Abstract
Background
Antibiotic-laden calcium sulfate beads are gaining popularity in the treatment of orthopaedic infections such as fracture-related infection and osteomyelitis. Calcium sulfate beads have several advantages over polymethylmethacrylate (PMMA) beads as they are bioabsorbable, have demonstrated improved elution characteristics, and have lower peak polymerization temperatures than seen in PMMA. The ability to make and store antibiotic beads for later use has the potential to standardize dosing and decrease operating room times and healthcare costs. This study aims to determine the antibiotic efficacy of premade, antibiotic-laden calcium sulfate beads.
Methods
Calcium sulfate beads containing vancomycin or tobramycin were molded to 4.8 mm in diameter and stored for shelf-life durations of three and six months at 20 °C. A subset of beads was tested immediately after creation. At the designated time points, beads were placed into a buffer solution and incubated at 37 °C with agitation. Antibiotic eluent was collected at 1-hour, 4-hour, 24-hour, and 48-hour timepoints. Eluent concentrations were inferred from a prior study implementing the same calcium sulfate bead model. Eluent was used in microbroth dilution assays to determine its minimum inhibitory concentration (MIC) against methicillin-sensitive Staphylococcus aureus.
Results
MIC assays for tobramycin and vancomycin against S. aureus yielded concentrations consistent with previously reported ranges. MIC results across different bead shelf lives also remained consistent without an increase in MIC with increasing shelf life for either antibiotic.
Conclusions
Shelf life up to six months does not impact the efficacy of tobramycin or vancomycin eluent from calcium sulfate beads in vitro compared to beads made and tested immediately. These results provide preliminary evidence that tobramycin and vancomycin retain their antimicrobial activity in calcium sulfate beads for at least six months stored at room temperature. Additional studies on sterilization techniques are necessary prior to considering use of prefabricated antibiotic-loaded calcium sulfate beads in clinical practice.
Keywords: Orthopaedic infection, Antibiotic beads, Calcium sulfate, Shelf life, Local antibiotic therapy
1. Introduction
Osteomyelitis, or infection of the bone, can occur due to contamination in open fractures or spread of microbes from nearby infected tissue.1 Orthopaedic infections in the setting of metal implants, such as after fracture fixation or arthroplasty, are especially difficult to treat given the tendency of implants to harbor bacteria on implant surfaces. Staphylococcus, Enterobacteriaceae, and Pseudomonas are some of the most common organisms identified in osteomyelitis, deep tissue infection, and implant-related infections.2, 3, 4 In addition to adequate surgical debridement and systemic treatment, local antibiotic therapy is a mainstay of treatment of such infections. The most common method of local drug delivery is antibiotic-laden polymethylmethylacrylate (PMMA) bone cement, first used in the 1970s.5 Vancomycin, as well as aminoglycoside antibiotics such as tobramycin, have been thoroughly studied within PMMA and are considered the gold standard of antibiotics for local delivery due to their stability during exothermic cement polymerization and at prolonged human physiological temperature.6 Local delivery of aminoglycosides is preferential to systemic treatment as it circumvents potential systemic side effects seen with this class of drugs such as ototoxicity and nephrotoxicity.3
Though commonplace, antibiotic-loaded PMMA beads present several drawbacks. As PMMA is not bioabsorbable, additional surgery for bead removal is necessary. Furthermore, the high polymerization temperature needed to create PMMA beads limits the options of antibiotic classes that can be used in bead creation. Alternatively, calcium-sulfate (CaSO4) cement beads are a drug delivery vehicle that is bioabsorbable, does not require surgical removal, and polymerizes at much lower temperatures, thus allowing a wider range of antibiotic selection.
Antibiotic beads are typically made during the procedure in the operating room under sterile conditions and require an entire package of cement be used. The option to prepare, sterilize, and package the beads in different quantities in advance would yield multiple benefits, such as decreased operative time, reduced wasted material and antibiotics, and decreased overall costs of surgery. In addition, prefabrication of beads would expand the opportunity for usage beyond the hospital operating room setting. One prior study has investigated the shelf life of calcium sulfate beads used in setting of diabetic foot wounds with and without osteomyelitis.7 The utility of premade calcium sulfate beads can be broadened to include combat settings and other instances of orthopaedic fractures beyond diabetic care. In this study, we sought to determine the impact of shelf life of pre-made calcium sulfate beads containing tobramycin or vancomycin on antibiotic elution and antimicrobial efficacy up to six months.
2. Methods
2.1. Antibiotic beads
Calcium sulfate beads were prepared using a commercial 4.8 mm diameter bead mold (Stimulan Rapid Cure; Biocomposites, Ltd.; Staffordshire, UK) under sterile and ambient conditions (20 °C, 1 atm pressure, 35% humidity). Beads were mixed with either 5% dry weight vancomycin (MP Biomedicals, Solon, OH) or 2.5% dry weight tobramycin (MP Biomedicals, Solon, OH) powder; antibiotic powder was mixed with the calcium-sulfate powder before adding the monomer. A lower percentage of antibiotic was used in the tobramycin beads to allow proper hardening of beads for usage.8 Five sets of four beads were made for each antibiotic to test shelf life at different time intervals (immediately after creation [0-day control], 3 months, 6 months). Beads were stored in sterile 15 mL conical tubes at 20 °C until interval time was reached for experimentation.1
2.2. Antibiotic elution
At each respective shelf life, one bead was placed into a 2 mL Eppendorf tube with 500 μL phosphate-buffer saline (PBS) and incubated at 37 °C with agitation to allow elution of the antibiotic. Antibiotic eluent was collected at specific timepoints (1 h, 4 h , 24 h, 48 h ) correlating to maximum expected elution. Beads were washed with 50 μL PBS in the same collecting tube they were incubated in and then transferred to a new tube with 500 μL PBS to continue antibiotic elution until the next time point.8 Collected eluent was used to perform microbroth dilution assays to determine the minimum inhibitory concentration (MIC) against methicillin-sensitive Staphylococcus aureus (American Type Culture Collection 12600; Microbiologics Inc.; St. Cloud, MN). Antibiotic eluent concentrations were estimated via high-performance liquid chromatography (HPLC) data from a previous study using the same in vitro model.8 Based on these estimations, collected eluent was subsequently diluted with PBS to achieve target concentrations to perform microbroth dilution assays.
2.3. Microbroth dilution assays
Microbroth dilution assays were performed to determine the antimicrobial efficacy of each antibiotic after its elution from the calcium-sulfate beads against methicillin-sensitive S. aureus. Vancomycin and tobramycin stock solutions were preliminarily tested to obtain baseline MIC values as a relative control for comparison to eluent efficacy. In a 96-well plate, cation-adjusted Mueller Hinton Broth (CAMHB) media (BD #212322) and diluted antibiotic eluent were added together at a 1:1 ratio in the first column and serially diluted across columns at a factor of two to yield ten different testing concentrations. The last two columns were designated for a positive control (S. aureus without antibiotic) and a negative control (CAMHB media only).
Bacterial overnight cultures of S. aureus were grown in tryptic soy broth (BD #211825) media and incubated at 37 °C, 250 rpm. Overnight cultures were diluted 1:50 in media and re-incubated at 37 °C, 250 rpm to achieve mid-logarithmic growth phase, designated at an optical density of 600 nm on spectrophotometry (SPECTRAmax PLUS384 Microplate, Molecular Devices; San Jose, CA). To confirm proper bacterial inoculation concentrations used for the dilution assays, the bacterial culture was diluted to yield countable colony forming units (CFU) on spread plating.
2.5 × 105 CFU of bacterial inoculation was added to each well, and the plate was then incubated at 37 °C for 24 h. Plates were read for quantitative absorbance via spectrophotometry at 600 nm (SoftMax Pro 6.2, Molecular Devices; San Jose, CA) to determine the MIC90, defined as the first concentration inhibiting 90% of bacterial growth compared to the positive control. All samples were tested in quadruplicate.
3. Results
The average MIC90 values for tobramycin and vancomycin across all elution times for each shelf time are reported in Table 1. Baseline tobramycin and vancomycin MIC90 values obtained with antibiotic stock solutions were 2.375 μg/mL and 1.00 μg/mL, respectively. Average MIC90 for tobramycin at time points of 1, 4, and 24 h were 2.00 ± 0.00 μg/mL, 0.55 ± 0.58 μg/mL, and 0.46 ± 0.64 μg/mL, respectively. Average MIC90 for vancomycin at time points of 1, 4, 24, and 48 h were 0.92 ± 0.20 μg/mL, 1.00 ± 0.00 μg/mL, 0.50 ± 0.18 μg/mL, and 0.42 ± 0.12 μg/mL, respectively. Bacterial inoculation concentrations used for the microbroth dilution assays were also confirmed via spread plating. Counts on spread plating across all experiments was 193 ± 28 CFU, confirming bacterial inoculation concentrations used for microbroth dilution assays.
Table 1.
Average MIC90 across Shelf Times
Average MIC90 across shelf times accounting for all incubation timepoints (1 h, 4 h, 24 h, 48 h). Each incubation timepoint MIC90 value represents the average of quadruplicate tests.
| Shelf Time | Average MIC90 (μg/mL) ± S.D. |
|
|---|---|---|
| Tobramycin | Vancomycin | |
| 0 day | 1.46 ± 0.56 | 0.72 ± 0.25 |
| 3 months | 0.87 ± 0.82 | 0.66 ± 0.35 |
| 6 months | 0.82 ± 0.86 | 0.75 ± 0.25 |
MIC90 values at each elution time point for three months and six months shelf-life samples were compared to the MIC90 values of the set of beads that was not stored to calculate a relative MIC90 value (Fig. 1). A relative MIC90 of 1.0 indicated that the MIC90 between the stored and non-stored beads was the same. A relative MIC90 > 1.0 indicated that the stored beads had an MIC90 value greater than that of the non-stored beads, while a relative MIC90 < 1.0 indicated the stored bead had a lower MIC90 value than that of the non-stored beads. Relative MIC90 values at all elution time points and shelf times are comparable to non-stored calcium sulfate beads. Importantly, the MIC90 values did not increase at any shelf life time point for either antibiotic.
Fig. 1.
MIC90 against methicillin-sensitive S. aureus via microbroth dilution assay. The 0-day Control subset of beads was tested immediately after creation. The 48 hour elution timepoint was excluded for tobramycin due to insufficient eluent concentrations for MIC testing based on values inferred from a previous study.8
4. Discussion
Shelf life of calcium sulfate beads containing antibiotics has not been fully investigated. Preparation of beads prior to surgery outside of the operating room yields multiple cost saving advantages including decreased operating room time and less material waste. Additionally, the use of pre-made beads broadens the indications of bead usage to settings outside of the operating room, including combat and austere settings as well as outpatient use for chronic wounds.
Our data suggest that shelf life up to six months does not impact efficacy of antibiotic eluent from calcium sulfate beads compared to that of beads made and allowed to elute immediately after creation. Because the eluent concentrations were inferred from data from a prior study and were not reconfirmed via HPLC testing, the primary aim of this study was to determine and elucidate any possible inter-shelf-life differences in MIC90 trends rather than the absolute MIC90 values. With respect to our preliminary baseline MIC90 values obtained from antibiotic stock testing, all of the MIC90 values obtained within our study are consistent with previously reported clinical ranges. This study investigates the impact of shelf life on antibiotic efficacy of calcium sulfate beads up to six months, the longest analysis period reported in literature thus far.
This study has several limitations. Eluent concentrations used to guide eluent dilutions for the microbroth assay were drawn from quantitative HPLC data obtained utilizing the same experimental model. This study did not directly quantify eluent concentrations at the tested time points, which may explain the decreasing MIC90 trend shown in the data. However, the MIC90 values obtained from each bead set yielded concentrations that were within reported ranges, verifying our estimated calculations of the antibiotic eluent concentrations. Our study only investigates antibiotic eluent efficacy of tobramycin and vancomycin against one strain of S. aureus and does not evaluate the effect of particular sterilization techniques on elution kinetics or antibiotic efficacy.1,7,9, 10, 11, 12, 13
5. Conclusion
This study demonstrates that antibiotic eluent from pre-made calcium sulfate antibiotic beads retains antibiotic efficacy for at least six months after creation. Future studies with longer shelf lives will be needed to determine a maximum safety limit for storage duration. Additional studies may also help define ideal storage conditions accounting for temperature and humidity. Implementation of the same model should also be completed for alternative antibiotic options against other common pathogens found in the setting of orthopaedic trauma to determine if these antibiotics maintain their integrity after long-term storage. Furthermore, the impact of sterilization techniques on premade beads will be necessary prior to clinical use. Overall, this study provides preliminary evidence that prefabricated calcium sulfate beads embedded with tobramycin and vancomycin maintain their antimicrobial activity for at least six months when stored at room temperature.
Guardian/patient consent
This research did not involve any human subjects and therefore does not require guardian/patient consent.
Ethical statement
This research did not involve any human subjects and is therefore exempt from IRB approval. Informed consent for this study is not required. The authors agree that this research work represents honest and original work.
Funding
We acknowledge funding from the National Institutes of Health (NIH) Grant NIAID T35 AI125220 and from AO Trauma North America Young Investigator Research Development Award.
CRediT authorship contribution statement
Samuel R. Shing: Conceptualization, Methodology, Validation, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization, Funding acquisition. Aaron K. Hoyt: Formal analysis, Writing – review & editing, Visualization, Supervision. Ashley E. Levack: Conceptualization, Methodology, Validation, Resources, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition.
Declarations of competing interest
None.
Acknowledgements
We would like to thank Dr. John Callaci for helpful discussion and experimental support.
References
- 1.Shaw J., Gary J., Baker A., et al. Effects of sterilization techniques on bioactivity of polymethyl methacrylate antibiotic beads containing vancomycin and tobramycin. J Orthop Trauma. 2020 Apr;34(4):e109–e113. doi: 10.1097/BOT.0000000000001729. [DOI] [PubMed] [Google Scholar]
- 2.Levack A.E., Cyphert E.L., Bostrom M.P., Hernandez C.J., von Recum H.A., Carli A.V. Current options and emerging biomaterials for periprosthetic joint infection. Curr Rheumatol Rep. 2018 Apr 30;20(6):33. doi: 10.1007/s11926-018-0742-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Gogia J.S., Meehan J.P., Di Cesare P.E., Jamali A.A. Local antibiotic therapy in osteomyelitis. Semin Plast Surg. 2009 May;23(2):100–107. doi: 10.1055/s-0029-1214162. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Masri B.A., Duncan C.P., Beauchamp C.P. Long-term elution of antibiotics from bone-cement: an in vivo study using the prosthesis of antibiotic-loaded acrylic cement (PROSTALAC) system. J Arthroplasty. 1998 Apr;13(3):331–338. doi: 10.1016/s0883-5403(98)90179-6. [DOI] [PubMed] [Google Scholar]
- 5.Buchholz H.W., Elson R.A., Heinert K. Antibiotic-loaded acrylic cement: current concepts. Clin Orthop Relat Res. 1984 Nov;190:96–108. [PubMed] [Google Scholar]
- 6.Lawrie C.M., Jo S., Barrack T., et al. Local delivery of tobramycin and vancomycin in primary total knee arthroplasty achieves minimum inhibitory concentrations for common bacteria causing acute prosthetic joint infection. Bone Joint Lett J. 2020 Jun;102-B(6_Supple_A):163–169. doi: 10.1302/0301-620X.102B6.BJJ-2019-1639.R1. [DOI] [PubMed] [Google Scholar]
- 7.Armstrong D.G., Stephan K.T., Espensen E.H., Lipsky B.A., Boulton A.J. What is the shelf life of physician-mixed antibiotic-impregnated calcium sulfate pellets? J Foot Ankle Surg. 2003 Sep-Oct;42(5):302–304. doi: 10.1016/s1067-2516(03)00307-7. [DOI] [PubMed] [Google Scholar]
- 8.Levack A.E., Turajane K., Driscoll D.A., et al. Identifying alternative antibiotics that elute from calcium sulfate beads for treatment of orthopedic infections. J Orthop Res. 2022 May;40(5):1143–1153. doi: 10.1002/jor.25135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Báez L.A., Langston C., Givaruangsawat S., McLaughlin R. Evaluation of in vitro serial antibiotic elution from meropenem-impregnated polymethylmethacrylate beads after ethylene oxide gas and autoclave sterilization. Vet Comp Orthop Traumatol. 2011;24(1):39–44. doi: 10.3415/VCOT-10-05-0070. [DOI] [PubMed] [Google Scholar]
- 10.Trencart P., Elce Y.A., Rodriguez Batista E., Michaud G. Sterilization by gamma radiation of antibiotic impregnated polymethylmethacrylate and plaster of Paris beads. A pilot study. Vet Comp Orthop Traumatol. 2014;27(2):97–101. doi: 10.3415/VCOT-13-03-0037. [DOI] [PubMed] [Google Scholar]
- 11.Ramos J.R., Howard R.D., Pleasant R.S., et al. Elution of metronidazole and gentamicin from polymethylmethacrylate beads. Vet Surg. 2003 May-Jun;32(3):251–261. doi: 10.1053/jvet.2003.50024. [DOI] [PubMed] [Google Scholar]
- 12.Noor S., Gilson A., Kennedy K., et al. Pre-packing of cost effective antibiotic cement beads for the treatment of traumatic osteomyelitis in the developing world - an in-vitro study based in Cambodia. Injury. 2016 Apr;47(4):805–810. doi: 10.1016/j.injury.2016.01.038. [DOI] [PubMed] [Google Scholar]
- 13.Durham M.E., Elfenbein J.R. Evaluation of vaporized hydrogen peroxide sterilization on the in vitro efficacy of meropenem-impregnated polymethyl methacrylate beads. Am J Vet Res. 2019 Jan;80(1):45–50. doi: 10.2460/ajvr.80.1.45. [DOI] [PubMed] [Google Scholar]