Use of Xylitol To Enhance the Therapeutic Efficacy ofPolymethylmethacrylate-Based Antibiotic Therapy in Treatment ofChronic Osteomyelitis

Karen E. Beenken,a Laura Bradney,a William Bellamy,b Robert A. Skinner,c Sandra G. McLaren,c M. Johannes Gruenwald,c

Horace J. Spencer,d James K. Smith,e Warren O. Haggard,e and Mark S. Smeltzera,b,c

Department of Microbiology and Immunology,a Department of Pathology,b Department of Orthopaedic Surgery,c and Department of Biostatistics,d University ofArkansas for Medical Sciences, Little Rock, Arkansas, USA, and Department of Biomedical Engineering, University of Memphis, Memphis, Tennessee, USAe

Using a rabbit model of postsurgical osteomyelitis, we demonstrate that incorporation of xylitol into polymethylmethacry-late (PMMA) bone cement enhances the elution of daptomycin under in vivo conditions. We also demonstrate that this canbe correlated with an improved therapeutic outcome in the treatment of a chronic bone infection following surgicaldebridement.

Bone infections are intrinsically resistant to antimicrobial ther-apy even when caused by bacteria that are not clinically de-

fined as resistant to the preferred antibiotics. One contributingfactor to this is the difficulty in achieving a definitive diagnosisbefore the infection has progressed to a chronic stage, which ischaracterized by compromise of the local vasculature to an extentthat limits antibiotic delivery using systemic methods (10). A sec-ond contributing factor is formation of a bacterial biofilm, whichconfers a level of intrinsic antibiotic resistance that further com-promises the efficacy of conventional antibiotic therapy (4). Thesefactors necessitate an integrated therapeutic approach to chronicbone infection that includes long-term, systemic antimicrobialtherapy and surgical debridement. Debridement is often accom-panied by some form of local, matrix-based antibiotic delivery, theobjective being to achieve a concentration of antibiotic at the siteof infection that is high enough to overcome this intrinsic resis-tance without systemic toxicity (5, 16). Even then, the failure ratecan be as high as 10%, particularly in patients suffering from someform of underlying systemic compromise (5).

A number of delivery matrices have been employed for localantibiotic delivery, but one of the most common is polymethyl-methacrylate (PMMA). While widely used, PMMA has severaldisadvantages, including the fact that it is stable and can itselfprovide a substrate for bacterial colonization, particularly as thelevel of eluted antibiotic falls over time (6). A second disadvantageis that antibiotic recovery from PMMA is inefficient, thus makingit difficult to achieve and maintain the desired concentrations ofantibiotic for the desired length of time. One solution is to incor-porate large amounts of antibiotics either alone or in combinationwith each other (1), thus increasing the porosity of PMMA. How-ever, this can be prohibitively expensive, particularly given the sizeof the defect sometimes created by debridement and the need forfull coverage of the wound environment.

In an effort to overcome this problem, we have examined al-ternative methods of increasing the porosity of PMMA and en-hancing antibiotic elution. This is not a novel idea, and in fact,gentamicin-impregnated PMMA beads containing glycine for thispurpose are available in Europe under the trade name Septopal(Biomet Europe, Dordrecht, The Netherlands). However, wedemonstrated that xylitol enhances antibiotic elution fromPMMA to a greater extent than does glycine (13, 14), and xylitol

itself has been shown to have antibacterial properties and to pre-vent biofilm formation (2, 8, 9, 17, 20). In a rat model, xylitol wasalso found to have a positive impact on bone density (11, 18).

Based on these considerations, we carried out experimentsaimed at optimizing the use of xylitol as a means of enhancing theantibiotic elution profile from PMMA (21). We placed a primaryemphasis on daptomycin in these studies because we have foundthat it exhibits greater therapeutic efficacy than other commonlyused antibiotics, including vancomycin, in the context of an es-tablished biofilm. The specific parameters that we used to definean optimal elution profile were a maximum concentration at least100 times higher than the breakpoint MIC for daptomycin (�1.0�g/ml) and a sustained concentration, defined at 10 days postelu-tion under in vitro conditions, at least 5 times this breakpoint MIC(21). These experiments confirmed that incorporation of xylitolinto PMMA increases elution to the point that both of these pa-rameters can be achieved using an amount of daptomycin that isotherwise insufficient, particularly with respect to sustained elu-tion (21). However, these studies were limited to defining elutionprofiles in vitro, and it remains unknown whether this can becorrelated with an improved therapeutic outcome. To addressthis, we used a rabbit model of postsurgical bone infection (19) tocompare the therapeutic efficacies of PMMA formulations con-taining equivalent amounts of daptomycin with and withoutxylitol.

MATERIALS AND METHODSPMMA formulations and characterization. The PMMA (Palacos-R) for-mulations evaluated in these experiments included PMMA with no addi-tives, PMMA with xylitol (22 g per 40-g packet), PMMA with daptomycin(4 g per packet), and PMMA with both xylitol and daptomycin (22 and 4g, respectively). All formulations were prepared according to the manu-facturer’s instructions (Palacos-R; Zimmer Inc., Dover, OH) without ad-

Received 29 May 2012 Returned for modification 2 August 2012Accepted 22 August 2012

Published ahead of print 4 September 2012

Address correspondence to Mark S. Smeltzer, smeltzermarks@uams.edu.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.01127-12

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justment for the inclusion of additives. After mixing, PMMA pastes werepacked into a 1-ml tuberculin syringe with the Luer lock top cut off andallowed to harden. They were then extruded and cut to 10-mm lengthsusing a sterile saw. This resulted in a cylindrical pellet with dimensions of4 mm by 10 mm, which approximates the size of the excised radial seg-ment as detailed below.

To examine the impact of incorporating xylitol on the elution of dap-tomycin, in vitro elution comparisons were done by placing 1 pellet ofeach formulation into 4 ml of sterile phosphate-buffered saline (PBS).After overnight incubation at 37°C, the buffer was removed and replacedwith an equal volume of PBS. This was repeated for 7 consecutive days, atwhich time the amount of daptomycin present in each sample was deter-mined by high-pressure liquid chromatography (HPLC) using a C8 col-umn and an acidic acetonitrile-salt mobile phase (21). Pellets were alsorecovered after the in vivo treatment period (see below) and placed into 4ml PBS for 24 h at 37°C, and the amount of daptomycin in the eluate wasdetermined by HPLC. Representative pellets were also imaged by micro-computed tomography (microCT) to visually assess structural properties.

Rabbit osteomyelitis model. All in vivo experiments were done inaccordance with the policies of the Public Health Service (PHS) on careand use of laboratory animals, the Animal Welfare Act, and the NIH Guidefor the Care and Use of Laboratory Animals in an AAALAC (Association forAssessment and Accreditation of Laboratory Animal Care) International-accredited facility. All animal procedures were reviewed and approved bythe Institutional Animal Care and Use Committee of the University ofArkansas for Medical Sciences. Therapeutic efficacy was assessed using anestablished rabbit model of postsurgical osteomyelitis (7, 19). Briefly, a1-cm midradial segment was surgically excised from each of 24 male, NewZealand White rabbits. The infection was initiated in all rabbits by injec-tion into the intramedullary canal of 106 CFU of the Staphylococcus aureusosteomyelitis isolate UAMS-1 in a total volume of 10 �l of sterile phos-phate-buffered saline (PBS). The segment was then returned to the radialdefect in its original orientation, and the wound was closed. After 3 weeks,radiographs were obtained from all rabbits prior to opening the incisionsite and performing a minimal debridement limited to removal of the1-cm infected bone segment and irrigation with 50 ml of sterile PBS.Samples for bacteriological analysis were taken from the bone and sur-rounding soft tissue before and after debridement as previously described(7, 19). Postdebridement, the defect was filled with a single pellet modifiedusing sterile sandpaper as necessary to fit snugly into the defect (Fig. 1).Rabbits were randomized by treatment group, with only a single techni-cian in the operating suite aware of the PMMA formulation placed intoeach rabbit.

Assessment of relative therapeutic efficacy. Pellets were left in placefor 3 weeks without any additional form of antibiotic treatment, at whichtime rabbits were humanely euthanized and the surgical limb was har-vested for X-ray, histological, and bacteriological analysis as previouslydescribed (19). For bacteriological analysis, samples were collected byswab from the infection site and used to inoculate tryptic soy agar (TSA)without antibiotic selection. To achieve a quantitative comparison, swabswere used to comprehensively inoculate the first quadrant of a TSA plate,which was then struck for isolated colonies using standard bacteriologicaltechniques. The relative amount of growth was scored after 24 h at 37°Cbased on growth in the first quadrant only (1�) to growth across all fourquadrants (4�). The scores obtained with all swabs from each experimen-tal animal were then averaged to obtain a single bacteriological score. Thesame scoring protocol was employed immediately before and after de-bridement, thus yielding three bacteriological scores for each rabbit.

X rays were scored by an orthopedic surgeon blinded to the infectionstatus of each rabbit. Scores were based on evidence of periosteal eleva-tion, sequestration, architectural deformation, and deformation of softtissue as previously described (19). Each parameter was scored on a5-point scale (0 to 4), with 4 representing the most severe evidence ofdisease. Scores were then averaged to obtain a single radiographic scorefor each rabbit.

After collection of the final samples for bacteriological analysis, thesurgical limb was removed and processed for histological analysis by he-matoxylin and eosin (H&E) staining and by Gram stain as previouslydescribed (19). However, the histopathological scoring system was mod-ified from our previous studies, which were limited to studies evaluatingthe progression of disease, to address the inclusion of a treatment phase inthese experiments (Fig. 2). A separate score was also derived for eachrabbit based on Gram stain and the relative abundance of intraosseousGram-positive cocci.

Statistical analysis. Lowess curves were used to provide visual sum-maries of the effect of xylitol on in vitro elution over time, with linearregression models used to determine whether xylitol significantly affectedelution. Kruskal-Wallis tests were employed to determine whether vari-ous PMMA formulations differed with respect to bacteriology, X ray,histology, and Gram stain scores. If significant, Wilcoxon rank sum testswere used to perform pairwise testing, in which case the Simes methodwas used to adjust P values for multiple comparisons. Statistical signifi-cance for all analyses was determined using an � level of 5%.

RESULTS

In our in vitro studies, we arbitrarily defined an optimal formula-tion of PMMA and xylitol as one that required the minimumamount of daptomycin to achieve an elution profile characterizedby a maximum concentration at least 100 times (100�) higherthan the breakpoint MIC for a daptomycin-resistant strain ofStaphylococcus aureus (1.0 �g per ml) and a sustained concentra-tion at least 5� higher than this breakpoint MIC (21). This for-mulation consisted of 2 g of daptomycin and 22 g of xylitol per40-g packet of PMMA. Elution studies evaluating this formulationwere done using 7-mm spherical beads (21), which allowed us touse a commercially available bead mold (Wright Medical Tech-

FIG 1 X-ray images before and after debridement. Images were taken 3 weekspostinfection. The predebridement image (top) confirms soft tissue deforma-tion, periosteal elevation, new bone formation, and displacement of the in-fected segment. The postdebridement image (bottom) confirms illustratesplacement of the PMMA pellet into the defect created by debridement.

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nology, Arlington, TN). However, this architecture was not ap-propriate for our rabbit model because the beads were too largeand unstable within the postdebridement defect. For this reason,we switched to a 4-mm by 10-mm cylindrical pellet as describedabove. These pellets were smaller by weight, with a single pelletbeing on average 94.2% of the weight of a single 7-mm bead (datanot shown). Additionally, architectural changes can be expectedto affect the elution characteristics based on differences in thesurface area-to-volume ratios. Based on this, we first comparedthe in vitro elution characteristics of a single 4- by 10-mm pelletwith that of a single 7-mm bead.

While the overall elution profiles were very similar (data notshown), the maximum concentration achieved with a single bead(107 �g per ml) exceeded that achieved with a single pellet (80 �gper ml). The latter is below the desired 100� standard of 100 �gper ml (based on a breakpoint MIC for daptomycin of �1.0 �g perml). More importantly, when we carried out a preliminary rabbitexperiment comparing pellets made with this formulation, wefound no difference between any of the treatment groups, with allrabbits exhibiting a level of infection consistent with that observedat debridement (data not shown). One interpretation is that xyli-tol offers no therapeutic benefit, but the fact that all treatmentgroups were equivalent, including those treated with PMMAalone, suggests that the overall treatment regimen used in this

study was inadequate. Based on this, we altered the PMMA for-mulation to include 4 g of daptomycin per 40-g packet of PMMA.Additionally, the postdebridement treatment period in the pre-liminary trial was limited to 1 week, and in this second trial, it wasextended to 3 weeks based on the possibility that 1 week was in-sufficient to observe any therapeutic benefit.

Elution studies done in vitro confirmed that incorporation ofadditional daptomycin significantly enhanced the overall elutionprofile in comparison to our earlier studies (21) and that this wasfurther enhanced by the inclusion of xylitol (Fig. 3). In fact, whilethe 100� standard was easily achieved even without the additionof xylitol, only the inclusion of xylitol allowed us to achieve the 5�standard for sustained elution (Fig. 3). Additionally, in vitro elu-tion studies done with pellets retrieved from rabbits after the treat-ment period confirmed that the amounts of daptomycin elutedfrom pellets containing xylitol were significantly larger than theamounts eluted from pellets lacking xylitol (Fig. 4). In fact, theamounts eluted from PMMA pellets containing xylitol were �4-to 5-fold larger than the breakpoint MIC for daptomycin, whilethe amount eluted from pellets formulated without xylitol fell wellbelow this breakpoint MIC (Fig. 4). Structural analysis of thesepellets by microCT also demonstrated that, while the incorpora-tion of daptomycin was associated with increased porosity incomparison with PMMA alone, the greater effect in this regardwas achieved by the incorporation of xylitol irrespective of theinclusion of daptomycin (Fig. 5).

Bacteriological analysis confirmed infection in all 24 experi-mental animals at the time of debridement, and the overall bacte-riological scores were comparable across all experimental groupsboth before and after debridement (Fig. 6). However, when post-treatment bacteriological scores were compared, the combinationof xylitol and daptomycin was found to offer a statistically signif-icant therapeutic advantage in comparison with all other treat-ment groups (Fig. 6). Perhaps most importantly, only 1 of 6 rab-bits (16.7%) in the daptomycin treatment group was cleared ofinfection based on bacteriological analysis, while 5 of 6 (83.3%) inthe xylitol-daptomycin treatment group were cleared. None of the

FIG 2 Histological scoring parameters. Overall histopathological score ��(all scores)/5. Overall Gram stain score � F1 � F2.

FIG 3 In vitro elution profiles with and without xylitol. Duplicate samplesfrom PMMA beads containing equivalent amounts of daptomycin with andwithout the inclusion of xylitol were collected over the indicated time period,and the amount of daptomycin was determined by HPLC.

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rabbits in either of the other two treatment groups were cleared ofinfection based on bacteriological analysis.

Analysis of X rays, histological scores, and Gram stain resultsall revealed downward trends correlated with the combination ofxylitol and daptomycin, but none of the differences observedbased on any of these parameters were statistically significant(Fig. 7).

DISCUSSION

The successful treatment of an established, chronic bone infectionis a clinical challenge. Hallmarks of the current clinical approachinclude intensive, long-term systemic antibiotic therapy, adequatedebridement, and in most cases local antibiotic therapy usingsome form of matrix-based delivery (1, 5, 16). The last is critical inthat it provides for a concentration of antibiotic at the site ofinfection that is high enough to overcome any residual infectionremaining after debridement without the concern of systemic tox-icity. This is important because adequate debridement presentssomething of a paradox in that the more extensive the debride-ment, the greater the likelihood of clearing the infection, but thiscan also result in structurally unstable defects that require stabili-zation. Given the unique characteristics of bone infection, includ-ing its association with formation of a bacterial biofilm, it is un-likely that any advances in diagnosis and/or the development of

new antibiotics will preclude the need for debridement, and thisaccounts for the need for improved methods of local antibioticdelivery that would potentially allow orthopedic surgeons to con-fidently limit the extent of debridement in cases in which thisparadox is in play. Achieving this was the overall focus of theexperiments that we describe.

With respect to PMMA as a delivery matrix, the goal would be tomodify its elution characteristics to achieve both higher maximumconcentrations and more-sustained antibiotic release. One way to dothis is to incorporate large amounts of antibiotic either alone or incombination with each other. The use of multiple antibiotics canserve several purposes, including antibiotic synergy, increased cover-age for alternative bacterial pathogens, and a reduced likelihood ofemergent antibiotic resistance. Synergy between conventional antibi-otics is likely more theoretical than actual, particularly under in vivoconditions (3), and increased coverage is a concern that is primarilylimited to empirical therapeutic decisions. Reducing the likelihood ofemergent resistance is an important consideration, particularly whenconsidering the inclusion of rifampin (16), but combination therapyhas not proven superior to monotherapy when the latter is appropri-ately targeted to the offending pathogen. Moreover, the inclusion ofone antibiotic can sometimes limit rather than enhance the elutionof another antibiotic, thus potentially negating this therapeutic ben-efit (1).

FIG 4 Elution of daptomycin after in vivo recovery. Individual pellets wererecovered after the in vivo treatment period and subjected to in vitro elution for24 h, with the amount of daptomycin eluted from each pellet determined byHPLC.

FIG 5 Structural characteristics after in vivo recovery. Pellets recovered after the in vivo treatment period were imaged by microCT.

FIG 6 Therapeutic efficacy based on bacteriological results. Bacteriologyscores for individual rabbits in each treatment group are indicated for resultsobtained before and immediately after debridement and at the end of thetreatment period.

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While the microbiological benefits of employing multiple an-tibiotics are unclear, particularly in cases for which there is a con-firmed diagnosis that includes susceptibility testing, what is clearis that incorporating multiple antibiotics, or increased amounts ofa single antibiotic, increases the porosity of PMMA to an extentthat enhances antibiotic elution (13–15). However, what is equallyclear is that the use of antibiotics for this purpose is expensive and,if increased porosity is the critical goal, unnecessary. Our grouphas explored the use of inexpensive and biologically inert com-pounds as a means of accomplishing this goal, and the results ofthe in vitro studies done to date have led us to focus on the use ofxylitol (12–15). Indeed, we have confirmed that incorporationof xylitol can be used to enhance the elution of functionally dis-tinct antibiotics, including daptomycin, gentamicin, and vanco-mycin (21). However, to date, there is no evidence to suggest thatthis can be correlated with an improved therapeutic outcome inthe context of chronic, biofilm-associated bone infection.

The results that we present here address this deficiency by com-paring the therapeutic efficacies of equivalent amounts of dapto-mycin with and without xylitol using a preclinical rabbit model ofchronic, postsurgical osteomyelitis. The results confirm that xyli-tol enhances the elution of daptomycin under in vitro conditionsas reflected in both the maximum concentration attainable andprolonged elution of antibiotic at levels that exceed the breakpointMIC. More importantly, they demonstrate that, even 3 weeks afterplacement in vivo, therapeutically relevant levels of daptomycincan still be recovered from PMMA beads containing xylitol butnot those containing daptomycin alone. The most compelling ev-idence that this is clinically relevant comes from the observationthat viable bacteria were obtained from only 1 of 6 rabbits treatedwith daptomycin-xylitol and from 4 of 6 rabbits treated with dap-tomycin alone.

Although the differences did not reach statistical significance,average scores for radiographic and histological evidence of dis-ease were also lowest in the daptomycin-xylitol treatment group.It is important to emphasize in this respect that our experimentalprotocol employed a 3-week infection period, which we have pre-viously demonstrated is sufficient to achieve a well-established,chronic infection (19). Thus, a significant degree of bone and tis-

sue damage had occurred prior to initiating treatment. Based onthis, it is perhaps not surprising that differences between the ex-perimental groups were less evident in the context of these param-eters than in the context of microbiological culture. Similarly,while overall bacterial burden as assessed by the presence of in-traosseous Gram-positive cocci was lowest in the daptomycin-xylitol treatment group, in no case were intraosseous cocci absentfrom any experimental animal. However, assessment using thismethod does not distinguish between viable and killed bacteria,and this too would be expected to limit differences between exper-imental groups using the chronic infection model employed inthese experiments. Thus, when taken together, the results that wepresent provide strong support for the hypothesis that xylitol canbe used as an inexpensive agent to increase the porosity of PMMAand enhance antibiotic elution to an extent that can be correlatedwith an improved therapeutic outcome in the clinically challeng-ing context of chronic bone infections requiring surgical debride-ment.

ACKNOWLEDGMENTS

This work was supported by grant AI069087 (M.S.S.) from the NationalInstitute of Allergy and Infectious Diseases. Support was also obtainedfrom resources provided through the Clinical and Translational SciencesAward (RR0298884) to the University of Arkansas for Medical Sciences.

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FIG 7 Therapeutic efficacy based on radiographic and histological results.Scores based on X-ray analysis, histopathological analysis, and Gram stainingare indicated for individual rabbits in each treatment group.

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