Local Antibiotic Delivery with OsteoSet, DBX, and Collagraft

Transcription

1 CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Number 451, pp Lippincott Williams & Wilkins Local Antibiotic Delivery with OsteoSet, DBX, and Collagraft Andras Heijink, MD * ; Michael J. Yaszemski, MD, PhD *, ; Robin Patel, MD, ; Mark S. Rouse, BS ; David G. Lewallen, MD * ; and Arlen D. Hanssen, MD * Biodegradable local antibiotic delivery systems have gained interest for prophylaxis and treatment of musculoskeletal infections. We studied the biodegradable materials Osteo- Set, DBX and Collagraft for local delivery of vancomycin and gentamicin in vitro. We determined the antimicrobial activity of vancomycin and gentamicin after mixing with each biodegradable material and determined the release of each antimicrobial from each material in an intermittent flow chamber. Antimicrobial activity was expressed as percent of antimicrobial loaded into each sample that was detected; antimicrobial release was expressed as concentration (µg/ml) after timed intervals of chamber flow, peak concentration, area under the curve and percent antimicrobial recovered. Activity of vancomycin after mixing with Osteo- Set, DBX and Collagraft was > 73%. Activity of gentamicin after mixing with DBX was 100%; after mixing with OsteoSet and Collagraft it was reduced to < 61%. AUC 0 48hrs of vancomycin was 469, 426 and 432 µg hr/ml, and the AUC 0 48hrs of gentamicin was 368, 306 and 301 µg hr/ml after release from OsteoSet, DBX, and Collagraft, respectively. Recovered percentages of vancomycin were 39%, 11% and 25%, and recovered percentages of gentamicin were 39%, 9% and 23% after release from OsteoSet, DBX, and Collagraft, respectively. OsteoSet, DBX and Collagraft may be suitable for local delivery of vancomycin and gentamicin. Optimal prophylaxis and treatment of orthopedic infections remain controversial. Local antibiotic delivery systems, introduced by Buchholz et al, 3 have gained great From the * Department of Orthopedic Surgery; the Department of Biomedical Engineering; the Division of Infectious Diseases, Department of Medicine; and the Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN. Each of the authors certifies that he or she has no commercial associations that might pose a conflict of interest in connection with the submitted manuscript. Correspondence to: Arlen D. Hanssen, MD, Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN Phone: ; Fax: ; hanssen.arlen@mayo.edu. DOI: /01.blo interest, as high local concentrations can be achieved while the risk of systemic toxicity is minimized. High concentrations are crucial to the treatment of avascular areas, resistant microorganisms, and sessile microorganisms (ie, biofilms). 5,6 Polymethylmethacrylate (PMMA) is commonly used in clinical orthopaedic practice for local antibiotic delivery. 14 However, a large percentage of the incorporated antibiotic is not released and surgical removal may be recommended after antimicrobial treatment has ceased. 16 In situations where no load bearing is required, biodegradable materials may be superior to PMMA because most of the antibiotic loaded into the material could be released and it would not have to be surgically removed. Various biodegradable materials are available or being developed as local antibiotic delivery systems; some available materials are being used for local antibiotic delivery as a Clinician Directed Application. 14 We report in vitro observations on compatibility with and release of vancomycin and gentamicin from the biodegradable materials OsteoSet, DBX, and Collagraft. MATERIALS AND METHODS OsteoSet (Wright Medical, Arlington, TN) is medical grade calcium-sulfate; it is available as a powder that, after it has been mixed to a paste with sterile water, sets over time. DBX (Musculoskeletal Transplant Foundation, Edison, NJ) is composed of morselized, demineralized bone and hyaluronic acid; it is available as a paste. Collagraft (Zimmer Inc, Warsaw, IN) is composed of hydroxyapatite, tricalcium phosphate and fibrillar collagen; it is available in sterile strips. Initially, we determined the activity of vancomycin and gentamicin after mixing with OsteoSet, DBX and Collagraft. OsteoSet beads, 6 mm in diameter, were prepared by adding vancomycin hydrochloride (USP, Novaplus, Harrisburg, PA) or gentamicin sulfate (USP, Hawkins, Inc, Minneapolis, MN) to the powder component of OsteoSet to a concentration of 7.5% (weight/weight, w/w) of powder and liquid component combined. Subsequently the liquid component was added according 29

2 30 Heijink et al Clinical Orthopaedics and Related Research to the manufacturer s recommendations. DBX-putty was mixed with 7.5% (w/w) vancomycin or gentamicin. An arbitrary amount of DBX-putty was weighed, and the appropriate amounts of vancomycin and gentamicin were added based on weight. The samples were slightly larger than the OsteoSet beads. Collagraft strips were ground to a fine powder which was then mixed with sterile H 2 O 1:1 (w/w) to obtain a suspension that had a paste-like consistency. Sample size was similar to the OsteoSet beads. Vancomycin or gentamicin was added to the paste to a concentration of 7.5% (w/w). Polymethylmethacrylate (PMMA) (Surgical Simplex, Limerick, Ireland) beads, 3 mm in diameter, were prepared by adding vancomycin or gentamicin to the powder component of PMMA to a concentration of 7.5% (w/w). Subsequently the liquid component was added according to the manufacturer s recommendations. After preparation, all antibiotic-loaded samples were allowed to set for 2 hours at room temperature. Twelve beads (OsteoSet and PMMA) or samples (Collagraft and DBX-putty ) were independently prepared. After preparation, each specimen was homogenized in 5 ml of sterile saline using a homogenizer (Ultra- Turrax T 25 basic, IKA Werke Staufen, Germany) at rpm to yield 50 m particles. Subsequently, the homogenate was assayed in triplicate for antimicrobial concentration by antimicrobial assay. Vancomycin and gentamicin assays were performed using tryptic soy agar seeded with Bacillus subtilis (ATCC 6633) and Enterobacter agglomerans (IDRL 3860) L of five standard solutions (range, 2 32 g/ml) or test sample was pipetted onto 6 mm filter paper discs and placed on the surface of the seeded agar plates. After incubation, zones of inhibition around the filter paper discs were measured and the concentrations derived using linear regression. The lower limit of detection was 2 g/ml of vancomycin and 2 g/ml of gentamicin. The mean percent active drug recovered by assay was calculated from the amount of drug loaded into each sample and averaged for the twelve samples of each combination. This percentage of recovered antibiotic activity is a measure of compatibility of vancomycin or gentamicin with either OsteoSet, DBX or Collagraft ; a percentage of 100% indicates complete biocompatibility, with smaller percentages suggesting some degree of loss of antimicrobial activity. The mean percentage of vancomycin or gentamicin activity after mixing with the three biodegradable materials was compared using two-way analysis of variance (ANOVA). Post hoc comparisons, where indicated, were made using the Student s t-test. Significance was set at the p < 0.05 level. Subsequently, the release kinetics of vancomycin and gentamicin from OsteoSet, DBX and Collagraft were determined in an intermittent flow chamber. Three beads (OsteoSet ) or samples (Collagraft and DBX ) of each formulation were independently loaded with 7.5% vancomycin or gentamicin as described, weighed and placed in a reservoir containing 1 ml of sterile water. This reservoir was placed in an incubator (Barnstead International, Thermolyne, Dubuque, IA) to maintain a 37ºC temperature. Two hundred microliter samples were collected hourly for the first 24 hours and every 2 hours up to 48 hours. Each time a 200 L sample was collected, 200 L of sterile H 2 O was pipetted into the chamber to replace the sampled volume and maintain the volume in the chamber constant at 1 ml. Subsequently, the eluates were assayed in triplicate for antimicrobial concentration by microbiological assay; the average of the three samples was calculated for each formulation. The concentration of vancomycin and gentamicin in the eluate at each hour was plotted against time to profile the release of vancomycin or gentamicin from OsteoSet, DBX and Collagraft. The peak concentration, time of peak concentration, duration of detection, area under the curve (AUC) and recovered percentage of antibiotic were determined from the release data for each sample. RESULTS Vancomycin and gentamicin were detectable after mixing with all three biodegradable materials (Tables 1 and 2). The activity of vancomycin after mixing with Collagraft was greater (p < 0.05) than the activity of vancomycin after mixing with OsteoSet and DBX ; the activity of vancomycin after mixing with OsteoSet and DBX were not different. The activity of gentamicin after mixing with DBX was greater (p < 0.05) than the activity of gentamicin after mixing with Collagraft ; and the activity of gentamicin after mixing with Collagraft was in turn greater (p < 0.05) than the activity of gentamicin after mixing with OsteoSet. The release of vancomycin and gentamicin from all studied materials showed a rapid initial release followed by a slower sustained release (Figs 1A F, Table 3). The peak concentration in the release profile occurred within the first 5 hours for all formulations. The highest peak concentration of vancomycin was observed with release from Collagraft ; the highest peak concentration of gentamicin was observed with release from OsteoSet. The duration of vancomycin and gentamicin release was longest from DBX. OsteoSet showed the largest AUC for vancomycin and gentamicin. The largest percentage of vancomycin and gentamicin was recovered after release from OsteoSet. Variation between samples within each formulation was small, as indicated by small standard deviations. The exception was release of vancomycin from Collagraft, where larger standard deviations were observed due to the lower concentrations measured with one of three samples. TABLE 1. Activity of Vancomycin After Mixing with the Studied Materials Mean (±s.d.) (percent) 95% CI OsteoSet 81.0 (±16.4) DBX 73.6 (±13.6) Collagraft 98.1 (±20.5)

3 Number 451 October 2006 Local Antibiotic Delivery Systems 31 TABLE 2. Activity of Gentamicin After Mixing with the Studied Materials DISCUSSION Mean (±s.d.) (percent) 95% CI OsteoSet 28.8 (±20.6) DBX (±17.8) Collagraft 61.0 (±12.9) Treatment of musculoskeletal infections classically involves surgical débridement of necrotic tissue, followed by a 4 to 6 weeks course of parenteral antibiotics. To obtain adequate antibiotic concentrations at the site of infection, high serum concentrations have to be obtained, which carries the risk of systemic toxicity. Local antibiotic delivery systems, first introduced by Buchholz et al, 3 have become increasingly popular for treatment and prophylaxis of orthopedic infections. Such systems yield local antibiotic concentrations higher than possible with parenteral antimicrobial administration, while reducing the risk of systemic side effects; they can either be used to supplement or replace the use of systemic antibiotics. 5,9,12,18,19 High local antibiotic concentrations are especially beneficial in the treatment of relatively avascular areas, and organisms resistant to antibiotic concentrations obtained with parenteral administration, including organisms in biofilms. 6,8,14 Fig 1A F. This series of graphs shows the vancomycin concentration (µg/ml) with standard deviation in eluate sample after release from (A) OsteoSet, (B) DBX and (C) Collagraft ; and the gentamicin concentration (µg/ml) with standard deviation in eluate sample after release from (D) OsteoSet, (E) DBX and (F) Collagraft.

4 32 Heijink et al Clinical Orthopaedics and Related Research TABLE 3. Combination Parameters of Release for the Studied Combinations Peak Concentration (±s.d.) (µg/ml) Time of Peak Value (±s.d.) (hours) Under Lower Detection Limit (±s.d.) (hours) AUC (±s.d.) (µg/ml/hour) Recovered (±s.d.) (percent) DBX /Vancomycin 31.5 (±4.0) 3.7 (±1.5) 43.3 (±14.2) (±19.3) 10.9 (±2.7) Collagraft /Vancomycin 50.2 (±24.2) 4.3 (±1.5) 23.7 (±6.0) (±28.4) 24.8 (±10.6) OsteoSet /Vancomycin 49.2 (±8.3) 3.3 (±1.2) 27.7 (±0.6) (±29.1) 38.9 (±8.5) DBX /Gentamicin 35.3 (±6.6) 3.7 (±0.6) 22.0 (±1.7) (±47.8) 8.6 (±2.9) Collagraft /Gentamicin 63.1 (±19.0) 2.0 (±0.0) 19.0 (±0.0) (±98.1) 22.8 (±14.0) OsteoSet /Gentamicin 80.9 (±7.5) 2.0 (±0.0) 19.7 (±0.6) (±38.1) 39.0 (±5.0) We acknowledge certain limitations of this study. Antimicrobial plate assays have been reported to show 3 17% variability. 13 This likely explains why the amount of gentamicin recovered from DBX and PMMA samples was measured as greater than 100% of the amount initially incorporated into the samples. We are not aware of any potentiating interactions between vancomycin or gentamicin and any of the carriers tested. We chose to take a 200 L sample to assess release kinetics. This is an arbitrary amount, and arguments could be made to take smaller or larger samples. Since the tissue fluid flow in vivo is unknown, we do not know the extent to which this data models in situ release of vancomycin or gentamicin from the materials studied. Our interpretation of this data is relative only to other similar in vitro test systems. Due to technical reasons the size of the samples, and hence the surface to volume ratio, was not equal for each combination. This could influence the release rate, although it would not change the release profile. Successful materials for local antibiotic delivery are biocompatible, do not inactivate the antibiotic, and release the antibiotic for a sufficient period of time. As local antibiotic delivery is often a Clinician Directed Application (CDA), an additional requirement is the material be easy for the surgeon to manipulate. Since the introduction of antibiotic-loaded acrylic cement by Buchholz et al, 3 PMMA has become the standard for local antibiotic delivery. However, PMMA has some disadvantages as a carrier material. During the polymerization process temperature rises up to 100ºC, limiting the incorporation of heat-labile antibiotics. 8 Most of the loaded antibiotic remains trapped in the PMMA. 2,16 Antibiotic release decreases fairly rapidly. PMMA is not biodegradable and functions as any other foreign body; after antibiotic release has ceased it is thus susceptible to bacterial colonization and consequently often has to be removed. 5,12 In cases where no load bearing or spacing is required, biodegradable materials for local antibiotic delivery are an attractive alternative for PMMA. 1,5,14,17 Biodegradable materials do not require removal after antibiotic release has ceased. 14 Since characteristics of the carrier material largely determine the rate of elution, the release profile can be optimized according to the application, either by choice of material, or, in the case of synthetic polymers, by deliberately controlling material properties. 1,11,14 Some biodegradable materials have osseoinductive properties, or can be loaded with bone morphogenetic proteins (BMP-2, BMP-7) to introduce osseoinductivity. 4 Many biodegradable polymers are being developed for local antibiotic delivery; only a few have been proven safe for human use. 5,10,11 We studied three FDA approved biodegradable polymers currently used in orthopedic surgery for interaction with and release of vancomycin and gentamicin in vitro. Our data demonstrate activity of vancomycin and gentamicin after mixing with OsteoSet, DBX and Collagraft is variable. This implies biodegradable materials as local antibiotic delivery systems should be tested for interaction with particular antibiotics prior to clinical application. The activity of vancomycin after polymerizing with PMMA was 90.7% (±6.7%) and the activity of gentamicin after polymerizing with PMMA was 103% (±12.5%). The reduction of activity of gentamicin after mixing with OsteoSet might be inherent to our study design, rather than inactivation of gentamicin. Aminoglycosides inhibit bacterial protein synthesis by binding to the bacterial 30S ribosomal subunit. 7,15 Aminoglycosides are polycationic compounds that are water soluble and poorly lipid soluble; passive diffusion through membranes is limited. 7,15 Aminoglycosides diffuse through aqueous channels in the outer membrane of gram-negative organisms. Subsequent transport across the cytoplasmic (inner) membrane is energy dependent. 7,15 This phase of transport is rate limiting and can be inhibited by divalent cations. 7 Because OsteoSet is CaSO 4 hemihydrate and degrades into Ca 2+ and SO 4 2, the concentration of Ca 2+ in eluates after grinding OsteoSet beads may have been sufficiently high to inhibit gentamicin uptake by the bacterium used in our bioassay. To what extent this might occur in vivo is not known. The release kinetics observed with all three biodegradable materials appeared to be similar to the release patterns

5 Number 451 October 2006 Local Antibiotic Delivery Systems 33 of vancomycin and gentamicin from PMMA. 9,16,18 All samples appeared grossly intact at the end of 48 hours of sampling. This suggests additional amounts of vancomycin or gentamicin might be released with further degradation of the carrier material. Although the literature is replete with in vitro and in vivo antibiotic release studies, detailed comparison is difficult because of differences in experimental design and the lack of understanding of how in vitro data translate to the in vivo situation. There is a need for in vivo models of local antibiotic release. Interaction with and release of vancomycin and gentamicin from the biodegradable materials OsteoSet, DBX and Collagraft was studied. Activity of vancomycin and gentamicin after mixing with OsteoSet, DBX and Collagraft was variable. Vancomycin and gentamicin eluted from OsteoSet, DBX and Collagraft with a similar profile. OsteoSet, DBX and Collagraft may be suitable for local delivery of vancomycin and gentamicin. Acknowledgments The authors wish to thank Wright Medical (Arlington, TN) for providing OsteoSet ; the Musculoskeletal Transplant Foundation (Edison, NJ) for providing DBX-putty ; and Zimmer Inc. (Warsaw, IN) for providing Collagraft. References 1. Ambrose CG, Clyburn TA, Louden K, Joseph J, Wright J, Gulati P, Gogola GR, Mikos AG. Effective treatment of osteomyelitis with biodegradable microspheres in a rabbit model. Clin Orthop Relat Res. 2004;421: Anguita-Alonso P, Rouse MS, Piper KE, Jacofsky DJ, Osmon DR, Patel R. Comparative study of antimicrobial release kinetics from polymethylmethacrylate. Clin Orthop Relat Res. 2006;445: Buchholz HW, Elson RA, Engelbrecht E, Lodenkamper H, Rottger J, Siegel A. Management of deep infection of total hip replacement. J Bone Joint Surg Br. 1981;63: Geiger M, Li RH, Friess W. Collagen sponges for bone regeneration with rhbmp-2. Adv Drug Deliv Rev. 2003;55: Hanssen AD. Local antibiotic delivery vehicles in the treatment of musculoskeletal infection. Clin Orthop Relat Res. 2005;437: Hanssen AD, Osmon DR, Patel R. Local antibiotic delivery systems: Where are we and where are we going? Clin Orthop Relat Res. 2005;437: Hardman JG, Limbird LE, Gilman AG, eds. Goodman & Gilman s The Pharmacological Basis of Therapeutics, 10th ed. New York, NY: McGraw-Hill; Henry SL, Galloway KP. Local antibacterial therapy for the management of orthopaedic infections. Pharmacokinetic considerations. Clin Pharmacokinet. 1995;29: Hoff SF, Fitzgerald RH Jr, Kelly PJ. The depot administration of penicillin G and gentamicin in acrylic bone cement. J Bone Joint Surg Am. 1981;63: Holtom PD, Patzakis MJ. Newer methods of antimicrobial delivery for bone and joint infections. Instr Course Lect. 2003;52: Kanellakopoulou K, Giamarellos-Bourboulis EJ. Carrier systems for the local delivery of antibiotics in bone infections. Drugs. 2000; 59: Klemm KW. Antibiotic bead chains. Clin Orthop Relat Res. 1993; 295: Lorian V. Antibiotics in Laboratory Medicine, 4th ed. Baltimore, MA: Williams & Wilkins; Nelson CL. The current status of material used for depot delivery of drugs. Clin Orthop Relat Res. 2004;427: Nightingale CH, Murakawa T, Ambrose PG. Antimicrobial Pharmacodynamics in Theory and Clinical Practice, 1st ed. New York: Marcel Dekker, Inc; Perry AC, Rouse MS, Khaliq Y, Piper KE, Hanssen AD, Osmon DR, Steckelberg JM, Patel R. Antimicrobial release kinetics from polymethylmethacrylate in a novel continuous flow chamber. Clin Orthop Relat Res. 2002;403: Rutledge B, Huyette D, Day D, Anglen J. Treatment of osteomyelitis with local antibiotics delivered via bioabsorbable polymer. Clin Orthop Relat Res. 2003;411: Wahlig H, Dingeldein E, Bergmann R, Reuss K. The release of gentamicin from polymethylmethacrylate beads: an experimental and pharmacokinetic study. J Bone Joint Surg Br. 1978;60: Walenkamp GH, Vree TB, van Rens TJ. Gentamicin-PMMA beads. Pharmacokinetic and nephrotoxicological study. Clin Orthop Relat Res. 1986;205: