Double-layered antibiotic-loaded cement spacer as a novel alternative for managing periprosthetic joint infection: an in vitro study
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Previous studies comparing antibiotic-loaded calcium phosphate cement to polymethylmethacrylate cement reported that although the former has higher elution volumes over a longer period, it is mechanically weak when used alone. To counter this problem, a double-layered antibiotic-loaded cement spacer in which calcium phosphate cement is coated with polymethylmethacrylate cement was created.
In this study, we compared the double-layered spacer to the polymethylmethacrylate cement spacer in terms of eluent antibiotic concentration, bioactivity against methicillin-resistant Staphylococcus aureus, and mechanical strength. Double-layered and polymethylmethacrylate cement spacers that were loaded with vancomycin (VCM) were prepared and immersed in phosphate buffer for 84 days. To facilitate VCM elution from calcium phosphate cores in double-layered spacers, we also drilled multiple holes into the calcium phosphate layer from the spacer surface.
We found that VCM concentrations in double-layered spacer eluents were higher than those in polymethylmethacrylate cement spacer eluents. The double-layered spacer also had higher bioactivity than the polymethylmethacrylate cement spacer. Although the polymethylmethacrylate cement spacer eluent lost the ability to inhibit bacterial growth on day 56, the double-layered spacer eluent maintained this ability for the duration of our study. Finally, the double-layered spacer retained high mechanical strength throughout the study period.
The beneficial biomechanical and drug-eluting properties of the double-layered spacer might qualify it to serve as a promising biomaterial that could be used for managing periprosthetic joint infections.
KeywordsSpacer Antibiotic Bone cement Periprosthetic joint infection Vancomycin Methicillin-resistant Staphylococcus aureus
Calcium phosphate cement
- D-L spacer
High-performance liquid chromatography
Minimum inhibitory concentration
Methicillin-resistant Staphylococcus aureus
Periprosthetic joint infection
D-L spacers used in the clinical setting are not spherical, and those are with stems. Spherical spacers without stems may have different mechanical strengths compared to spacers with stems. However, the transition from the head to the stem of D-L spacer used in the clinical setting was used of only PMMA. Therefore, we considered that past data regarding strength of the transition from the head to the stem of PMMA spacers  can be applied to D-L spacers. In this study, we only used spherical spacer models without stems to compare the amount of VCM elution and mechanical strength of the two types of spacers.
Preparation of the spherical femoral head spacer models
We prepared two types of femoral head spacer models: D-L spacers (five samples) and PMMA spacers (five samples). Models were prepared using CPC (Biopex; HOYA Technosurgical Corporation, Tokyo, Japan) and PMMA (Cemex RX; Exactech, Gainesville, Florida, USA) in a clean operation room. Hemispherical molds with two different diameters (40 mm and 29 mm) were also prepared using silicone resin (EXAFINE; GC Corporation, Tokyo, Japan). Then, they were sterilized and preserved.
VCM-containing eluent collection
Each sample was placed in sterile phosphate-buffered saline (PBS) (1.5 mL of PBS per gram of spacer) and incubated at 37 °C for 84 days; PBS was changed every 24 h. Eluent samples were taken on days 1, 3, 7, 14, 28, 56, and 84, with the same sample used to measure VCM concentrations and bioactivity at each time point. Eluents were stored at − 30 °C until analysis, which was performed in a clean environment.
Measuring VCM concentrations using high-performance liquid chromatography
VCM concentrations in collected eluents were measured using high-performance liquid chromatography (HPLC). Briefly, the frozen eluents were milled. Then, 150 μL of each eluent and 100 μL of VCM in MeOH (50 μg/mL) as an internal standard were vortex-mixed in a micro-tube. Then, 30 μL of supernatant was injected in the HPLC system. The peak height ratios (VCM/internal standard) obtained from the standard solution were plotted against the VCM concentration to obtain a calibration curve, and the concentrations of VCM in the eluents were calculated. The HPLC conditions were as follows: mobile phase, 50 mmol/L ammonium acetate (pH 5.0)/CH3CN = 9/1; flow rate, 1.0 mL/min; column, TSK gel ODS–80 TM (250 mm × 4.6 mm internal diameter, 5 μm; TOSOH, Tokyo, Japan); column temperature, 40 °C; and detector, ultraviolet (246 nm).
Evaluating bioactivity using the broth microdilution method
Compressive strength test
After all spacers had been incubated in PBS at 37 °C for 84 days, we performed the compressive strength test. We took the samples out of PBS just before the test. Then, we performed the test immediately in the wet state. A uniaxial compressive load was applied to each spacer by the Universal Testing Machine (Instron model no. 33R 4467; Instron Corporation, Norwood, Massachusetts, USA). Spacers were positioned on the testing machine so that the drilled holes did not contact the loading plate. Compressive tests were performed with a cross-head speed of 5-mm/min, atmospheric pressure, and room temperature in the wet state. Compressive loads were applied until spacer failure occurred, and compressive strength was recorded as the maximum load applied before spacer failure.
Eluent VCM concentrations and compressive strength were compared for D-L and PMMA spacers using the Mann-Whitney U test, with two-sided P < 0.05 regarded as statistically significant. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R Commander designed to add statistical functions frequently used for biostatistics .
VCM concentrations (mean ± SD) and minimum VCM bioactivities
measured by HPLC
measured by HPLC
Compressive strength test
The D-L spacer was developed to achieve high-concentration and long-term elution of antibiotics. We made the following important observations regarding the D-L spacer in this study: (1) it released more antibiotics, (2) it was able to maintain bioactivity against MRSA, and (3) it was able to maintain high mechanical strength.
Regarding our first observation, we found that VCM concentrations of D-L spacer eluents significantly exceeded those of PMMA spacer eluents. This is consistent with the results of previous studies that showed that although PMMA allows only partial release of loaded antibiotics [15, 16], CPC allows the most loaded antibiotics to be released [8, 17, 18].
Sasaki et al. reported that the VCM elution volume from CPC on day 7 was 62.6 times that from PMMA, but that the rate thereafter decreased to 6.7 times on the day 13 . However, in this study, the VCM elution volume from the D-L spacer on day 7 was 2.3 times that from the PMMA spacer, but the rate continued to increase to 11.9 times until day 84. This indicated that VCM could be released slowly and over a long period by covering CPC with PMMA. It has been reported that this antibiotic release is initially controlled to some extent by surface phenomena, whereas long-term elution depends on penetration depth, as determined by the bulk porosity of the cement used . As such, we drilled multiple holes into CPC cores from the spacer surface to further facilitate VCM elution. Cement porosity, and thus the elution of antibiotics, can be increased with non-antibiotic fillers such as xylitol [20, 21]. However, fillers may degrade the mechanical properties of spacers, and the ideal amount of filler has not been established.
We also found that both D-L spacers and PMMA spacers maintained bioactivity throughout our study. Before the test, we expected that polymerization in these materials might have affected VCM activity. Specifically, because CPC does not generate the substantial amount of heat that is observed with PMMA polymerization, it does not cause the heat-induced antibiotic denaturation that may occur. It has also been reported that some organic solvents affect the stability of VCM complexes and peptide ligands , meaning the organic solvent contained in PMMA may have had some effect on VCM bioactivity. Considering these points, we expected to observe decreased bioactivity in the PMMA spacer. In contrast, inactivation or denaturation of loaded antibiotics, which was indicated by the presence of different peaks in HPLC analysis, was not observed. However, eluted antibiotics that show the same peak as the original loaded antibiotic do not necessarily have the same bioactivity. Therefore, we compared HPLC and bioactivity test results and found that they were comparable, and antibiotic inactivation/denaturation could be ruled out in our study.
VCM bioactivity can be affected by other factors not replicated in our study. First, biofilms that are formed by bacteria on implant surfaces can prevent the penetration of antibiotics. Nishimura et al. reported that even though antibiotics are effective against planktonic bacteria, the minimum bactericidal concentrations for biofilm bacteria of all antibiotics are high . In addition, it has been reported that the protein non-binding rate of VCM in serum is only approximately 45–50% . Although we could not obtain data regarding its protein-binding rate in joint fluid, the results of these studies suggest that the effect of VCM might be lost earlier in the living body than it was in our study.
It has also been reported that the MIC of VCM against MRSA is gradually increasing in several countries [25, 26, 27], which is a phenomenon known as MIC creep. We found that the minimum VCM bioactivity in PMMA barely exceeded 2 μg/mL until day 28, and it was lost by at least day 56. However, the minimum VCM bioactivity in the D-L spacer was higher than 2 μg/mL until at least day 84. If MIC creep with MRSA is considered (i.e., higher methicillin resistance levels, such as MIC of 2 μg/mL), then VCM concentrations in PMMA spacer eluents would only be effective until day 28 and are not guaranteed afterward; however, those in D-L spacer eluents would remain effective until at least day 84. This demonstrated the unreliability of the PMMA spacer and the possibility that it may be disadvantageous, especially when used for longer durations.
Finally, we found that the D-L spacer retained high mechanical strength throughout the study period, confirming our hypothesis that coating the mechanically weak CPC  with the stronger PMMA would result in a spacer with better mechanical properties than only CPC. This is important because the mechanical properties of cement are a primary clinical problem. Patients who are scheduled to undergo two-exchange arthroplasty for PJI are instructed to not place mechanical loads on the spacer-containing extremity while the spacer remains in the body (usually for 2 weeks to several months). However, it is possible that loads may be unconsciously placed on the affected extremity. When the patient stumbles, there could be a hip joint force of 7.2 times the body weight . Although we found that maximum compressive loads were significantly lower for D-L spacers than for PMMA spacers, even the weakest spacer could withstand 5.64 kN, or a load of 575 kg. This is the value that can withstand the hip joint force when an 80-kg patient stumbles. However, even if the spherical D-L spacer can withstand an 80-kg person stumbling, there is no guarantee that the strength of the D-L spacer will be sufficient when applied clinically. Future studies are necessary.
Despite the insights provided by this study, there were some limitations. First, because we changed the solution around the spacers every day, the experimental fluid dynamics differed from actual synovial fluid dynamics in the living body. Therefore, the possibility that the dissolution kinetics in our study differed from that in a real-life environment is high. Dissolution kinetics also vary with the amount of solvent present, which we did not consider during our comparison of D-L and PMMA spacers. Furthermore, because the spacer models used in this study were smaller than spacers used clinically, it is expected that the elution amount and period are different. Second, D-L spacers that are actually used in the clinical setting are not spherical; they also have a stem. Although cement spacer fractures sometimes occur, these tend to be localized mainly in the spacer stem and neck , and not on the spacer head. However, in this study, we have only studied the biomechanical properties of spherical femoral head spacer models without a stem. Third, it has been reported that the amount of antibiotics, the type of cement, and the method used to create the cement are correlated with the strength of the cement . Drilling several large holes can have a negative impact because these holes can act as stress concentration sites under loading. However, this was not investigated in our study. Fourth, we tested load failure once. We did not investigate the influence of repeated biomechanical loading as it happens during the clinical application. Therefore, future studies to further verify spacer strength are necessary.
We compared D-L and PMMA spacer properties in this study and found that the D-L spacer was superior to the PMMA spacer in terms of elution volume and maintenance of VCM bioactivity. The D-L spacer also maintained high mechanical strength during our study duration. Therefore, we concluded that due to the beneficial biomechanical and drug-eluting properties of the D-L spacer, it might be a promising biomaterial that could potentially be used for managing PJI.
The authors thank Mr. Satoshi Kishino, Mr. Ken Sugo, and Mr. Hidero Kitasato for their assistance.
This research did not receive any specific funding from any public or commercial entity.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
SI, KU, and MT were involved in the design of the study. KO measured the VCM concentrations. MN performed the bioactivity test. KY performed the compressive strength test. SI and YM were responsible for the data analysis. The manuscript was drafted by SI and revised by KU, KF, NT, and MT. All authors approved the final manuscript draft.
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Consent for publication
The authors declare that they have no competing interests.
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