Use of Xylitol To Enhance the Therapeutic Efficacy of Polymethylmethacrylate-Based Antibiotic Therapy in Treatment of Chronic Osteomyelitis

Abstract

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

INTRODUCTION

Bone infections are intrinsically resistant to antimicrobial therapy even when caused by bacteria that are not clinically defined as resistant to the preferred antibiotics. One contributing factor to this is the difficulty in achieving a definitive diagnosis before the infection has progressed to a chronic stage, which is characterized by compromise of the local vasculature to an extent that limits antibiotic delivery using systemic methods (10). A second contributing factor is formation of a bacterial biofilm, which confers a level of intrinsic antibiotic resistance that further compromises the efficacy of conventional antibiotic therapy (4). These factors necessitate an integrated therapeutic approach to chronic bone infection that includes long-term, systemic antimicrobial therapy and surgical debridement. Debridement is often accompanied by some form of local, matrix-based antibiotic delivery, the objective being to achieve a concentration of antibiotic at the site of infection that is high enough to overcome this intrinsic resistance without systemic toxicity (5, 16). Even then, the failure rate can be as high as 10%, particularly in patients suffering from some form of underlying systemic compromise (5).

A number of delivery matrices have been employed for local antibiotic delivery, but one of the most common is polymethylmethacrylate (PMMA). While widely used, PMMA has several disadvantages, including the fact that it is stable and can itself provide a substrate for bacterial colonization, particularly as the level of eluted antibiotic falls over time (6). A second disadvantage is that antibiotic recovery from PMMA is inefficient, thus making it difficult to achieve and maintain the desired concentrations of antibiotic for the desired length of time. One solution is to incorporate large amounts of antibiotics either alone or in combination with each other (1), thus increasing the porosity of PMMA. However, this can be prohibitively expensive, particularly given the size of the defect sometimes created by debridement and the need for full coverage of the wound environment.

In an effort to overcome this problem, we have examined alternative methods of increasing the porosity of PMMA and enhancing antibiotic elution. This is not a novel idea, and in fact, gentamicin-impregnated PMMA beads containing glycine for this purpose are available in Europe under the trade name Septopal (Biomet Europe, Dordrecht, The Netherlands). However, we demonstrated that xylitol enhances antibiotic elution from PMMA to a greater extent than does glycine (13, 14), and xylitol itself has been shown to have antibacterial properties and to prevent biofilm formation (2, 8, 9, 17, 20). In a rat model, xylitol was also found to have a positive impact on bone density (11, 18).

Based on these considerations, we carried out experiments aimed at optimizing the use of xylitol as a means of enhancing the antibiotic elution profile from PMMA (21). We placed a primary emphasis on daptomycin in these studies because we have found that it exhibits greater therapeutic efficacy than other commonly used antibiotics, including vancomycin, in the context of an established biofilm. The specific parameters that we used to define an optimal elution profile were a maximum concentration at least 100 times higher than the breakpoint MIC for daptomycin (≥1.0 μg/ml) and a sustained concentration, defined at 10 days postelution under in vitro conditions, at least 5 times this breakpoint MIC (21). These experiments confirmed that incorporation of xylitol into PMMA increases elution to the point that both of these parameters can be achieved using an amount of daptomycin that is otherwise insufficient, particularly with respect to sustained elution (21). However, these studies were limited to defining elution profiles in vitro, and it remains unknown whether this can be correlated with an improved therapeutic outcome. To address this, we used a rabbit model of postsurgical bone infection (19) to compare the therapeutic efficacies of PMMA formulations containing equivalent amounts of daptomycin with and without xylitol.

MATERIALS AND METHODS

PMMA formulations and characterization.

The PMMA (Palacos-R) formulations evaluated in these experiments included PMMA with no additives, 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 4 g, respectively). All formulations were prepared according to the manufacturer's instructions (Palacos-R; Zimmer Inc., Dover, OH) without adjustment for the inclusion of additives. After mixing, PMMA pastes were packed into a 1-ml tuberculin syringe with the Luer lock top cut off and allowed to harden. They were then extruded and cut to 10-mm lengths using a sterile saw. This resulted in a cylindrical pellet with dimensions of 4 mm by 10 mm, which approximates the size of the excised radial segment as detailed below.

To examine the impact of incorporating xylitol on the elution of daptomycin, in vitro elution comparisons were done by placing 1 pellet of each formulation into 4 ml of sterile phosphate-buffered saline (PBS). After overnight incubation at 37°C, the buffer was removed and replaced with an equal volume of PBS. This was repeated for 7 consecutive days, at which time the amount of daptomycin present in each sample was determined by high-pressure liquid chromatography (HPLC) using a C₈ column and an acidic acetonitrile-salt mobile phase (21). Pellets were also recovered after the in vivo treatment period (see below) and placed into 4 ml PBS for 24 h at 37°C, and the amount of daptomycin in the eluate was determined 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 in accordance with the policies of the Public Health Service (PHS) on care and use of laboratory animals, the Animal Welfare Act, and the NIH Guide for the Care and Use of Laboratory Animals in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) International-accredited facility. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Therapeutic efficacy was assessed using an established rabbit model of postsurgical osteomyelitis (7, 19). Briefly, a 1-cm midradial segment was surgically excised from each of 24 male, New Zealand White rabbits. The infection was initiated in all rabbits by injection into the intramedullary canal of 10⁶ CFU of the Staphylococcus aureus osteomyelitis isolate UAMS-1 in a total volume of 10 μl of sterile phosphate-buffered saline (PBS). The segment was then returned to the radial defect in its original orientation, and the wound was closed. After 3 weeks, radiographs were obtained from all rabbits prior to opening the incision site and performing a minimal debridement limited to removal of the 1-cm infected bone segment and irrigation with 50 ml of sterile PBS. Samples for bacteriological analysis were taken from the bone and surrounding soft tissue before and after debridement as previously described (7, 19). Postdebridement, the defect was filled with a single pellet modified using sterile sandpaper as necessary to fit snugly into the defect (Fig. 1). Rabbits were randomized by treatment group, with only a single technician in the operating suite aware of the PMMA formulation placed into each rabbit.

Fig 1.

X-ray images before and after debridement. Images were taken 3 weeks postinfection. The predebridement image (top) confirms soft tissue deformation, periosteal elevation, new bone formation, and displacement of the infected segment. The postdebridement image (bottom) confirms illustrates placement of the PMMA pellet into the defect created by debridement.

Assessment of relative therapeutic efficacy.

Pellets were left in place for 3 weeks without any additional form of antibiotic treatment, at which time rabbits were humanely euthanized and the surgical limb was harvested for X-ray, histological, and bacteriological analysis as previously described (19). For bacteriological analysis, samples were collected by swab from the infection site and used to inoculate tryptic soy agar (TSA) without antibiotic selection. To achieve a quantitative comparison, swabs were used to comprehensively inoculate the first quadrant of a TSA plate, which was then struck for isolated colonies using standard bacteriological techniques. The relative amount of growth was scored after 24 h at 37°C based on growth in the first quadrant only (1+) to growth across all four quadrants (4+). The scores obtained with all swabs from each experimental animal were then averaged to obtain a single bacteriological score. The same scoring protocol was employed immediately before and after debridement, thus yielding three bacteriological scores for each rabbit.

X rays were scored by an orthopedic surgeon blinded to the infection status of each rabbit. Scores were based on evidence of periosteal elevation, sequestration, architectural deformation, and deformation of soft tissue as previously described (19). Each parameter was scored on a 5-point scale (0 to 4), with 4 representing the most severe evidence of disease. Scores were then averaged to obtain a single radiographic score for each rabbit.

After collection of the final samples for bacteriological analysis, the surgical limb was removed and processed for histological analysis by hematoxylin and eosin (H&E) staining and by Gram stain as previously described (19). However, the histopathological scoring system was modified from our previous studies, which were limited to studies evaluating the progression of disease, to address the inclusion of a treatment phase in these experiments (Fig. 2). A separate score was also derived for each rabbit based on Gram stain and the relative abundance of intraosseous Gram-positive cocci.

Fig 2.

Histological scoring parameters. Overall histopathological score = Σ(all scores)/5. Overall Gram stain score = F1 + F2.

Statistical analysis.

Lowess curves were used to provide visual summaries of the effect of xylitol on in vitro elution over time, with linear regression models used to determine whether xylitol significantly affected elution. Kruskal-Wallis tests were employed to determine whether various PMMA formulations differed with respect to bacteriology, X ray, histology, and Gram stain scores. If significant, Wilcoxon rank sum tests were used to perform pairwise testing, in which case the Simes method was used to adjust P values for multiple comparisons. Statistical significance for all analyses was determined using an α level of 5%.

RESULTS

In our in vitro studies, we arbitrarily defined an optimal formulation of PMMA and xylitol as one that required the minimum amount of daptomycin to achieve an elution profile characterized by a maximum concentration at least 100 times (100×) higher than the breakpoint MIC for a daptomycin-resistant strain of Staphylococcus aureus (1.0 μg per ml) and a sustained concentration at least 5× higher than this breakpoint MIC (21). This formulation consisted of 2 g of daptomycin and 22 g of xylitol per 40-g packet of PMMA. Elution studies evaluating this formulation were done using 7-mm spherical beads (21), which allowed us to use a commercially available bead mold (Wright Medical Technology, Arlington, TN). However, this architecture was not appropriate for our rabbit model because the beads were too large and unstable within the postdebridement defect. For this reason, we switched to a 4-mm by 10-mm cylindrical pellet as described above. These pellets were smaller by weight, with a single pellet being on average 94.2% of the weight of a single 7-mm bead (data not shown). Additionally, architectural changes can be expected to affect the elution characteristics based on differences in the surface area-to-volume ratios. Based on this, we first compared the in vitro elution characteristics of a single 4- by 10-mm pellet with that of a single 7-mm bead.

While the overall elution profiles were very similar (data not shown), the maximum concentration achieved with a single bead (107 μg per ml) exceeded that achieved with a single pellet (80 μg per ml). The latter is below the desired 100× standard of 100 μg per ml (based on a breakpoint MIC for daptomycin of ≥1.0 μg per ml). More importantly, when we carried out a preliminary rabbit experiment comparing pellets made with this formulation, we found no difference between any of the treatment groups, with all rabbits exhibiting a level of infection consistent with that observed at debridement (data not shown). One interpretation is that xylitol offers no therapeutic benefit, but the fact that all treatment groups were equivalent, including those treated with PMMA alone, suggests that the overall treatment regimen used in this study was inadequate. Based on this, we altered the PMMA formulation to include 4 g of daptomycin per 40-g packet of PMMA. Additionally, the postdebridement treatment period in the preliminary trial was limited to 1 week, and in this second trial, it was extended to 3 weeks based on the possibility that 1 week was insufficient to observe any therapeutic benefit.

Elution studies done in vitro confirmed that incorporation of additional daptomycin significantly enhanced the overall elution profile in comparison to our earlier studies (21) and that this was further enhanced by the inclusion of xylitol (Fig. 3). In fact, while the 100× standard was easily achieved even without the addition of xylitol, only the inclusion of xylitol allowed us to achieve the 5× standard for sustained elution (Fig. 3). Additionally, in vitro elution studies done with pellets retrieved from rabbits after the treatment period confirmed that the amounts of daptomycin eluted from pellets containing xylitol were significantly larger than the amounts eluted from pellets lacking xylitol (Fig. 4). In fact, the amounts eluted from PMMA pellets containing xylitol were ∼4- to 5-fold larger than the breakpoint MIC for daptomycin, while the amount eluted from pellets formulated without xylitol fell well below this breakpoint MIC (Fig. 4). Structural analysis of these pellets by microCT also demonstrated that, while the incorporation of daptomycin was associated with increased porosity in comparison with PMMA alone, the greater effect in this regard was achieved by the incorporation of xylitol irrespective of the inclusion of daptomycin (Fig. 5).

Fig 3.

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

Fig 4.

Elution of daptomycin after in vivo recovery. Individual pellets were recovered after the in vivo treatment period and subjected to in vitro elution for 24 h, with the amount of daptomycin eluted from each pellet determined by HPLC.

Fig 5.

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

Bacteriological analysis confirmed infection in all 24 experimental animals at the time of debridement, and the overall bacteriological scores were comparable across all experimental groups both before and after debridement (Fig. 6). However, when posttreatment bacteriological scores were compared, the combination of xylitol and daptomycin was found to offer a statistically significant therapeutic advantage in comparison with all other treatment groups (Fig. 6). Perhaps most importantly, only 1 of 6 rabbits (16.7%) in the daptomycin treatment group was cleared of infection based on bacteriological analysis, while 5 of 6 (83.3%) in the xylitol-daptomycin treatment group were cleared. None of the rabbits in either of the other two treatment groups were cleared of infection based on bacteriological analysis.

Fig 6.

Therapeutic efficacy based on bacteriological results. Bacteriology scores for individual rabbits in each treatment group are indicated for results obtained before and immediately after debridement and at the end of the treatment period.

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

Fig 7.

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

DISCUSSION

The successful treatment of an established, chronic bone infection is a clinical challenge. Hallmarks of the current clinical approach include intensive, long-term systemic antibiotic therapy, adequate debridement, and in most cases local antibiotic therapy using some form of matrix-based delivery (1, 5, 16). The last is critical in that it provides for a concentration of antibiotic at the site of infection that is high enough to overcome any residual infection remaining after debridement without the concern of systemic toxicity. This is important because adequate debridement presents something of a paradox in that the more extensive the debridement, the greater the likelihood of clearing the infection, but this can also result in structurally unstable defects that require stabilization. Given the unique characteristics of bone infection, including its association with formation of a bacterial biofilm, it is unlikely that any advances in diagnosis and/or the development of new antibiotics will preclude the need for debridement, and this accounts for the need for improved methods of local antibiotic delivery that would potentially allow orthopedic surgeons to confidently limit the extent of debridement in cases in which this paradox is in play. Achieving this was the overall focus of the experiments that we describe.

With respect to PMMA as a delivery matrix, the goal would be to modify its elution characteristics to achieve both higher maximum concentrations and more-sustained antibiotic release. One way to do this is to incorporate large amounts of antibiotic either alone or in combination with each other. The use of multiple antibiotics can serve several purposes, including antibiotic synergy, increased coverage for alternative bacterial pathogens, and a reduced likelihood of emergent antibiotic resistance. Synergy between conventional antibiotics is likely more theoretical than actual, particularly under in vivo conditions (3), and increased coverage is a concern that is primarily limited to empirical therapeutic decisions. Reducing the likelihood of emergent resistance is an important consideration, particularly when considering the inclusion of rifampin (16), but combination therapy has not proven superior to monotherapy when the latter is appropriately targeted to the offending pathogen. Moreover, the inclusion of one antibiotic can sometimes limit rather than enhance the elution of another antibiotic, thus potentially negating this therapeutic benefit (1).

While the microbiological benefits of employing multiple antibiotics are unclear, particularly in cases for which there is a confirmed diagnosis that includes susceptibility testing, what is clear is that incorporating multiple antibiotics, or increased amounts of a single antibiotic, increases the porosity of PMMA to an extent that enhances antibiotic elution (13–15). However, what is equally clear is that the use of antibiotics for this purpose is expensive and, if increased porosity is the critical goal, unnecessary. Our group has explored the use of inexpensive and biologically inert compounds as a means of accomplishing this goal, and the results of the in vitro studies done to date have led us to focus on the use of xylitol (12–15). Indeed, we have confirmed that incorporation of xylitol can be used to enhance the elution of functionally distinct antibiotics, including daptomycin, gentamicin, and vancomycin (21). However, to date, there is no evidence to suggest that this can be correlated with an improved therapeutic outcome in the context of chronic, biofilm-associated bone infection.

The results that we present here address this deficiency by comparing the therapeutic efficacies of equivalent amounts of daptomycin with and without xylitol using a preclinical rabbit model of chronic, postsurgical osteomyelitis. The results confirm that xylitol enhances the elution of daptomycin under in vitro conditions as reflected in both the maximum concentration attainable and prolonged elution of antibiotic at levels that exceed the breakpoint MIC. More importantly, they demonstrate that, even 3 weeks after placement in vivo, therapeutically relevant levels of daptomycin can still be recovered from PMMA beads containing xylitol but not those containing daptomycin alone. The most compelling evidence that this is clinically relevant comes from the observation that viable bacteria were obtained from only 1 of 6 rabbits treated with daptomycin-xylitol and from 4 of 6 rabbits treated with daptomycin alone.

Although the differences did not reach statistical significance, average scores for radiographic and histological evidence of disease were also lowest in the daptomycin-xylitol treatment group. It is important to emphasize in this respect that our experimental protocol employed a 3-week infection period, which we have previously demonstrated is sufficient to achieve a well-established, chronic infection (19). Thus, a significant degree of bone and tissue damage had occurred prior to initiating treatment. Based on this, it is perhaps not surprising that differences between the experimental groups were less evident in the context of these parameters than in the context of microbiological culture. Similarly, while overall bacterial burden as assessed by the presence of intraosseous Gram-positive cocci was lowest in the daptomycin-xylitol treatment group, in no case were intraosseous cocci absent from any experimental animal. However, assessment using this method does not distinguish between viable and killed bacteria, and this too would be expected to limit differences between experimental groups using the chronic infection model employed in these experiments. Thus, when taken together, the results that we present provide strong support for the hypothesis that xylitol can be used as an inexpensive agent to increase the porosity of PMMA and enhance antibiotic elution to an extent that can be correlated with an improved therapeutic outcome in the clinically challenging context of chronic bone infections requiring surgical debridement.