S. aureus. The total number of E. coli was reduced by cylinders containing 1.32 mg cephalothin, but after 4 hr the bacteria multiplied exponentially. Cylinders containing 12.6 mg cephalothin reduced the total number and inhibited multiplication but were not completely bactericidal. Cylinders containing 28.15 mg were bactericidal to E. coli within 20 hr. P. aeruginosa was not tested, because it was not sensitive to cephalothin. The effect of cylinders containing gentamicin on staphylococci is shown in Figure 5. There was a marked reduction in bacteria after 4 hr contact with cylinders containing 0.85 mg gentamicin, but after 4-6 hr they multiplied exponentially. Cylinders containing larger amounts of gentamicin were bactericidal in 45 min t o 4 hr. Similar results were obtained with E. coli and P. aeruginosa (Fig. 6) except that cylinders containing 0.85 mg gentamicin were not effective in controlling growth of these organisms. However, cylinders containing larger amounts were bactericidal in 4-6 hr.
WELCH
690
21.-
/
20-1918-
I
8- I
7-
1
A combination of cephalothin and gentamicin incorporated in the bone cement produced very nearly the same effect on P. aeruginosa as did cylinders containing gentamicin alone. Results obtained using cylinders prepared with a combination of the antibiotics in cultures containing equal numbers of S. epidermidis and P. aeruginosa (Fig. 7 )demonstrated that the staphylococcuswas slightly more sensitive to a combination of the antibiotics than when the antibiotics were used individually. There was no appreciable difference in inhibition of the pseudomonas. Similar results were obtained by using a combination of S. aureus and P. aeruginosa (Fig. 8 ) in that the staphylococcus was slightly more sensitive to a combination of the antibiotics. However, the pseudomonas was not completely inhibited by a combination of 4.2 mg gentamicin plus 7.0 mg cephalothin per cylinder, as it had been when S. epidermidis and P. aeruginosa were used in the same culture.
Microorganism
P. aeruginosa
E. coli
S. aureus
S. epidermidis 0.05 0.5 Cephalothin 0.5 Gentamicin sulfate 0.005 0.05 0.5 Cephalothin 0.5 Gentamicin sulfate 0.005 0.05 0.5 Cephalothin 0.05 Gentamicin Sulfate 0.005 0.05 0.5 Cephalothin 0.5
Gentamicin sulfate 0.005
Grams Antibiotic per 10 g PMMA Powder
2
1
2
...
5
...
None None 25 X complete None
... ...
... 14 X 17
1
...
3 4
...
...
None 14 X 22 30X complete
32 X 28
...
... 2 3
1
4 2
... ...
None
None None None
14 X 17
None None 30 X 40
32 X 28
29 X 39 Complete
17 X 22
...
Complete 17 X 22 29 X 39 Complete
Complete
...
1
45 X complete Complete
3
2 1
22 x 22
Final Zone of Inhibition (mm) (Ends X Length)
45 X complete Complete
...
22 x 22
2
Day a t Which Daya t Which Maximum Distance from Maximum Zone Cylinder to Zone of Inhibition Edge of Zone (mm) of Inhibition Decreased WasObserved (Ends X Length)
TABLE 111
3
a
k5
Q
3
0
m
z
5
P Q
2
8
2 2 0 2
i2
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-S -aureus
HOURS
HOURS
Fig. 4. Effect of Surgical Simplex containing Keflin on broth cultures of 5'. aut-ezhs and E. coli. ( 0 )Controls without antibiotic. Cylinders prepared with 10 g PMMA powder and (A)0.05 g Keflin (1.32 mg Keflin per cylinder), ( W ) 0.5 g (12.6 mg per cyl1.0 g (18.15 mg per cylinder). inder), and (0)
DISCUSSION AND CONCLUSION Antibiotics incorporated into PMMA bone cement for prophylactic use in arthroplastic joint replacement should have the following characteristics: 1. Effective against a wide variety of bacteria 2. Show a rapid release from the acrylic cement soon after transplantation 3. Continue to be released from the cement for a long period of time in effective concentrations 4. Be nontoxic and nonallergenic under these conditions 5. Be effective in small doses 6. Have a marked bactericidal action 7. Physicochemical properties should include: Be highly soluble in aqueous solutions Diffuse well from the poly(methy1) methacrylate Be thermostable under conditions required for polymerization Be stable for prolonged periods at 37°C
ANTIBIOTICS IN ACRYLIC BONE CEMENT II\
693
-S -aureus
5 epidermidis
HOURS
HOURS
Fig. 5. Effect of Surgical Simplex containing gentamicin on broth cultures of bacteria. ( 0 ) Controls without antibiotic. Cylinders prepared with 10 g PMMA powder and (A)0.05 g gentamicin sulfate (0.85 mg active gentamicin per cylinder), (H) 0.5 g (8.38 mg gentamicin per cylinder), and (0) 1.0 g (16.88 mg gentamicin per cylinder).
Have a low bulk volume 8. Produce little or no change in mechanical properties of the cement. Gentamicin and cephalothin were selected for this study because they do have many of these properties and are commonly used in orthopedic surgery for irrigation of the wound during surgery or parenterally as prophylaxis against infection. In our experiments, the powder form of the antibiotics was mixed thoroughly with the polymer prior to addition of the liquid monomer. In preliminary experiments we attempted to use aqueous solutions of antibiotics mixed with both the powder and the liquid monomer and also attempted to add powdered antibiotics to the monomer. None of these alternate procedures proved satisfactory to obtain a homogeneous mixture. Gentamicin powder is not packaged aseptically for clinical use in this country. However, it has been shown that little or no loss of potency occurred after sterilization of the powder by exposure to ethylene oxide gas.1°
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I
LCO"
P aaruginosa
HOURS
HOURS
Fig. 6. Effect of Surgical Simplex containing gentamicin on broth cultures of bacteria. (@) Controls without antibiotic. Cylinders prepared with 10 g PMMA powder and ( A ) 0.05 g gentamicin sulfate (0.85 mg active gentamicin per cylinder), (m) 0.5 g (8.38 mg gentamicin per cylinder), and (0) 1.0 g (16.88 mg gentamicin per cylinder).
Results of the quantitative elution of both cephalothin and gentamicin from cylinders of Surgical Simplex cement demonstrated that the largest amount of antibiotic was eluted within the first 24-48 hr after cylinders were placed in PBS solution. The antibiotic on, or very near, the surface diffused into solution within a short period of time. Subsequently, increasingly smaller amounts were eluted with time as the solution penetrated into the pores of the cement. The observation of the initial elution of large amounts of antibiotics from the Surgical Simplex cement is in agreement with previous results obtained by using a variety of antibiotics and acrylic cements.3,5,6,8,9,11,12 Gentamicin is the one antibiotic that has been studied by most investigators, and there is considerable disagreement in the literature regarding elution rates. Hessert and Ruckdesche14reported release of 50% of the amount of gentamicin incorporated into Palacos R after 2 hr incubation and 80%after 24 hr. Chapman and Hadleyll stated that, when incubated in saline, there was a rapid leaching of the antibiotics from Surgical Simplex cement and 80% may be removed
ANTIBIOTICS IN ACRYLIC BONE CEMENT
S_ epidermidis __
695
and
P ceruginosa
.i
0
/
2
20
6 HOURS
Fig. 7. Effect of combination of antibiotics in Surgical Simplex on a combination of organisms in broth cultures. Cylinders prepared with 10 g PMMA powder and ( 0 ) P. aeruginosa 0.025 g gentamicin sulfate 0.025 g Keflin (0.42 mg gentamicin 0.7 mg Keflin per cylinder), (0) S. epidermidis response to concentrations listed above, ( A ) P. aeruginosa 0.25 gentamicin sulfate 0.25 g Keflin powder (4.2 mg gentamicin 7.0 mg Keflin per cylinder).
+
+
+
+
during the first 48 hr and no activity could be detected after 7 days. However, they did use relatively small conical pellets (weight, 0.19 g) in 10 ml saline. Elson et al.9 also reported a larger quantity of gentamicin was released from Palacos than from CMW, Simplex, and Sulfix cement and over longer periods of time. Experimental conditions used by these investigators were comparable with ours, except they used a larger volume of eluting fluid (50 ml). They recovered less than 1 pg gentamicidml after 4 days from Simplex cement, and measurements were terminated after 25 days. Marks et aL8 reported a release rate of gentamicin such that less than 0.4pg was recovered after 18 days from Surgical Simplex cement, whereas they were able
-S -oureus
P aeruginoso
and
lo--
/
Fig. 8. Effect of combination of antibiotics in Surgical Simplex on a combination of organisms in broth culture. Cylinders prepared with 10 g PMMA powder and ( 0 ) 0.025 g gentamicin sulfate 0.025 g Keflin (0.4 mg gentamicin 0.7 mg Keflin per cylinder), ( A ) 0.25 g gentamicin sulfate 0.25 g Keflin (4.2 mg gentamicin 7.0 mg Keflin per cylinder), (m) 0.5 g gentamicin sulfate 0.5 g Keflin (8.24 mg gentamicin 13.9 mg Keflin per cylinder), (0) 1.0 g gentamicin sulfate 1.0 g Keflin (15.9 mg gentamicin 28.1 mg Keflin per cylinder).
+
+
+
+
+
+
+
+
to measure release of a comparable amount from Palacos R for 61 days. However, these authors used disks 12 mm in diameter and failed to mention thickness and weight. Also, they measured zones of inhibition of the disks placed directly on seeded agar plates and compared with standard zones of inhibition produced by antibiotics on 6-mm filter-paper disks. Therefore, these results should not be compared with those obtained by using different techniques. In our tests, gentamicin was released from Surgical Simplex over a prolonged period of time. Amounts greater than 2 pglml were still being measured at 175 days, when the measurements were terminated. We also demonstrated the effect of volume of eluting fluid, surface area, and amount of antibiotic used. Although a greater amount of gentamicin was released per ml in a 2-ml volume of eluting fluid than in a 10-ml volume, the total amount released is greater than in the larger volume, which indicates a function of the diffusion rate. Gentamicin was eluted from the disks for a longer period of time than
ANTIBIOTICS IN ACRYLIC BONE CEMENT
697
the cylinders when the same amount of antibiotic was used to prepare the different shaped pieces of bone cement. This was probably due to the larger surface area of the disks as compared with the cylinders. Also, antibiotics were eluted in greater amounts and over a longer period of time from cylinders prepared with larger amounts of antibiotics. Our results correlated more closely with those reported for Palacos R by Wahlig et al.5 He calculated the rate of release as varying from 2.5 to 17.5% after 20-22 weeks. In our experience a slightly higher percentage (22.7-26.3%) was released in the same time period. Our cylinders were smaller, had less surface area, and weighed less than those used by Wahlig et al. The greater percentage of elution from our cylinders could be due to the properties of the polymer itself, the larger amount of antibiotic per unit, and the frequency with which we changed the elution fluid. We changed the fluid three times per week after the first 10 days, whereas Wahlig et al. changed the fluid weekly and then monthly. There is a fluctuation in concentration of the antibiotic in solution as a result of every change in fluid during the incubation period. Concentration changes take place during the time the antibiotic-bone cement remains in contact with the fluid, and a new diffusion process starts each time there is a change of the fluid. Wahlig et al. also stated that the release of antibiotic would be much larger when the fluid is changed more often and, therefore, would be greater in the bloodstream with a continuous removal of released antibiotic. To date, there have been no previous reports of elution rates of cephalothin from acrylic bone cement. The pattern of elution is similar to that of gentamicin, in that the largest amount was eluted within the first 24 hr and then increasingly smaller amounts were eluted with time. Also, the largest amount of cephalothin eluted was from cylinders prepared with the largest amount of antibiotic. However, in contrast, only approximately one-third as much cephalothin was eluted from the cylinders as compared with gentamicin. Also, the level of cephalothin fell below 2 pg/ml in 21-29 days. This may be due, at least in part, to the relative instability of cephalothin in solution at 37OC. Standard cephalothin solutions in low concentrations lost almost all activity within 24 hr and in high concentrations within 4 days, whereas gentamicin solutions retain more than 80% activity at 37OC for at least 15 days. Also, it is possible that gentamicin is more stable to heat of polymerization than cephalothin.
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However, from our recovery rates we may conclude that cephalothin is stable for a period of time when combined with the Surgical Simplex. As the solution comes in contact, the cephalothin diffuses out into solution and then gradually loses activity. Nevertheless, we were able to measure the release of considerable quantities of cephalothin. In these studies, the measurements were arbitrarily terminated when the amount of antibiotic in solution was less than 2 pg/ml, although lower quantities can be measured by the technique used. It was felt that measuring extremely low levels of antibiotic would not be of any practical significance. Results obtained by placing cylinders containing antibiotics in agar plates seeded with microorganisms demonstrated that antibiotics were eluted in sufficient quantities to inhibit the microorganisms. This test does not permit strictly quantitative interpretations and does not indicate if this is a bacteriostatic or bactericidal effect. However, reports on efficacy by other investigators6V8J0-l2are based on this test. It cannot be equated with the Bauer-Kirby test14 for sensitivity of organisms to antibiotics, because the amounts of antibiotics used and shapes of the cement used are not comparable with the standard filter-paper disks used in the Bauer-Kirby procedure. Placing cylinders containing antibiotics in broth cultures of microorganisms permitted a more quantitative determination of the bactericidal effect of the antibiotics. The numbers of organisms used for this test exceeded the number that would reasonably be expected to contaminate a wound. Our results demonstrated that gentamicin combined with Surgical Simplex was bactericidal to the organism used within 45 min to 6 hr, depending on the type of organism and concentration of gentamicin. Cephalothin was bactericidal to the sensitive organisms within 20 hr, but not to the resistant P. aeruginosa. Again, this was dependent on the organism and concentration of cephalothin. Chapman and Hadleyll added pellets containing cephalothin and gentamicin sulfate to broth cultures of bacteria and reported 100%inhibition of bacterial growth after 24 hr incubation. Hill et al.17 also incubated disks of antibiotic-containing cement in peptone water containing bacteria and demonstrated inhibition of growth for various periods of time, depending on the type of antibiotic and bacteria. However, none of these reports indicated approximate concentrations of bacteria, and it is well known that sensitivity to antibiotics is dependent in part on numbers of organisms present. Since P. aeruginosa is so frequently very difficult to control with
ANTIBIOTICS IN ACRYLIC BONE CEMENT
699
antibiotics, the effect of using a combination of antibiotics in bone cement was tested. This combination produced very nearly the same results as using gentamicin alone. A combination of antibiotics was also tested on a combination of organisms, since infections that may occur are not necessarily due to one single type of organism. When used in combination with P. aeruginosa, S. epidermidis and S. aureus were both slightly more sensitive to a combination of the antibiotics than when the antibiotics were used individually. However, there was no marked difference with P. aeruginosa. Combinations of two or more unrelated antibiotics are sometimes used in clinical medicine to prevent emergence of resistant forms, and it is essential that both antibiotics be present at inhibitory levels at the site of infection. It is possible that a more suitable combination of antibiotics for use in bone cement could be found. These experiments indicated that gentamicin and cephalothin combined with Surgical Simplex bone cement may be useful for certain orthopedic conditions. An initial high release of antibiotics followed by a longer rate of release is desirable. The initial high release would be effective in controlling bacteria found near the site of infection. Clinically, infections that occur following arthroplastic hip replacements are most often found at the distal portion of the prosthesis. Persistence of pathogenic bacteria can be responsible for relapse if therapy is discontinued too early, and, therefore, a prolonged release of the antibiotic is desirable. However, these results should not be interpreted to an in vivo situation, since the amount of fluid present influences the amount of antibiotic eluted from the bone cement. In a closed system in uitro localized microorganisms are in constant contact with the eluted antibiotics. Nevertheless, these experiments demonstrated that sufficient quantities of antibiotics are eluted to have bactericidal effects in a relatively short time. This research was supported in full by USPHS Research Grant 1R01 AM 18794from the Department of Health, Education and Welfare, National Institutes of Health, Bethesda, Md. The author wishes to thank Howmedica,Inc., for supplying the Surgical Simplex, Schering Corp. for the gentamicin, and Eli Lilly & Co. for the cephalothin. I also thank Richard Stutte and Deborah Hassler for technical assistance.
References 1. R. Koschmieder, W. Ritzerfeld, and H. Kleymann, Z . Orthop., 111,244 (1973). 2. G. Hunter and D. Dandy, J . Bone Jt. Surg., 59-B, 293 (1977). 3. H. W. Buchholz and H. Engelbrecht, Chirurg, 41,511 (1970). 4. G. R. Hessert and G. Ruckdeschel, Arch. Orthop. Unfall-Chir.,68,249 (1970).
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5. H. Wahlig, W. Hameister, and A. Grieben, Langenbecks Arch. Chir., 331, 169 (1972). 6. W. P. Gartenmann, H. Brodhage, J. Zippel, and L. Badeira, in Arthroplasy of the Hip, G. Chapchal, Ed., Intercontinental Medical Book Corp., New York, 1973, p. 21. 7. G. Ruckdeschel, G. R. Hessert, and T. Schollhammer, Arch. Orthop. Unfall-Chir., 74,291 (1973). 8. K. E. Marks, C. L. Nelson, and E. P. Lautenschlager, J . Bone Jt. Surg., 58-A, 358 (1976). 9. R. A. Elson, A. E. Jephcott, D. B. McGechie, and D. Verettas, J . Bone Jt. Surg., 59-B, 200 (1977). 10. P. D. Levin, J . Bone Jt. Surg., 57-B, 234 (1975). 11. M. W. Chapman and W. K. Hadley, J . Bone J t . Surg., 58-A, 76 (1976). 12. A. L. Rosenthal, J. Znt. Med. Res., 4,296 (1976). 13, L. D. Sabath and J. M. Matsen in Manual of Clinical Microbiology, E. H. Lennette, E. H. Spaulding, and J. P. Truant, Eds., American Society for Microbiology, Washington, D.C., 1974, p. 428. 14. A. W. Bauer, W. M. Kirby, J. C. Sherris, and M. Turck, Am. J . Clin. Pathol., 45, 493 (1966). 15. J. A. Washington I1 and A. L. Barry in Manual of Clinical Microbiology, E. H. Lennette, E. H. Spaulding, and J. P. Truant, Eds., American Society for Microbiology, Washington, D.C., 1974, p. 410. 16. A. L. Barry and L. D. Sabath, in Manual of Clinical Microbiology, E. H. Lennette, E. H. Spaulding, and J. P. Truant, Eds., American Society for Microbiology, Washington, D.C., 1974, p. 431. 17. J. Hill, L. Klenerman, S. Trustey, and R. Blowers, J . Bone Jt. Surg., 59-B, 197 (1977).
Received August 26,1977 Revised January 4,1978
Summary The effect of combining gentamicin and cephalothin with Surgical Simplex bone cement was tested in uitro. Experiments were designed to determine (1) quantitative elution rates and period of time the antibiotics were eluted and (2) effective bactericidal qualities. Both antibiotics were eluted from polymerized acrylic cement in large quantities within 24-48 hr after coming in contact with fluid and continued to be released in smaller amounts for 21 days to more than 175 days. This varied with the antibiotic, concentration of antibiotic in the cement, surface area of cement, and volume of elution fluid. Quantitative determinations of numbers of bacteria in broth cultures demonstrated that sufficient quantities of antibiotics were released to be bactericidal to microorganisms within 45 min to 20 hr. This was dependent on the type of antibiotic, type of bacteria, concentration of antibiotic in the cement, and time of contact.
INTRODUCTION Arthroplastic joint replacement is an accepted procedure for surgical correction of various orthopedic diseases and congenital malformations. In the majority of total hip and knee replacements poly(methy1methacrylate) (PMMA) is used as a filler to provide mechanical fixation of the prosthetic components to bone. Infection is one of the most serious complications following arthroplastic joint replacement, with reported rates varying from 11to less than 1 % . l y 2 Various techniques have been advocated to reduce infection rates, including use of clean-air surgical rooms, impermeable drapes and gowns, prophylactic systemic antibiotics pre- and postoperative, and intraoperative irrigation of the wound with antibiotics. However, infections may occur, in spite of the most rigid precautions, from exogenous sources or from undetected foci of infection. The observation that a PMMA bone cement, Palacos R (manu-
Journal of Biomedical Materials Research, Vol. 12,679-700 (1978) 0 1978John Wiley & Sons, Inc. 0021-9304/78/0012-0679$01.00
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factured by Kulzer, Bad Homburg), released residual monomer over an extended period of time gave Buchholz and Engelbrecht3 the idea of mixing antibiotics with the cement. The reasoning was that antibiotics should be released from the cement for a prolonged period after surgery, which should reduce the possibility of infection a t the implant-bone boundary. They reported a decrease in deep infection in over 1000 operations by using antibiotic additives. Antibiotics introduced a t the site where infections may develop have the advantage of being more effective than parenteral or oral therapy. It may be possible that a lower dose of antibiotics would be effective, thereby eliminating some of the undesirable side effects of large doses administered over long periods of time. This technique could also eliminate the possibility of altering normal flora of the body by prolonged systemic treatment. Until recently the only PMMA bone cement licensed for use in the United States was Surgical Simplex, which has a slightly different composition from other PMMA bone cements, The majority of reports in the literature regarding release of antibiotics from acrylic cements in uitro are based on use of Palacos R, Palacos E, or CMW.3-9 Wahlig et al.,5using Palacos R, Palacos E, and two other PMMA cements, demonstrated that release of antibiotics varies with the composition of the PMMA. Similar results were reported by Elson et al.9 Wahlig et al.5 stated that usability of an antibiotic-bone cement mixture must be demonstrated by detailed experimental tests, not only with respect to suitability of the antibiotic, but also with respect to the properties of the cement. Results obtained with one antibiotic and one bone cement cannot be transferred to other antibiotics or bone cements. There is a need for a better understanding of the effects of adding antibiotics to Surgical Simplex, since there has been a limited amount of research in this The present study was undertaken to determine long-term release rates of gentamicin and cephalothin quantitatively from Surgical Simplex cement under carefully controlled laboratory conditions. These antibiotics were selected since they have many properties that would be desirable in antibiotic-bone cement combination and they are frequently used in orthopedic surgery. Also, bone cement combined with antibiotics was tested quantitatively for effective inhibition of microorganisms commonly reported to be the cause of infections following orthopedic surgery.
ANTIBIOTICS IN ACRYLIC BONE CEMENT
681
MATERIALS AND METHODS Antibiotics Gentamicin sulfate powder, potency 580 ,ug/mg, was supplied by Schering Corp., Bloomfield, N.J. Concentrations reported in this article are distinguished as being based on weight of the gentamicin sulfate powder and on potency of the gentamicin. Sterile sodium cephalothin was supplied by Eli Lilly and Co., Indianapolis, Ind.
Preparation of Cylinders and Disks
A teflon mold, 55 X 65 mm, was constructed containing 20 cylindrical holes 6 mm in diameter and 12 mm deep. Commercially available PMMA bone cement, Surgical Simplex P Radiopaque (manufactured by North Hill Plastics, Ltd., 49 Grayling, London N16, England), supplied by Howmedica, Inc., Medical Division, Rutherford, 'N.J., was prepared in accordance with the manufacturer's directions at a ratio of 40 g powder and 20 ml liquid monomer. Varying amounts of gentamicin sulfate and cephalothin, either separately or in combination, were thoroughly mixed with the PMMA powder in a ceramic dish. Liquid monomer was added to the dry mixture and stirred gently with a teflon-coated spatula for approximately 11/2 to 2 min until just prior to the dough stage. The viscous mixture was placed in the holes of the teflon mold, being careful to have each hole well packed so that no air bubbles were formed inside the cylinders. The sides of the mold were scraped with the spatula to remove any excess PMMA in order to form even ends of cylinders. The mold was allowed to stand at room temperature until the mixture had polymerized, and then cylinders were punched out of the mold. Disks were prepared by rolling the material in the dough stage to a sheet 2 mm thick and cutting circular disks 15 mm in diameter. All procedures were carried out using sterile equipment and aseptic technique. Calculation of Theoretical Amount of Antibiotic in Cylinders A large number of samples of PMMA powder and liquid monomer were weighed, and it was determined that 1ml of liquid monomer weighed 0.93 g. The liquid was mixed with PMMA powder at the prescribed ratio of 1 ml to 2 g powder. Weights were determined
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immediately after polymerization and at intervals up to 18 hr at 235°C and relative humidity of 44%. By determining the loss of weight in the polymerized bone cement due to volatilization of the monomer, the theoretical amount of antibiotic could be calculated. Accordingly, each cylinder was weighed prior to using the calculated amount of antibiotic in each cylinder determined.
Antibiotic Standards and Quantitative Procedures Stock solutions of antibiotics at concentrations of 1000 pg/ml were prepared in sterile distilled water, dispensed into small portions, and frozen at -20°C. Working standards, ranging from 1.0 to 20 pg/ml were prepared by diluting the stock solutions in 0.1M phosphate buffer plus 0.85% NaCl (PBS). Gentamicin sulfate was diluted in PBS pH 7.4, since this pH approximates that found in normal mammalian blood. Cephalothin was diluted in PSB pH 7.2, because it is the sodium salt and is not ionized between pH 6.8 and 7.2. Preliminary tests using phosphate buffer, PBS, phosphate buffer plus 5%normal human serum, and normal human serum as diluents produced almost identical results. Working solutions of gentamicin sulfate were stored at 4°C and used within 4 days, whereas working solutions of cephalothin were prepared fresh daily. Antibiotics were measured quantitatively by an agar diffusion method similar to that described by Sabath and Matsen.13 Antibiotic medium agar # 5 (Difco Laboratories, Detroit, Mich.) was dispensed in 25 ml amounts into 150 X 15 mm plastic petri dishes in a laminar flow hood with a level bench and allowed to set until the surface of the agar was dry (approximately '/z hr). An overnight broth culture of Staphylococcus epidermidis ATCC 12228 was diluted to approximately 1X lo6 organisms/ml and 0.1 ml spread evenly on the surface of the agar. Filter-paper disks, 6.35 mm diameter (#740-E, Schleicher & Schuell, Inc., Keene, N.J.), were placed in sterile plastic pertri dishes. For each assay, 0.02-ml portions of standard antibiotic or unknown test solutions were pipetted with a MLA pipette (Medical Laboratories Automation, Inc., Mt. Vernon, N.Y.) fitted with sterile disposable tips onto each of two filter-paper disks. Four standard antibiotic concentrations, ranging from 1or 2.5 to 20 pg/ml, were used on each plate. The disks were then placed in pairs on the agar surface, with members of the same pair placed directly opposite each other. After overnight incubation at 35"C, zone diameters were measured
ANTIBIOTICS IN ACRYLIC BONE CEMENT
683
with a vernier calipers and recorded to the nearest 0.1 mm. Zone diameters of each antibiotic standard were averaged, and a dose response curve was plotted on semilog graph paper using point-to-point connection relating the concentration of antibiotic in the standard (log scale) to the diameter (in mm) of the zone of inhibition produced. A separate graph was prepared for each of the plates. Diameters of the zones of the test samples were measured, determined individually from the graph, and then averaged. Quantitative Measurement of Elution of Antibiotics
Cylinders prepared with 0.5, 1.0, and 2.0 g gentamicin sulfate or cephalothin per 10 g PMMA powder were placed individually in sterile screw-cap tubes. Five cylinders of each concentration were used. Five ml PBS was added to each tube as eluting fluid, and the tubes were incubated at 37OC. At intervals cylinders were removed, dried on filter paper, and placed in fresh solutions. The elution fluid was quantitatively tested for amount of antibiotic by the assay procedure. Standard working solutions of cephalothin were also incubated at the same time and tested at intervals to determine stability of this antibiotic under the same conditions. In order to test the influence of volume of eluting fluid on amount of antibiotic eluted, cylinders were prepared with 0.5 g gentamicin sulfate per 10 g PMMA powder. Cylinders that differed no more than 0.00048 g in weight were used. Two ml PBS were added to each cylinder in one set and 10 ml PBS to each cylinder in the other set. The amount of gentamicin eluted at 2 days and a t 7 days was determined. The effect of the surface area of the bone cement used was determined by preparing disks, 15.0 mm in diameter and 2.0 mm thick, with 0.5 g gentamicin sulfate per 10 g PMMA powder. Two ml PBS were added to each tube containing individual disks, and the amount of gentamicin eluted was determined at intervals for a total period of 65 days. Microorganisms Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and a clinical isolate of Pseudomonas aeruginosa (DEW) were used to test efficacy of anti-
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biotics eluted from PMMA cylinders. These microorganisms were selected as representative strains of bacteria most frequently reported to be isolated from orthopedic infections. Each organism was tested for susceptibility to gentamicin and cephalothin by the standard disk diffusion meth0d.1~ Minimal inhibitory concentrations (MIC) were determined by the tube dilution method,15using Antibiotic medium # 3 (Difco Laboratories, Detroit, Mich.). After 18 hr incubation at 37°C the lowest concentration of antibiotics resulting in complete inhibition of visible growth was considered to be the MIC. All tubes not showing visible growth were subcultured on nutrient agar plates to determine minimal lethal concentrations (MLC).16 The lowest concentration of antibiotic resulting in 99.9% reduction in total bacterial count after 18 hr incubation of the broth dilution series was considered to be the MLC. In addition, the MIC and MLC resulting from a combination of the two antibiotics were determined by the method of Barry and Sabath.16
Agar Plates Seeded with Microorganisms Cylinders were prepared using a ratio of 0.005,0.05, and 0.5 g gentamicin sulfate and 0.5 g Keflin to 10 g PMMA powder. Cylinders were kept a t room temperature for 2 hr before using. Forty ml of Mueller-Hinton agar was cooled to 50°C, 0.1 ml of the individual microorganisms a t a concentration of approximately 1X lo7 added, mixed, and poured into a 100 X 15 mm petri dish. One cylinder was placed in each plate, the agar allowed to solidify, and incubated a t 37°C. Triplicate sets were prepared and results averaged. Plates were observed for 7 weeks.
Efficacy Testing with Broth Cultures of Microorganisms Cylinders were prepared using a ratio of 0.05, 0.5, 1.0, and 2.0 g gentamicin sulfate or cephalothin to 10 g PMMA powder 2 hr prior to using. Broth cultures of the microorganisms were diluted to approximately 1X lo5 to 1 X lo6 microorganisms per ml in Trypticase soy broth (TSB) and incubated approximately 20-30 min in order to obtain organisms in the exponential growth phase. Individual cylinders were placed in 5 ml of the broth cultures and incubated a t 37°C. At intervals numbers of viable organisms were determined by plating the cultures on Trypticase soy agar (TSA).
ANTIBIOTICS IN ACRYLIC BONE CEMENT
685
In another set of experiments cylinders were prepared using a combination of 0.025,0.25,0.5, or 1.0 g each of gentamicin sulfate and cephalothin per 10 g PMMA powder. These were tested with broth cultures of P. aeruginosa, a combination of P. aeruginosa and S. epidermidis, and a combination of P. aeruginosa and S. aureus.
RESULTS Cylinders and Disks Dough and set time of the bone cement varied slightly, depending on the temperature and relative humidity of the room. Usually the mixture reached the dough stage approximately 3 min after addition of the monomer and was polymerized a t approximately 13 min. There was no extreme variation due to addition of the antibiotics in the concentrations used. Average measurements of cylinders were weight 382.356 mg f 15.937, diameter 6.0071 mm f 0.03048, length 12.0777 mm f 0.237, volume 339.3 mm3, and surface area 282.7 mm2. Measurements for disks were weight 254.2 mg f 34.5, diameter 15.0 mm, thickness 2.0 mm, volume 353.4 mm3, and surface area 447.6 mm2. The greatest variation among individual samples was in weight, but this was not correlated with amount of antibiotic used. The greatest weight loss of the combined PMMA powder and monomer occurred immediately after or during polymerization and was 10.35%. At 18 hr the total weight loss was calculated to be 12.45%.
Antibiotic Susceptibility of Microorganisms Susceptibility of the microorganisms to gentamicin and cephalothin is shown in Table I. All are within the range considered to be sensitive, except P. aeruginosa, which was resistant to cephalothin. There were no marked synergistic or antagonistic effects when organisms were tested with a combination of the two antibiotics.
Elution of Antibiotics Results of the quantitative elution of antibiotics from cylinders are presented in Figure 1 (cephalothin) and Figure 2 (gentamicin). The graphs have been plotted to show additive values of the percentage
686
WELCH TABLE I Susceptibility of Microorganisms to Antibiotics
Microorganism
S. epidermidis S. aureus E. coli P. aeruginosa
Zone Sizea MICb MLC' Genta- Cephalo- Genta- Cephalo- Genta- Cephalomicin thin micind thin micind thin 29.0 20.7 20.7 22.7
34.6 31.3 19.3 None
0.10 0.15 3.5 1.5
0.25 0.25 20.0 >50.0
0.3 1.5 5.0 3.0
2.0 2.0 50.0 >50.0
a Inhibition zone diameter in millimeters by the disk diffusion method using standard GM lO-Kg disks of gentamicin and CF 30-unit disks of cephalothin (BBL, Division of Becton, Dickinson & Co.). Minimal inhibitory concentration in gg/ml. Minimal lethal concentration in pg/ml. Based on potency of gentamicin.
eluted. The amount of antibiotic per ml was determined by the assay procedure, multiplied by ml of elution fluid, and divided by the calculated amount of antibiotic in the cylinder. Each point represents an average of five individual cylinders. The largest amount of antibiotic was eluted within the first 24-48 hr after placing cylinders in the fluid, and proportionally smaller amounts were eluted with time. The assays were terminated a t 175 days or when the amount of antibiotic in solution measured less than 2 pg/ml. Amounts of gentamicin greater than 2 pg/ml were eluted after 175 days from cylinders prepared with 1and 2 g per 10 ml PMMA powder. In the preceding determinations the volume of PBS used for each cylinder was 5 ml. The effect of volume of fluid in which cylinders were placed is shown in Table 11. The smaller amount of eluting fluid contained more pg/ml of antibiotic, but a larger total amount was eluted into the larger volume. The 2 ml volume of fluid contained 105 pg gentamicin per ml, whereas the 10 ml volume contained only 49 pg/ml after 2 days incubation. If the total volumes are considered, the 10 ml volume contained only 2.3 times as much gentamicin as the 2 ml volume. A comparison of results obtained using the disks and cylinders is shown in Figure 3. The volume of eluting fluid was 5 ml with the cylinders and 2 ml with the disks. The total percentage of gentamicin eluted at 40 days was the same, although a slightly greater percentage was eluted from the cylinders from the second to the thirty-fifth day. A larger percentage was eluted from the disks within the first 24 hr.
ANTIBIOTICS IN ACRYLIC BONE CEMENT
687
Elution of Keflin Additive Values
I : : :
: :
8h I 2 3 4
67
I1 1213 15
18
21
25 27 29
DAYS
Fig. 1. Elution of Keflin from Surgical Simplex cement. Additive values of the percentage eluted from cylinders. Cylinders prepared with 10 g PMMA powder and ( 0 )0.5 g Keflin (13.9 mg Keflin per cylinder), (A)1g (28.1 mg per cylinder), and (m) 2 g (56.4 mg per cylinder).
After 40 days the amount of gentamicin per ml was less than 2 pglml with the cylinders, but larger amounts were eluted from the disks until the assays were terminated at 65 days.
Agar Plates Seeded with Microorganisms Results shown in Table I11 demonstrated that gentamicin was eluted from all the cylinders in sufficient quantities to inhibit growth
WELCH
688
7
28.. 27.-
26.25. 24-
23-
/
22-
21
.-
20-
6.-
/
f
5.-
gentamicin per cylinder).
of S. epidermidis and S. aureus. However, cylinders prepared with 0.5 g gentamicin sulfate per 10 g PMMA powder were required to produce a zone of inhibition against E. coli that remained without being overgrown by the bacteria after 5 days. The same concentration inhibited growth of P. aeruginosa, but this organism grew into the zone of inhibition after 5 days.
ANTIBIOTICS IN ACRYLIC BONE CEMENT
689
TABLE I1 Effect of Volume of Fluid on Elution of Gentamicin Calculated Amount Gentamicin Gentamicin Gentamicin in Total Weight of per Calculated Cylinder Recovery Volume Cylinder per ml Volume of PBS (ml) PBS (Fg) PBS (pg) (mg) (ms) (%) 2 days 7 days
2.0 10.0 1.5 9.5
105 49 370 117
210 490 555 1111.5
391.99 392.47 391.99 392.47
8.57 8.58 8.57 8.58
2.45 5.71 6.48 12.95
Efficacy in Broth Cultures The effect of cylinders containing cephalothin on broth cultures of S. aureus and E. coli is shown in Figure 4. Control cultures without antibiotics multiplied in a normal exponential fashion. There was a marked reduction in the total number of S. aureus after addition of a cylinder containing 1.32 mg cephalothin which was even more pronounced a t 20 hr. Cylinders containing 12.6 mg cephalothin or greater were bactericidal within 20 hr. Results obtained using S. epidermidis are not shown, since they so closely paralleled those obtained with S. aureus. The total number of E. coli was reduced by cylinders containing 1.32 mg cephalothin, but after 4 hr the bacteria multiplied exponentially. Cylinders containing 12.6 mg cephalothin reduced the total number and inhibited multiplication but were not completely bactericidal. Cylinders containing 28.15 mg were bactericidal to E. coli within 20 hr. P. aeruginosa was not tested, because it was not sensitive to cephalothin. The effect of cylinders containing gentamicin on staphylococci is shown in Figure 5. There was a marked reduction in bacteria after 4 hr contact with cylinders containing 0.85 mg gentamicin, but after 4-6 hr they multiplied exponentially. Cylinders containing larger amounts of gentamicin were bactericidal in 45 min t o 4 hr. Similar results were obtained with E. coli and P. aeruginosa (Fig. 6) except that cylinders containing 0.85 mg gentamicin were not effective in controlling growth of these organisms. However, cylinders containing larger amounts were bactericidal in 4-6 hr.
WELCH
690
21.-
/
20-1918-
I
8- I
7-
1
A combination of cephalothin and gentamicin incorporated in the bone cement produced very nearly the same effect on P. aeruginosa as did cylinders containing gentamicin alone. Results obtained using cylinders prepared with a combination of the antibiotics in cultures containing equal numbers of S. epidermidis and P. aeruginosa (Fig. 7 )demonstrated that the staphylococcuswas slightly more sensitive to a combination of the antibiotics than when the antibiotics were used individually. There was no appreciable difference in inhibition of the pseudomonas. Similar results were obtained by using a combination of S. aureus and P. aeruginosa (Fig. 8 ) in that the staphylococcus was slightly more sensitive to a combination of the antibiotics. However, the pseudomonas was not completely inhibited by a combination of 4.2 mg gentamicin plus 7.0 mg cephalothin per cylinder, as it had been when S. epidermidis and P. aeruginosa were used in the same culture.
Microorganism
P. aeruginosa
E. coli
S. aureus
S. epidermidis 0.05 0.5 Cephalothin 0.5 Gentamicin sulfate 0.005 0.05 0.5 Cephalothin 0.5 Gentamicin sulfate 0.005 0.05 0.5 Cephalothin 0.05 Gentamicin Sulfate 0.005 0.05 0.5 Cephalothin 0.5
Gentamicin sulfate 0.005
Grams Antibiotic per 10 g PMMA Powder
2
1
2
...
5
...
None None 25 X complete None
... ...
... 14 X 17
1
...
3 4
...
...
None 14 X 22 30X complete
32 X 28
...
... 2 3
1
4 2
... ...
None
None None None
14 X 17
None None 30 X 40
32 X 28
29 X 39 Complete
17 X 22
...
Complete 17 X 22 29 X 39 Complete
Complete
...
1
45 X complete Complete
3
2 1
22 x 22
Final Zone of Inhibition (mm) (Ends X Length)
45 X complete Complete
...
22 x 22
2
Day a t Which Daya t Which Maximum Distance from Maximum Zone Cylinder to Zone of Inhibition Edge of Zone (mm) of Inhibition Decreased WasObserved (Ends X Length)
TABLE 111
3
a
k5
Q
3
0
m
z
5
P Q
2
8
2 2 0 2
i2
692
WELCH
-S -aureus
HOURS
HOURS
Fig. 4. Effect of Surgical Simplex containing Keflin on broth cultures of 5'. aut-ezhs and E. coli. ( 0 )Controls without antibiotic. Cylinders prepared with 10 g PMMA powder and (A)0.05 g Keflin (1.32 mg Keflin per cylinder), ( W ) 0.5 g (12.6 mg per cyl1.0 g (18.15 mg per cylinder). inder), and (0)
DISCUSSION AND CONCLUSION Antibiotics incorporated into PMMA bone cement for prophylactic use in arthroplastic joint replacement should have the following characteristics: 1. Effective against a wide variety of bacteria 2. Show a rapid release from the acrylic cement soon after transplantation 3. Continue to be released from the cement for a long period of time in effective concentrations 4. Be nontoxic and nonallergenic under these conditions 5. Be effective in small doses 6. Have a marked bactericidal action 7. Physicochemical properties should include: Be highly soluble in aqueous solutions Diffuse well from the poly(methy1) methacrylate Be thermostable under conditions required for polymerization Be stable for prolonged periods at 37°C
ANTIBIOTICS IN ACRYLIC BONE CEMENT II\
693
-S -aureus
5 epidermidis
HOURS
HOURS
Fig. 5. Effect of Surgical Simplex containing gentamicin on broth cultures of bacteria. ( 0 ) Controls without antibiotic. Cylinders prepared with 10 g PMMA powder and (A)0.05 g gentamicin sulfate (0.85 mg active gentamicin per cylinder), (H) 0.5 g (8.38 mg gentamicin per cylinder), and (0) 1.0 g (16.88 mg gentamicin per cylinder).
Have a low bulk volume 8. Produce little or no change in mechanical properties of the cement. Gentamicin and cephalothin were selected for this study because they do have many of these properties and are commonly used in orthopedic surgery for irrigation of the wound during surgery or parenterally as prophylaxis against infection. In our experiments, the powder form of the antibiotics was mixed thoroughly with the polymer prior to addition of the liquid monomer. In preliminary experiments we attempted to use aqueous solutions of antibiotics mixed with both the powder and the liquid monomer and also attempted to add powdered antibiotics to the monomer. None of these alternate procedures proved satisfactory to obtain a homogeneous mixture. Gentamicin powder is not packaged aseptically for clinical use in this country. However, it has been shown that little or no loss of potency occurred after sterilization of the powder by exposure to ethylene oxide gas.1°
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I
LCO"
P aaruginosa
HOURS
HOURS
Fig. 6. Effect of Surgical Simplex containing gentamicin on broth cultures of bacteria. (@) Controls without antibiotic. Cylinders prepared with 10 g PMMA powder and ( A ) 0.05 g gentamicin sulfate (0.85 mg active gentamicin per cylinder), (m) 0.5 g (8.38 mg gentamicin per cylinder), and (0) 1.0 g (16.88 mg gentamicin per cylinder).
Results of the quantitative elution of both cephalothin and gentamicin from cylinders of Surgical Simplex cement demonstrated that the largest amount of antibiotic was eluted within the first 24-48 hr after cylinders were placed in PBS solution. The antibiotic on, or very near, the surface diffused into solution within a short period of time. Subsequently, increasingly smaller amounts were eluted with time as the solution penetrated into the pores of the cement. The observation of the initial elution of large amounts of antibiotics from the Surgical Simplex cement is in agreement with previous results obtained by using a variety of antibiotics and acrylic cements.3,5,6,8,9,11,12 Gentamicin is the one antibiotic that has been studied by most investigators, and there is considerable disagreement in the literature regarding elution rates. Hessert and Ruckdesche14reported release of 50% of the amount of gentamicin incorporated into Palacos R after 2 hr incubation and 80%after 24 hr. Chapman and Hadleyll stated that, when incubated in saline, there was a rapid leaching of the antibiotics from Surgical Simplex cement and 80% may be removed
ANTIBIOTICS IN ACRYLIC BONE CEMENT
S_ epidermidis __
695
and
P ceruginosa
.i
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Fig. 7. Effect of combination of antibiotics in Surgical Simplex on a combination of organisms in broth cultures. Cylinders prepared with 10 g PMMA powder and ( 0 ) P. aeruginosa 0.025 g gentamicin sulfate 0.025 g Keflin (0.42 mg gentamicin 0.7 mg Keflin per cylinder), (0) S. epidermidis response to concentrations listed above, ( A ) P. aeruginosa 0.25 gentamicin sulfate 0.25 g Keflin powder (4.2 mg gentamicin 7.0 mg Keflin per cylinder).
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during the first 48 hr and no activity could be detected after 7 days. However, they did use relatively small conical pellets (weight, 0.19 g) in 10 ml saline. Elson et al.9 also reported a larger quantity of gentamicin was released from Palacos than from CMW, Simplex, and Sulfix cement and over longer periods of time. Experimental conditions used by these investigators were comparable with ours, except they used a larger volume of eluting fluid (50 ml). They recovered less than 1 pg gentamicidml after 4 days from Simplex cement, and measurements were terminated after 25 days. Marks et aL8 reported a release rate of gentamicin such that less than 0.4pg was recovered after 18 days from Surgical Simplex cement, whereas they were able
-S -oureus
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Fig. 8. Effect of combination of antibiotics in Surgical Simplex on a combination of organisms in broth culture. Cylinders prepared with 10 g PMMA powder and ( 0 ) 0.025 g gentamicin sulfate 0.025 g Keflin (0.4 mg gentamicin 0.7 mg Keflin per cylinder), ( A ) 0.25 g gentamicin sulfate 0.25 g Keflin (4.2 mg gentamicin 7.0 mg Keflin per cylinder), (m) 0.5 g gentamicin sulfate 0.5 g Keflin (8.24 mg gentamicin 13.9 mg Keflin per cylinder), (0) 1.0 g gentamicin sulfate 1.0 g Keflin (15.9 mg gentamicin 28.1 mg Keflin per cylinder).
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to measure release of a comparable amount from Palacos R for 61 days. However, these authors used disks 12 mm in diameter and failed to mention thickness and weight. Also, they measured zones of inhibition of the disks placed directly on seeded agar plates and compared with standard zones of inhibition produced by antibiotics on 6-mm filter-paper disks. Therefore, these results should not be compared with those obtained by using different techniques. In our tests, gentamicin was released from Surgical Simplex over a prolonged period of time. Amounts greater than 2 pglml were still being measured at 175 days, when the measurements were terminated. We also demonstrated the effect of volume of eluting fluid, surface area, and amount of antibiotic used. Although a greater amount of gentamicin was released per ml in a 2-ml volume of eluting fluid than in a 10-ml volume, the total amount released is greater than in the larger volume, which indicates a function of the diffusion rate. Gentamicin was eluted from the disks for a longer period of time than
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the cylinders when the same amount of antibiotic was used to prepare the different shaped pieces of bone cement. This was probably due to the larger surface area of the disks as compared with the cylinders. Also, antibiotics were eluted in greater amounts and over a longer period of time from cylinders prepared with larger amounts of antibiotics. Our results correlated more closely with those reported for Palacos R by Wahlig et al.5 He calculated the rate of release as varying from 2.5 to 17.5% after 20-22 weeks. In our experience a slightly higher percentage (22.7-26.3%) was released in the same time period. Our cylinders were smaller, had less surface area, and weighed less than those used by Wahlig et al. The greater percentage of elution from our cylinders could be due to the properties of the polymer itself, the larger amount of antibiotic per unit, and the frequency with which we changed the elution fluid. We changed the fluid three times per week after the first 10 days, whereas Wahlig et al. changed the fluid weekly and then monthly. There is a fluctuation in concentration of the antibiotic in solution as a result of every change in fluid during the incubation period. Concentration changes take place during the time the antibiotic-bone cement remains in contact with the fluid, and a new diffusion process starts each time there is a change of the fluid. Wahlig et al. also stated that the release of antibiotic would be much larger when the fluid is changed more often and, therefore, would be greater in the bloodstream with a continuous removal of released antibiotic. To date, there have been no previous reports of elution rates of cephalothin from acrylic bone cement. The pattern of elution is similar to that of gentamicin, in that the largest amount was eluted within the first 24 hr and then increasingly smaller amounts were eluted with time. Also, the largest amount of cephalothin eluted was from cylinders prepared with the largest amount of antibiotic. However, in contrast, only approximately one-third as much cephalothin was eluted from the cylinders as compared with gentamicin. Also, the level of cephalothin fell below 2 pg/ml in 21-29 days. This may be due, at least in part, to the relative instability of cephalothin in solution at 37OC. Standard cephalothin solutions in low concentrations lost almost all activity within 24 hr and in high concentrations within 4 days, whereas gentamicin solutions retain more than 80% activity at 37OC for at least 15 days. Also, it is possible that gentamicin is more stable to heat of polymerization than cephalothin.
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However, from our recovery rates we may conclude that cephalothin is stable for a period of time when combined with the Surgical Simplex. As the solution comes in contact, the cephalothin diffuses out into solution and then gradually loses activity. Nevertheless, we were able to measure the release of considerable quantities of cephalothin. In these studies, the measurements were arbitrarily terminated when the amount of antibiotic in solution was less than 2 pg/ml, although lower quantities can be measured by the technique used. It was felt that measuring extremely low levels of antibiotic would not be of any practical significance. Results obtained by placing cylinders containing antibiotics in agar plates seeded with microorganisms demonstrated that antibiotics were eluted in sufficient quantities to inhibit the microorganisms. This test does not permit strictly quantitative interpretations and does not indicate if this is a bacteriostatic or bactericidal effect. However, reports on efficacy by other investigators6V8J0-l2are based on this test. It cannot be equated with the Bauer-Kirby test14 for sensitivity of organisms to antibiotics, because the amounts of antibiotics used and shapes of the cement used are not comparable with the standard filter-paper disks used in the Bauer-Kirby procedure. Placing cylinders containing antibiotics in broth cultures of microorganisms permitted a more quantitative determination of the bactericidal effect of the antibiotics. The numbers of organisms used for this test exceeded the number that would reasonably be expected to contaminate a wound. Our results demonstrated that gentamicin combined with Surgical Simplex was bactericidal to the organism used within 45 min to 6 hr, depending on the type of organism and concentration of gentamicin. Cephalothin was bactericidal to the sensitive organisms within 20 hr, but not to the resistant P. aeruginosa. Again, this was dependent on the organism and concentration of cephalothin. Chapman and Hadleyll added pellets containing cephalothin and gentamicin sulfate to broth cultures of bacteria and reported 100%inhibition of bacterial growth after 24 hr incubation. Hill et al.17 also incubated disks of antibiotic-containing cement in peptone water containing bacteria and demonstrated inhibition of growth for various periods of time, depending on the type of antibiotic and bacteria. However, none of these reports indicated approximate concentrations of bacteria, and it is well known that sensitivity to antibiotics is dependent in part on numbers of organisms present. Since P. aeruginosa is so frequently very difficult to control with
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antibiotics, the effect of using a combination of antibiotics in bone cement was tested. This combination produced very nearly the same results as using gentamicin alone. A combination of antibiotics was also tested on a combination of organisms, since infections that may occur are not necessarily due to one single type of organism. When used in combination with P. aeruginosa, S. epidermidis and S. aureus were both slightly more sensitive to a combination of the antibiotics than when the antibiotics were used individually. However, there was no marked difference with P. aeruginosa. Combinations of two or more unrelated antibiotics are sometimes used in clinical medicine to prevent emergence of resistant forms, and it is essential that both antibiotics be present at inhibitory levels at the site of infection. It is possible that a more suitable combination of antibiotics for use in bone cement could be found. These experiments indicated that gentamicin and cephalothin combined with Surgical Simplex bone cement may be useful for certain orthopedic conditions. An initial high release of antibiotics followed by a longer rate of release is desirable. The initial high release would be effective in controlling bacteria found near the site of infection. Clinically, infections that occur following arthroplastic hip replacements are most often found at the distal portion of the prosthesis. Persistence of pathogenic bacteria can be responsible for relapse if therapy is discontinued too early, and, therefore, a prolonged release of the antibiotic is desirable. However, these results should not be interpreted to an in vivo situation, since the amount of fluid present influences the amount of antibiotic eluted from the bone cement. In a closed system in uitro localized microorganisms are in constant contact with the eluted antibiotics. Nevertheless, these experiments demonstrated that sufficient quantities of antibiotics are eluted to have bactericidal effects in a relatively short time. This research was supported in full by USPHS Research Grant 1R01 AM 18794from the Department of Health, Education and Welfare, National Institutes of Health, Bethesda, Md. The author wishes to thank Howmedica,Inc., for supplying the Surgical Simplex, Schering Corp. for the gentamicin, and Eli Lilly & Co. for the cephalothin. I also thank Richard Stutte and Deborah Hassler for technical assistance.
References 1. R. Koschmieder, W. Ritzerfeld, and H. Kleymann, Z . Orthop., 111,244 (1973). 2. G. Hunter and D. Dandy, J . Bone Jt. Surg., 59-B, 293 (1977). 3. H. W. Buchholz and H. Engelbrecht, Chirurg, 41,511 (1970). 4. G. R. Hessert and G. Ruckdeschel, Arch. Orthop. Unfall-Chir.,68,249 (1970).
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5. H. Wahlig, W. Hameister, and A. Grieben, Langenbecks Arch. Chir., 331, 169 (1972). 6. W. P. Gartenmann, H. Brodhage, J. Zippel, and L. Badeira, in Arthroplasy of the Hip, G. Chapchal, Ed., Intercontinental Medical Book Corp., New York, 1973, p. 21. 7. G. Ruckdeschel, G. R. Hessert, and T. Schollhammer, Arch. Orthop. Unfall-Chir., 74,291 (1973). 8. K. E. Marks, C. L. Nelson, and E. P. Lautenschlager, J . Bone Jt. Surg., 58-A, 358 (1976). 9. R. A. Elson, A. E. Jephcott, D. B. McGechie, and D. Verettas, J . Bone Jt. Surg., 59-B, 200 (1977). 10. P. D. Levin, J . Bone Jt. Surg., 57-B, 234 (1975). 11. M. W. Chapman and W. K. Hadley, J . Bone J t . Surg., 58-A, 76 (1976). 12. A. L. Rosenthal, J. Znt. Med. Res., 4,296 (1976). 13, L. D. Sabath and J. M. Matsen in Manual of Clinical Microbiology, E. H. Lennette, E. H. Spaulding, and J. P. Truant, Eds., American Society for Microbiology, Washington, D.C., 1974, p. 428. 14. A. W. Bauer, W. M. Kirby, J. C. Sherris, and M. Turck, Am. J . Clin. Pathol., 45, 493 (1966). 15. J. A. Washington I1 and A. L. Barry in Manual of Clinical Microbiology, E. H. Lennette, E. H. Spaulding, and J. P. Truant, Eds., American Society for Microbiology, Washington, D.C., 1974, p. 410. 16. A. L. Barry and L. D. Sabath, in Manual of Clinical Microbiology, E. H. Lennette, E. H. Spaulding, and J. P. Truant, Eds., American Society for Microbiology, Washington, D.C., 1974, p. 431. 17. J. Hill, L. Klenerman, S. Trustey, and R. Blowers, J . Bone Jt. Surg., 59-B, 197 (1977).
Received August 26,1977 Revised January 4,1978
