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Xylitol and Glycine Fillers Increase Permeability of PMMA to Enhance Elution of Daptomycin

McLaren, Alex C, MD; McLaren, Sandra G, BS; Smeltzer, Mark, PhD

Section Editor(s): Garvin, Kevin MD, Guest Editor

Clinical Orthopaedics and Related Research®: October 2006 - Volume 451 - Issue - p 25-28
doi: 10.1097/01.blo.0000229321.53040.a1
SECTION I: SYMPOSIUM I: Papers Presented at the 2005 Meeting of the Musculoskeletal Infection Society
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The elution of antibiotics from hand mixed antibiotic-laden polymethylmethacrylate (PMMA) must be increased to achieve clinical performance equivalent to commercially manufactured antibiotic beads (not available in the USA) in the management of musculoskeletal infections. Adding fillers such as glycine and dextran to polymethylmethacrylate increases the elution of antibiotics from antibiotic-laden PMMA. We propose xylitol, a naturally occurring sweetener with direct antibiofilm properties, as a filler material. To compare the efficacy of xylitol and glycine as fillers on the elution of antibiotics from PMMA, elution studies were performed on mixtures of Palacos® polymethylmethacrylate and daptomycin (1 gm) with xylitol or glycine as the filler (28 g). Xylitol and glycine enhanced the daptomycin activity eluted from the polymethylmethacrylate. Xylitol was more effective than glycine, having a greater increase in daptomycin release at all data points; on day one xylitol increased the elution of daptomycin 2.67 times whereas glycine increased it 1.78 times also on day one. The eluant concentration of daptomycin remained higher longer for xylitol; 3.90 μg/mL for xylitol versus 2.25 μg/mL for glycine on day 9. Xylitol is inexpensive and readily available. It can be hand mixed with PMMA, and is more effective than glycine as a filler to enhance daptomycin release. Considering possible antibiofilm activity, xylitol may be a more advantageous choice.

From the Department of Orthopaedic Surgery, University of Arkansas Medical Sciences, Little Rock, AR.

Each author certifies that he or she has no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article.

Correspondence to: Sandra G. McLaren, Center for Orthopaedic Research, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Mail Slot 644, Little Rock, AR 72205. Phone: 501-686-7841; Fax: 501-686-8987; E-mail: sgm@uams.edu.

Various fillers such as dextran7 and glycine13 added to antibiotic-laden polymethylmethacrylate (PMMA) increase antibiotic delivery from the PMMA by causing interconnecting voids in the PMMA. The resultant porosity increases the permeability and thereby antibiotic elution. The ideal filler material and amount of filler are yet to be established. Xylitol is a naturally occurring sugar alcohol commonly purified from birch bark and many fruits and vegetables.16 It is used as a natural food sweetener. The five-carbon molecular structure cannot be metabolized by bacteria utilizing six-carbon sugars for energy. It has a suppressive effect on bacterial formation of biofilm.10 The dental profession recommends its use as a sweetener for preventing tooth decay.5,8 Clinically, it decreases dental caries9 and other infection-related ear, nose and throat conditions.18,19 It is used in oral care products such as toothpaste and mouthwash.3

If xylitol, in amounts compatible with being mixed in PMMA, is effective in augmenting the release of antibiotics from antibiotic-laden PMMA, then its additional antibiofilm activity may make it preferable to other fillers. The resultant antibiotic-laden PMMA with xylitol filler would not be usable for implant fixation because of compromised mechanical properties of the PMMA. Nonetheless, antibiotic-laden PMMA is an essential modality in the management of the surgically debrided wound in arthroplasty infections.

An antibiotic used for depot delivery should be bactericidal for the bacteria present or likely to be present. Increasing effectiveness with increasing concentration is desirable as is a slow tendency to develop bacterial resistance. Adverse effects from systemic administration should be eliminated by local delivery. When used in PMMA the ideal antibiotic for depot delivery should be heat stable and in powder form with small uniform crystal/particle size. Daptomycin is a cyclic lipopeptide, with a mechanism of action distinct from any other antibiotic. Its bactericidal activity is concentration-dependent. It binds to the bacterial cell membrane causing rapid depolarization of membrane potential. Loss of membrane potential leads to inhibition of protein, DNA, and RNA synthesis, resulting in bacterial cell death. Daptomycin retains potency against antibiotic-resistant gram-positive bacteria, including isolates resistant to methicillin, vancomycin, and linezolid.6,17 It is effective against aerobic and facultative gram-positive microorganisms including methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, and vancomycin-susceptible Enterococcus faecalis.6,17 A severe adverse effect of daptomycin is rhabdomyolysis.2 This is dosing interval dependent and can limit its therapeutic usefulness. For serious musculoskeletal infections, daptomycin is administered in a once a day dose (4 mg/kg/24 hours). Once a day dosing decreases the risk of rhabdomyolysis to less than 3%. The efficacy of lower doses less at risk for rhabdomyolysis is unreliable.6,15,17 For orthopaedic infections, depot delivery with low systemic levels while delivering high levels locally in bone may be a good therapeutic option.

We hypothesized xylitol added to PMMA is more effective than glycine in enhancing daptomycin elution from PMMA.

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MATERIALS AND METHODS

We used daptomycin elution to evaluate the release properties of daptomycin from daptomycin-laden PMMA, with and without filler and to compare xylitol as a filler with glycine, a material used in other investigations as a filler to enhance antibiotic release from PMMA. Two daptomycin PMMA filler test mixtures were prepared using 1 g of daptomycin and 28 g of xylitol or glycine. Three control mixtures were prepared using 1 g of daptomycin and no filler, or 0 g of daptomycin and 28 g of xylitol or glycine. All daptomycin and filler combinations were added per batch (40 g polymer, 20 mL monomer) of Palacos® PMMA (Biomet Inc, Warsaw, IN). Beads were made with the cement in the dough phase using 7 mm silicone molds. The beads were divided into five-bead groups. Three groups (15 beads) were used for each PMMA mixture studied. The beads were eluted in 20 mL of phosphate buffered saline (PBS) at 37°C. After swirling to mix the fluid, 5 mL of PBS was exchanged daily for 9 days. The eluant was stored at −80°C until assay.

To assess the level of daptomycin eluted from the beads, 3 mL of eluant was mixed with 1 mL of 4X tryptic soy broth. Serial two-fold dilutions were then prepared using sterile tryptic soy broth as diluent. Then 105 colony-forming units of the daptomycin-sensitive (minimum inhibitory concentration = 1.5 μg/mL) Staphylococcus aureus strain UAMS-1 was then added to each tube. After overnight incubation (18-24 hours), growth was scored by assessing turbidity. The last dilution to inhibit growth was recorded and used to calculate the daptomycin concentration in the undiluted eluant for each daily sample and each bead mixture.

The data were analyzed using one-way ANOVA, noting the sample size was small, leading to low power.

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RESULTS

Daptomycin eluted from PMMA without a filler (Fig 1). On day 1, the daptomycin concentration in the eluant was31.65 μg/mL. Glycine and xylitol did not have an independent inhibitory effect on UAMS-1 growth in tryptic soy broth.

Fig 1

Fig 1

Xylitol and glycine enhanced the daptomycin activity eluted from the PMMA. The increase in daptomycin release with xylitol was greater than the increase with glycine; 2.67 times higher for xylitol (84.375 μg/mL vs 31.65 μg/mL) versus 1.78 times higher for glycine (56.25 μg/mL versus 31.65 μg/mL ) on Day 1. The daptomycin concentration in the eluant for the two test fillers decreased with time in a similar pattern to daptomycin alone. The daptomycin concentration in the eluant decreased to ≤ 1 μg by Day 6, but remained higher for a longer period of time for the xylitol and glycine mixtures: 3.90 μg/mL for xylitol and 2.25 μg/mL for glycine on Day 9. The data were analyzed using one-way ANOVA with the Tukey posttest confirmation with p = 0.038. The measurement error of the assay is expected to be normally distributed about the true value, and the results of the assays in all samples were affected to the same extent by the general conditions present in the methods, as necessary assumptions for this analysis. The power of the study was 0.480 (less than 0.800), making false negative conclusions less reliable.

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DISCUSSION

It is clinically recognized that commercially manufactured antibiotic laden beads are more effective than hand mixed beads unless the performance of hand mixed antibiotic laden PMMA is enhanced.1,13 Identifying a method to enhance the elution of antibiotics from antibiotic laden PMMA continues through investigations looking for a filler/antibiotic combination that is therapeutically effective at an acceptable cost.

We note several limitations. The relationship between particle size and effect on elution enhancement has not been defined by this study and will necessitate future investigations before the optimal form of a filler can be made available for clinical use. Furthermore, the optimal amount of filler remains to be defined. This study investigates only one dose of filler added to PMMA and the power of the study is low necessitating confirmatory study with greater sample size. Xylitol is reported to have anti- biofilm properties10 independent of its possible role as a filler. Inhibition of biofilm is desirable in the management of orthopaedic implant infections, which makes xylitol the preferred filler compared with other materials that do not have an independent therapeutic effect. However, dental studies that have documented antibiofilm properties are derived from oral bacteria, most importantly Streptococcus mutans.9 It remains to be confirmed whether xylitol has a similar effect on a known biofilm-producing human osteomyelitis pathogen. The bacteria in our assays were planktonic (in the suspension). They were not a valid indicator of biofilm production or inhibition as bacteria produce biofilm only while in sessile growth. Our study was not valid to detect any possible bacterial growth limitation caused by xylitol. Our data did not suggest xylitol alone directly limited UAMS-1 growth. The trypsin broth used as a carrier for the 105 cfu inoculation of UAMS-1 was used specifically to maximize growth of the UAMS-1 in the assay irrespective of the available nutrients in the eluant.

Daptomycin is an antibiotic that has properties amenable to depot delivery in PMMA. Xylitol is a potential filler material with independent antimicrobial properties. This investigation was carried out to evaluate xylitol as a filler to enhance the elution of daptomycin from PMMA. The data confirm release of daptomycin from PMMA, which is consistent with data reported using a different elution model.4 The elution of daptomycin from PMMA was enhanced by adding fillers to the PMMA, which is consistent with previously published data on gentamicin- PMMA-glycine mixtures using a similar elution model.13 Our data established xylitol's efficacy as filler material to increase the release of daptomycin. The released daptomycin remained biologically active. Xylitol had a greater effect than the equivalent amount of glycine at each time period. One possible explanation of the difference is the xylitol had a smaller particle size than the glycine. Larger numbers of smaller particles could lead to a more effective porous formation in the PMMA. If larger amounts of glycine, the less effective filler, were to be used it may be possible to increase its effectiveness to be the same as smaller amounts of xylitol, the more effective filler.

The greatest enhancement is during the first few days of the elution study. The elutions were not carried out long term so how long the daptomycin levels in the eluant remained above the usual mean bactericidal concentration of 1 μg/mL is not known. Antibiotic levels in eluant from laboratory studies has not been correlated with wound fluid levels so the laboratory data are best used as relative comparators for different depot preparations. Fluid dynamics in vivo are unknown so the shape of the elution curve in vivo may be substantially different than obtained in the lab. Irrespective of these unknowns, the shape of the elution curve was not changed by adding a filler. Based on clinical experience with commercially manufactured antibiotic laden PMMA beads1,14 having a similarly shaped elution curve, one would expect improved clinical performance with the pattern of enhanced elution seen with the xylitol filler. Considering the minimal cost, availability, and ease of use, it is unlikely that there is a meaningful difference between xylitol and glycine based on their ability to augment the elution of daptomycin from PMMA if they are used in optimized amounts. At present these fillers are available as both scientific non-sterile lab reagents, both with no option in particle size.

xylitol is reported to increase bone mineral content and strength in animal models.11,12 Although this is a desirable effect in many orthopaedic situations, it has not been confirmed in vivo in humans. Xylitol is effective as a filler to augment the release of daptomycin from antibiotic-laden PMMA. The magnitude of augmentation with xylitol was greater than with glycine. The pilot data obtained in this study will be used to guide the design of a study with sufficient power to be statistically significant Other properties of xylitol that would make it a preferred filler material, such as biofilm inhibition and osteogenesis, remain to be validated. Study of biofilm inhibition of UAMS-1 staphylococcus is planned.

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Acknowledgment

The authors thank Katherine Palm for her technical contribution.

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References

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