Original Article Journal of Biomaterials Applications 0(0) 1–9 ! The Author(s) 2016 Elution profiles of tobramycin and Reprints and permissions: sagepub.co.uk/journalsPermissions.nav vancomycin from high-purity calcium DOI: 10.1177/0885328216663392 jba.sagepub.com sulphate beads incubated in a range of simulated body fluids JJ Cooper1, H Florance2,3, JL McKinnon1, PA Laycock1 and SS Aiken1 Abstract The aim of this study was to characterise the elution profiles of antibiotics in combination with pharmaceutical grade calcium sulphate beads in phosphate buffered saline and other physiological solutions which more closely mimic the in vivo environment. Synthetic recrystallised calcium sulphate was combined with vancomycin hydrochloride powder and tobramycin sulphate solution and the paste was formed into 3 mm diameter hemispherical beads. Then 2 g of beads were immersed in 2 ml of either phosphate buffered saline, Dulbecco’s Modified Eagle Medium or Hartmann’s solution and incubated at 37C for up to 21 days. At a range of time points, eluent was removed for analysis by liquid chromato- graphy-mass spectrometry (LC-MS). Tobramycin sulphate and vancomycin hydrochloride release was successfully quan- tified against standard curves from solutions eluted in all three physiological media (phosphate buffered saline, Dulbecco’s Modified Eagle Medium and Hartmann’s solution) during incubation with calcium sulphate beads. One hour eluate concentrations were high, up to 2602 mg/ml for tobramycin in phosphate buffered saline and 7417 mg/ml for vancomycin, whereas in DMEM, the levels of tobramycin were 2458 mg/ml and 4401 mg/ml for vancomycin. The levels in HRT were 2354 mg/ml for tobramycin and 5948 mg/ml for vancomycin. The results show highest levels of antibiotic elution over the first 24 h, which gradually diminish over the following 21 days. Keywords Antibiotics, calcium sulphate, elution Introduction now widely applied as an integral part of two-stage The clinical practice of application of antimicrobial treatment of revision arthroplasty as a result of infec- agents at the site of musculoskeletal infections is one tion, in the form of antibiotic-loaded articulated that is being increasingly reported. This ranges from the spacers or beads.5,6 direct infusion of antibiotic into intra-articular spaces Although providing essential load-bearing capabil- following total knee arthroplasty,1 to the intra-site ity, PMMA is a non-resorbing material requiring placement of antibiotic powder to prevent infection surgical removal. For indications where structural following spinal surgery.2,3 Due to the uncertainty over the duration of effective local antimicrobial concentrations with these methods, the combination 1Biocomposites Ltd., Keele Science Park, Staffordshire, UK of antibiotics with implantable materials in order to 2Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, UK enable predictable, longer-term, release profiles is 3 being widely investigated. Exeter Mass Spectrometry Facility, Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, UK The concept of local antibiotic release as part of an Corresponding author: infection management strategy is not new. Poly-methyl Phillip A Laycock, Biocomposites Ltd., Keele Science Park, Staffordshire methacrylate (PMMA) has been used in combination ST5 5NL, UK. 4 with antibiotics in this manner for over 40 years, and is Email: [email protected] Downloaded from jba.sagepub.com by guest on August 10, 2016 2 Journal of Biomaterials Applications 0(0) integrity is less important than the ability to predictably HRT, also known as Compound Sodium Lactate release antibiotic, the development of absorbable alter- (CSL), is a solution, isotonic with blood, commonly natives has gained momentum in recent years. used post-operatively to replace bodily fluids, especially Applications such as bone void fillers following surgical in situations of high blood loss. HRT has similar ion treatment of osteomyelitis,7–10 dead space management concentrations to those of human blood, hence the in soft tissue following joint revision surgery,11,12 and rationale for administering to patients suffering from diabetic wound care13,14 are amongst those reporting acute blood loss. It is also commonly used to irrigate promising outcomes. wounds during general surgery and arthroscopic proced- Fundamental to the potential utility of these mater- ures as it has compatible physiological properties.30,31 ials is the rate at which they release their antibiotic load while maintaining antimicrobial efficacy. As this is dif- Materials and methods ficult to accurately determine in vivo, the measurement of antibiotic elution is usually determined in vitro. The release of vancomycin hydrochloride and tobra- In the literature, a broad range of variations in mycin sulphate was evaluated to determine the effect in vitro experimental methods are used to determine of the chosen media on eluted levels of antibiotic. antibiotic elution. Methodologies vary in the nature The samples were prepared as beads, loaded with a by which the sample is presented to the solution, ran- fixed concentration of vancomycin hydrochloride and ging from single small beads15–17 to larger cast cylin- tobramycin sulphate. Beads were immersed in physio- ders.18,19 Also, variation occurs in the quantity of logical solutions at 37C, and the rate of antibiotic material tested, the volume into which the sample is elution was measured over time, up to a period of immersed, the volume removed for analysis and the three weeks using liquid chromatography mass spec- eluent sampling intervals.20,21 However, there is limited trometry (LC-MS). data available on the effect of the elution medium on The in vitro conditions executed were intended to the release of antibiotics. Frequently, elution experi- mimic the implantation of calcium sulphate beads in ments use phosphate buffered saline (PBS) as the a small bone defect. The volume fill of 2 g of calcium elution medium, but this has limited similarity to the sulphate beads is comparable to those used clinically in in-vivo environment with respect to composition and a small bone defect. viscosity. In this study, the antibiotics chosen were vanco- Preparation of beads mycin hydrochloride and tobramycin sulphate since these are frequently used together in clinical practice All bead/antibiotic combinations were prepared using as they provide good empiric coverage for a high pro- the SRCS, provided as a powder (alpha calcium sul- portion of both Gram-positive and Gram-negative phate hemihydrate, CaSO4 Á 1/2 H2O) together with pathogens frequently encountered in surgical site infec- an aqueous mixing solution. When mixed, the resultant tions (SSIs). These were combined with a synthetic paste hydrates and sets according to the following recrystallised calcium sulphate (SRCS), (Stimulan reaction Rapid Cure, Biocomposites Ltd, UK), a fully absorb- able implant material that has been reported previously CaSO4 Á 1=2H2O þ 11=2H2O ¼ CaSO4 Á 2H2O 11–13,22–28 in literature in combination with antibiotics. Calcium sulphate hemihydrate þ water The aim of this study was to characterise the elution ¼ profiles of antibiotics in three different media: phos- Calcium sulphate dihydrate phate buffered saline (PBS) and two physiological solutions which more closely mimic the in-vivo environ- The beads were prepared as follows: In a mixing ment, Dulbecco’s Modified Eagle’s medium (DMEM) bowl, vancomycin hydrochloride powder (1 g, Hospira and Hartmann’s Solution (HRT). UK) was blended with SRCS powder (20 g, powder Although DMEM is water based, the presence of contents of Stimulan Rapid Cure, 10cc pack size, bovine serum increases viscosity and the potential for Biocomposites Ltd, UK) for 1 min to produce a homo- protein binding, resulting in a medium more closely genous mixture. This particular calcium sulphate hemi- mimicking the in vivo environment. In a previous hydrate powder is prepared from high purity study where calcium sulphate was used in combination pharmaceutical grade reagents. When blended with with DMEM and Bone marrow stem cells (BMSC), no the hydrating solution, it has a physiologic pH and adverse effects were reported and it was concluded that contains no earth impurities or stearate additives. It is it was a successful combination for bone tissue engin- known that antibiotic solubility and performance can eering in the treatment of long-standing non-union of vary with pH and this is an important and unreported the Tibia.29 variable in many elution studies. Downloaded from jba.sagepub.com by guest on August 10, 2016 Cooper et al. 3 To the blended powders was added tobramycin sul- phase was held at 5% B for 30 s before increasing in phate solution (6 ml, 40 mg/ml, Hospira UK). The con- a linear fashion to 100% over 3.5 min, where it was tents of the bowl were then mixed together for 30 s to then held for 1 min. Column temperature was main- form a smooth paste. The paste was then transferred tained at 30C with a flow rate of 0.25 ml/min. Due into the mould mat to produce hemispherical beads to the size of the study, high throughput methods with a diameter of 3.0 mm. The beads were allowed to (i.e. short gradients with simultaneous detection of all set in the mould for 15 min before removal. compounds) were required to be able to maximise rep- The beads were extracted from the mould once com- lication and minimise the length of time the samples pletely set simply by flexing the mould and were then were left to stand at 4C. To this end, a compromise stored in sealed moisture proof pouches, to prevent on column length and flow rate was required. The pola- drying out, in a refrigerator at 5C overnight. Two rities of the two antibiotics are very different. grams of the moulded antibiotic beads contain 74 mg Tobramycin is a highly polar molecule and not well vancomycin and 18.5 mg tobramycin. retained on reverse phase columns, whereas vanco- mycin is non-polar and well retained.