Aim. The aim of this study is to evaluate if the gentamycin elution from bone cement is influenced by the timing of application of the antibiotic powder. Method. This was an experimental in vitro study that compared the elution properties of different formulation of gentamycin from a commercially available hip, knee and shoulder cement spacers. Four different experimental models were prepared. Five different spacers were prepared for each experimental mode and for each joint. We compared four different formulation of cement spacers: spacer #1, in which the spacer was prepared with a premixed bone-cement antibiotic mixture; spacer #2, in which the spacer was prepared by adding antibiotic powder to the bone cement at the time of spacer preparation; spacer #3, in which the spacer was prepared as spacer #2 but was stored for two months before starting the experiment; spacer #4, in addition to the gentamycin, other two antibiotics (tobramycin and vancomycin) were added to the bone cement. Gentamycin concentration was documented at seven intervals of time: T0 = 0h, T1 = 1h, T2 = 24h, T3 = 1W, T4 = 2W, T5 = 1M, T6 = 3M and T7 = 6M. The gentamycin elution at each interval of time was evaluated by using a T-student test. Results. Spacer #2, in which the gentamycin powder was added to the bone cement at the time of spacer preparation showed the higher gentamycin elution at each interval of time observed. In addition, Spacer #1, in which gentamycin powder was premixed with the bone cement showed a higher gentamycin elution when compared with spacer #3, in which the spacers were stored for two months to simulate the preformed spacers. Lastly, the addition of different antibiotic to the bone cement increases the gentamycin elution from the spacers (as demonstrated by spacer #4 model). Conclusions. a higher gentamycin elution was observed if spacer was prepared at the time of surgery when compared with preformed spacer. Lastly, our study confirmed the synergistic effect of adding one or more antibiotics with the aims to increase gentamycin elution

Abstract. Background. The aim of the present experimental study was to analyse vancomycin elution kinetics of nine bone fillers used in orthopaedic and trauma surgery over 42 consecutive days. Methods. Two allograft bone chips (carriers 1 and 2), a calcium-sulfate matrix (carrier 3), a hydroxyapatite/calcium-sulphate composite (carrier 4), four bone cements (carriers 5-8) and a pure tricalcium phosphate matrix (carrier 9), either already contained vancomycin, or were mixed with it following manufacturer's recommendations. Over 42 days, half of elution medium was substituted by the same amount of PBS at 9 distinct time points. Vancomycin concentration in obtained samples were measured with a kinetic microparticle immunoassay, and masses consecutively calculated. To enhance comparability between carriers analysed, vancomycin mass released related to overall mass within each probe was determined. Notably, elution kinetics of carriers 1 to 4 have been published previously. Results. All carriers initially released high vancomycin masses, followed by constant reduction later into the experiment. Mean initial vancomycin masses released after 4 hours were highest for carriers 1 (337.7 ± 76.2 mg), 9 (68.4 ± 4.9 mg), and 2 (49.0 ± 54.6 mg). From prefinal (35 days) to last measurement (42 days) carriers 2 (8.6 ± 4.8 mg), 1 (2.4 ± 1.0 mg), and 5 (0.1 ± 0.1 mg) had released highest vancomycin masses. Notably, all five bone cements tested only released a small percental amount of their total mass up to the last measurement (42 days; 2.1% – 9.3%), whilst allografts and resorbable synthetic bone fillers discarded high percental values (22.5% – 79.2%). Conclusions. Elution kinetics differ between 9 antibiotic-loaded bone fillers, with high vancomycin masses released by allografts and resorbable bone fillers over time. Transferred to clinical practice, these may be favoured over bone cements in case prolonged and high antibiotic release is warranted rather than mechanical stability

Prosthetic joint infection(PJI) still remains a concern in orthopaedic practice. Antibiotic-loaded acrylic-cement(ALAC) is a proven means of lowering the incidence of PJI. However, increasing antimicrobial resistance has complicated both prophylaxis and treatment, prompting the use of combination antimicrobial therapy, with the addition of vancomycin to gentamicin-containing ALAC commonly used. The new antimicrobial, daptomycin, has better activity than vancomycin and we studied its elution from ALAC in comparison with vancomycin, along with its impact on the co-elution of gentamicin. Cement beads were prepared from PalacosRG containing, 1g/2g daptomycin, 1g/2g vancomycin and without additional antibiotics. Six replicates of each combination were eluted in PBS at 37oC, at timed intervals, for up to 90days, the antibiotic loss was assessed using validated assays. The mean recovery of gentamicin after 90days was 1.1mg with half eluted within the first 6 hours. Recovery was significantly increased by 60% and 40% with addition of 1g&2g of daptomycin(two-tail t-test: p=0.004 and p=0.02), respectively. Although there was a slight increase in gentamicin recovery in vancomycin loaded samples, this was not statistically significant(p>0.05). The significant increases in gentamicin elution from Palacos RG when supplemented with daptomycin, along with a superior activity, may provide a better synergistic effect than PalacosRG supplemented with vancomycin in the management of PJI

Prosthetic joint infections are difficult to treat due to bacterial biofilm. Our group has developed a linezolid elution system by human cancellous bone delivering high concentrations the first 48 hours (Giannitsioti et al. 53rd ICAAC, 2013: A-1050). We tested the activity of this system to inhibit growth of one ica expressing isolate of Staphylococcus epidermidis (MRSE). At a first step, sterile mesh cylinders containing bone particles of the femoral head of healthy volunteers (MCB) were impregnated into 3mg/ml linezolid for 1, 24 and 48 hours. Then log-phase inocula of 103, 105 and 107cfu/ml were exposed to MCB at 370C for 8 days with regular readings of bacterial growth. MCB were transferred into fresh Muller-Hinton Broth (MHB) every 24h to avoid material corrosion. At a second step, to simulate the ability of the system against biofilm-coated MCB, MCB without linezolid were incubated with 103 and 105 cfu/ml for 1 and 24h. MCB were daily transferred into fresh MHB containing 100μg/ml on day 1, 15 μg/ml on day 2, 3 μg/ml on day 3 and 0.5 μg/ml on day 4. 24h linezolid impregnated MCB achieved rapid bacterial killing of the 105 cfu/ml bacterial suspension followed by re-growth (Figure, n= 5). Similar results were observed for 1h and 48h impregnation and for both tested inocula. When biofilm-coated MCB generated by 24h exposure to 105 cfu/ml were exposed to linezolid, rapid bacterial killing was achieved followed by re-growth. Linezolid local elution may inhibit biofilm-producing MRSE only when locally eluted concentrations exceed 10μg/ml

Aim. In vivo studies have shown a preventive and curative effect of using an injectable vancomycin containing biphasic ceramic in an osteomyelitis model. No clinical long term pharmacokinetic release study has been reported. Inadequate concentration in target tissues results in treatment failure and selection pressure for antibiotic-resistant organisms. Our hypothesis was that vancomycin in the first week would reach high local concentrations but with low systemic levels. Method. 9 patients (6 women, 3 men) with trochanteric hip fractures classified as A1 and A2 according to the AO-classification all had internal fixations. The mean age was 75.3 years (± S.D. 12.3 years, range 44–84y). An injectable ceramic with hydroxyapatite embedded in a calcium sulphate matrix containing 66mg vancomycin per mL augmented the fixation. A mean of 9.7 mL (± S.D. 0.7 mL, range 8–10mL) was used. The elution of vancomycin was followed by collecting drain fluid, blood (4 days) and urine (4 weeks). Results. The concentration of antibiotics in the drain showed an important burst during the first 12h after surgery, with a mean value of 709.9 µmol/L (± S.D. 383.9), which decreased linearly to a mean value of 60.9 µmol/L at 2.5 days. In the urine, the vancomycin concentration reached 68.9 µmol/L (± S.D. 34.4) during the first day, which was decreased logarithmic over the first two weeks to reach zero at 20 days (see Figure). The systemic concentration of vancomycin was constantly low, not exceeding 2.6 µmol/L. Conclusions. This is the first long term pharmacokinetic study reporting vancomycin release from a biphasic injectable ceramic bone substitute. The study shows initial high targeted local vancomycin levels (wound drainage), sustained and complete release at three weeks (verified by the urine concentrations), and systemic concentrations well below toxic levels. This system should be useful in preventing and treating bone infection

Aim. Local antibiotics released through a carrier is a commonly used technique to prevent infection in orthopaedic procedures. An interesting carrier in aseptic bone reconstructive surgery are bone chips impregnated with AB solution. Systemically administered Cefazolin (CFZ) is used for surgical site infection prophylaxis however in vitro study showed that fresh frozen and processed bone chips impregnated with CFZ solution completely release the CFZ within a few hours. On the other hand irradiated freeze-dried bone chips, treated with supercritical CO2 (scCO2) have been shown to be an efficient carrier for the antibiotics vancomycine or tobramycine. With this pilot study we wanted to investigate if CFZ solution impregnation of bone chips treated with scCO2 shows a more favorable release pattern of CFZ. Method. The bone chips were prepared using the standard scCO2 protocol and were impregnated with 100 mg/ml cefazolin at different timepoints during the process: before freeze drying (BC type A), after freeze drying (BC type B) and after gamma-irradiation. 0.5g of the impregnated bone grafts were incubated with 5ml of fetal calf serum (FCS) at 37°C. At 2, 4, 6, 8 and 24h of incubation 200µl of eluate was taken for analysis. After 24h the remaining FCS was removed, bone grafts were washed and new FCS (5ml) was added. Consecutive eluate samples were taken at 48, 72 and 96h of incubation. The concentration of CFZ in the eluates was measured with the validated UPLC-DAD method. Analysis was performed in triplicate. Results. The mean concentration of CFZ in the eluate obtained from BC type A incubated for 2h was higher compared to BC type B, respectively 581 mg/l and 297 mg/l. However, the elution profile is the same for both types: the CFZ concentration in the eluates drops within the first 24h from 581 mg/l to 365 mg/l (37%) for BC type A and from 297 mg/l to 132 mg/l (56%) for BC type B. After 24h no further significant CFZ release is seen. Impregnation of the bone chips before or after gamma irradiation did not affect this elution profile. Conclusions. Bone chips treated with scCO2 show a comparable elution pattern compared to non-scCO2 treated bone chips. AB release depends on the properties of the AB, making it impossible to copy the same impregnation protocol for different antibiotics. The stability of CFZ in solution at 37°C and its release are a major concern when establishing an impregnation protocol with CFZ

Objectives. Investigate the incorporation of an antibiotic in bone cement using liposomes (a drug delivery system) with the potential to promote osseointegration at the bone cement interface whilst maintaining antibiotic elution, anti-microbiological efficacy and cement mechanical properties. Prosthetic joint infection and aseptic loosening are associated with significant morbidity. Antibiotic loaded bone cement is commonly used and successfully reduces infection rates; however, there is increasing resistance to the commonly used gentamicin. Previous studies have shown gentamicin incorporated into bone cement using liposomes can maintain the cement's mechanical properties and improve antibiotic elution. The phospholipid phosphatidyl-l-serine has been postulated to encourage surface osteoblast attachment and in a liposome could improve osseointegration, thereby reducing aseptic loosening. Preliminary clinical isolate testing showed excellent antimicrobial action with amoxicillin therefore the study aims were to test amoxicillin incorporated into bone cement using liposomes containing phosphatidyl-l-serine in terms of antibiotic elution, microbiological profile and mechanical properties. Methods. Amoxicillin was encapsulated within 100nm liposomes containing phosphatidyl-L-serine and added to PMMA bone cement (Palacos R (Heraeus Medical, Newbury, UK)). Mechanical testing was performed according to Acrylic Cement standards (ISO BS 5833:2002). Elution testing was carried out along with microbiological testing utilising clinical isolates. Results. Liposomal encapsulated amoxicillin PMMA bone cement exceeded minimum ISO BS 5833:2002 standards, had better elution at 12.9% when compared with plain amoxicillin (p=0.036 at 48 hours) or commercial gentamicin cement (Palacos R+G, Heraeus Medical, Newbury, UK – previous studies showed 6% elution over the same time period). Amoxicillin showed superior antimicrobial action when compared with gentamicin of the same concentration. However, liposomal encapsulated amoxicillin in solution and liposomal encapsulated amoxicillin in PMMA were both less effective than free amoxicillin in bacterial growth inhibition. The liposomal amoxicillin also seemed to decrease the cement setting time. Conclusions. Phosphatidyl-l-serine containing liposomes maintained the cement's mechanical properties and seemed to have better antibiotic elution, however, had less effective antibacterial action than plain amoxicillin. This difference in antibacterial action requires further investigation along with investigation of osteoblast attachment to phosphatidyl-l-serine containing liposomes within cement. Plain amoxicillin, for those not penicillin allergic, seems to be a credible alternative to gentamicin for incorporation in PMMA bone cement. It has shown superior antibacterial action, which may improve infection rates, whilst maintaining the cement's mechanical properties

Aim. Allograft bone chips used in complex bone reconstruction procedures are associated with an increased infection risk. The perioperative use of systemic cefazolin is standard to prevent infection, but is less effective in the presence of avascular bone grafts. Bone chips have been described as a carrier for local delivery of antibiotics, but impregnation with cefazolin in a prophylactic setting has not been described. We aimed to obtain a prolonged cefazolin release from bone chips to maximize the prophylactic effect. Method. Three types of bone chips were evaluated: fresh frozen, decellularized frozen and decellularized lyophilized. Bone chips were incubated with 20 mg/ml cefazolin or treated with liquid hydrogel containing either 1 mg/ml fibrin or 1 mg/ml collagen and 20 mg/ml cefazolin. The cefazolin hydrogel was distributed in the porous structure by short vacuum treatment. Bone chips with cefazolin but without hydrogel were incubated for 20 min- 4h under atmospheric pressure or under vacuum. Cefazolin elution of bone chips was carried out in fetal bovine serum and analyzed by Ultra Performance Liquid Chromatography – Diode Array Detection. Results. Without hydrogel, cefazolin release was limited to 4 hours. When vacuum was applied during impregnation, elution of cefazolin exceeding the MIC (minimal inhibitory concentration) from decellularized lyophilized bone chips was obtained for 36 hours. Use of a collagen hydrogel and vacuum treatment resulted in a high concentration at 24 hours, but did not support prolonged release for any of the three types of tested bone chips. In contrast, combination of decellularized frozen bone chips with fibrin hydrogel resulted in an initial release of 533 μg/ml, declining to the MIC at 72 hours, while no longer measurable after 92 hours. Such elution profile is desirable, since high initial levels are important to maximize antibacterial action whereas the complete wash out prevents antibiotic resistance. By increasing the cefazolin concentration during impregnation, elution above the MIC could be obtained for 120 hours. Impregnated bone chips stored at −20° C for 3 months performed similarly to freshly impregnated bone chips. Conclusions. Bone chips processed with the described hydrogel-based impregnation protocol allows tunable delivery of cefazolin for a local prophylactic effect

High-dose antibiotic-loaded acrylic cement (ALAC) is used for managing periprosthetic joint infections (PJIs). The marked increase in resistant high-virulence bacteria is drawing the attention of physicians towards alternative antimicrobial formulations to the routinely used antibiotics. To date, few studies simultaneously investigated the elution properties of a broad range of antibiotics. The aim of thepresent in-vitrostudy was to determine the elution kinetics of 14 different high-dose ALACs. All the ALAC samples showed a burst release of antibiotics in the first hour, progressively decreasing overtime, and elution curves strictly adhered to a non-linear regression analysis formula. Among aminoglycosides, commonly addressed as the most appropriate antibiotics to be loaded into the bone cement, the highest elution rate was that of tobramycin. Among the glycopeptides, commonly used to treat PJIs because of the prevalence of aminoglycoside resistance, vancomycin showed better elution in comparison with teicoplanin. Clindamycin, that can be associated with aminoglycosides to prepare ALACsshowed the highest absolute and relative elutions among all the tested formulations. A noticeable elution was also detected for colistin, an antibiotic of last resort for treating multi-drug resistant bacteria. The current study demonstrates theoretical advantages in the preparation of ALAC for some antibiotics notroutinely used in the clinical setting for PJIs. The use of these antibiotics based on the infecting bacteria sensitivity may represent an useful option for physicians to eradicate PJIs. In vivo testing should be considered in the future to confirm the results of this study

Background. Additive manufacturing (AM) has created many new avenues for material and manufacturing innovation. In orthopaedics, metal additive manufacturing is now widely used for production of joint replacements, spinal fusion devices, and cranial maxillofacial reconstruction. Plastic additive manufacturing on the other hand, has mostly been utilized for pre-surgical planning models and surgical cutting guides. The addition of pharmaceuticals to additively manufactured plastics is novel, particularly when done at the raw material level. The purpose of this study was to prove the concept of antibiotic elution from additively manufactured polymeric articles and demonstrate feasibility of application in orthopaedics. Methods. Using patented processes, three heat-stable antibiotics commonly used in orthopaedics were combined with six biocompatible polymers (2 bioresorbable) into filament and powder base materials for fused deposition modeling (FDM) and selective laser sintering (SLS) AM processes. Raw materials of 1%, 2%, and 5% antibiotic concentrations (by mass) were produced as well as a blend of all three antibiotics each at 1% concentration. Thin disks of 25 mm diameter were manufactured of each polymer with each antibiotic at all concentrations. Disks were applied to the center of circular petri dishes inoculated with a bacterium as per a standard zone of inhibition, or Kirby-Bauer disk diffusion tests. After 72 hours incubation, the zone of inhibited bacterial growth was measured. Periprosthetic joint infection (PJI) of the knee was selected as the proof-of-concept application in orthopaedics. A series of tibial inserts mimicking those of a common TKR system were manufactured via SLS using a bioresorbable base material (Figure 1). Three prototype inserts were tested on a knee wear simulator for 333,000 cycles following ISO 14242–1:2014 to approximate 2–4 months of in vivo use between surgeries of a 2-stage procedure for PJI. Gravimetric measurement and visual damage assessment was performed. Results. Bacterial growth was inhibited to a mean diameter of 32.3 mm (FDM) and 42.2 mm (SLS) for nearly all combinations of polymers and concentrations of antibiotics. Prototype tibial inserts experienced an average of 200 mg of wear during testing and demonstrated no evidence of cracking, delamination or significant deformation (Figure 2). Conclusion. Bench-level testing of these novel antibiotic-eluting polymers demonstrates feasibility for their application in orthopaedic medicine. In particular, treatment of stubborn PJI with potential for increased and sustained antibiotic elution, patient-specific cocktailing, and maintenance of knee joint structure and function compared to existing PJI products and practices. Subsequent testing for these novel polymers will determine static and dynamic (wear-induced) antibiotic elution rates. For any figures or tables, please contact the authors directly

Aim. The preparation of antibiotic-containing polymethyl methacrylate (PMMA), as spacers generates a high polymerization heat, which may affect their antibiotic activity; it is desirable to use bone cement with a low polymerization heat. Calcium phosphate cement (CPC) does not generate heat on polymerization, and comparative elution testings are reported that vancomycin (VCM)-containing CPC (VCM-CPC) exceeded the antibiotic elution volume and period of PMMA (VCM-PMMA). Although CPC alone is a weak of mechanical property spacer, the double-layered, PMMA-covered CPC spacer has been created and clinically used in our hospital. In this study, we prepared the double-layered spacers: CPC covered with PMMA and we evaluated its elution concentration, antimicrobial activity and antibacterial capability. Method. We prepared spherical, double-layered, PMMA-coated (CPC+PMMA; 24 g CPC coated with 16 g PMMA and 2 g VCM) and PMMA alone (40 g PMMA with 2 g VCM) spacers (5 each). In order to facilitate VCM elution from the central CPC, we drilled multiple holes into the CPC from the spacer surface. Each spacer was immersed in phosphate buffer (1.5 mL/g of the spacer), and the solvent was changed daily. VCM concentrations were measured on days 1, 3, 7, 14, 28, 56, and 84. Antimicrobial activity against MRSA and MSSA was evaluated by the broth microdilution method. After measuring all the concentration, the spacers were compressed at 5 mm/min and the maximum compressive load up to destruction was measured. Results. The VCM concentration of the CPC+PMMA spacer exceeded that of the PMMA spacer at all-time points; in particular, it was approximately 7.3 times (109.30 vs. 15.03 μg/mL) and approximately 9.1 times (54.47 vs. 6.50 μg/mL) greater on days 14 and 28, respectively. Using the broth microdilution method, we found that the CPC+PMMA spacer had higher antimicrobial activity than the PMMA model. On day 56, the PMMA spacer lost the capability to inhibit bacterial growth, but the CPC+PMMA spacer maintained this ability. The average maximum compressive load for the CPC+PMMA was 7.28 kN, and that of PMMA was 16.21 kN. Conclusions. The CPC+PMMA spacer was superior to PMMA alone in VCM elution volume and duration, so CP- C+PMMA may be effective for the treatment of MRSA and MSSA infection. The double-layered, antibiotic-loaded cement spacer may maintain antibacterial capability and sufficient strength

Aim. The demand for a synthetic bone substitute that can build bone and at the same time kill bacteria is high. The aim of this study was to compare the elution of gentamicin from a new synthetic bone substitute in vitro with the performance in clinical applications. Method. Gentamicin release was measured from a synthetic bone graft substitute, comparing in vitro and clinical conditions:. 1). elution in Ringers solution. The bone graft substitute contained 175mg gentamicin per 10mL. The material was introduced either as paste or as pre-set beads with a high or low surface areas, >100cm. 2. and 24cm. 2. respectively. The gentamycin release was measured by daily collection of samples. 2). elution in patients treated for trochanteric hip fractures(n=6) or uncemented hip revisions(n=5) 7,3±1,1mL of substitute was implanted and drainage was collected at 6h,12h,24h,30h,36h post-op. Blood serum was collected every hour for the first 6h and thereafter every 6h until 4 days post-op, urine – daily for the first 7 days post-op. 3). elution in patients treated after bone tumor resection(n=8), 12,1±5,5mL of substitute was implanted and both drainage and blood serum were collected daily until 2 days post-op. Gentamicin concentrations were analyzed using antibody technique. Results. In the in vitro study, there was an initial peak in the gentamicin concentration (GC) for all the samples and at a level above 4mg/L, which is the MIC break point, during the whole test period of 28 days. All gentamicin was released during the test period and more than 95 % had been released after 2–4 days independently of the surface area of the material, or if it was pre-set or paste. In the clinical studies similar results were found. Gentamicin was detected in the drainage until 2 days post-op. and the hip patients 40% less GC – compared to the tumor patients. In the blood serum with higher GC in the tumor patients and non-detectable levels after 2 days post-op for the hip patients. The GC was significantly lower than maximum systemic level recommended of 12 mg/L. In the urine, GC was above the MIC of 4mg/L for the first seven days post-op. Conclusions. A reliable in vitro test method has been identified for the future development of additional new and effective antibiotic containing bone substitutes. The new bone regenerating carrier gives very high local antibiotic release for a controlled short time after surgery and high systemic serum concentrations are avoided

Vancomycin -impregnated bonechips from a human morselized femoral head allograft (BCs) are used in orthopaedic surgery to treat infections. Literature suggests that bonechips can be efficient vancomycin carriers, but due to the diversity in the type of bonechips, of impregnation and of method used to evaluate AB release, there are no uniform guidelines. We performed an in vitro study to examine the release of vancomycin from solution-impregnated deepfrozen processed bonechips. Quantification was performed using a fully validated chromatographic method. Results were compared with the elution-profile from Osteomycin®, a commercially available lyophilised processed bonegraft. Different vancomycine impregnation-concentrations and impregnation-durations of frozen processed bonechips were investigated. After impregnation, bonechips were rinsed with saline in order to determine only the absorbed vancomycin. Elution was performed in newborn calf serum at 37°C. Eluted vancomycin concentrations were determined using Ultra Performance Liquid Chromatography – Diode Array Detection (UPLC-DAD). In addition an elution study was performed on the commercially available Osteomycin®, bone chips containing vancomycine. Using processed frozen bonechips, an impregnation-concentration of minimum 100 mg/mL during 10 minutes delivers the desired local concentration (therapeutic window 25 – 1000 mg/L) for 3 days. Longer impregnation time at this concentration had no effect. Osteomycin®: delivers the desired local concentration for 8 days in our experimental setting. Literature suggests that freshfrozen BCs can be used as carrier for vancomycin through solution-impregnation [1,3]. There is however much less information on the carrier-capacities of frozen processed bonechips, a type used in our hospital. Our impregnation-protocol was based on that of Mathyssen et al., but with direct quantification of elution concentrations. Impregnation with vancomycine 100 mg/mL during 10 minutes results in a release above the desired concentration for 3 days, which seems too short when treating bone-infections. Osteomycin®, shows a substantially longer elution [2]. Vancomycin-solution impregnation of frozen processed bonechips may not be sufficient to obtain the desired release-characteristics for the treatment of bone-infections

Aim. Bone and implant-associated infections caused by microorganisms that grow in biofilm are difficult to treat because of persistence and recurrence. Systemic administration of antibiotics is often inefficient because the poor vascularization of the site of infection. This issue has led to the development of biomaterials capable to locally deliver high doses of therapeutic agents to the injured bone with minimal systemic effects. In this context, calcium sulphate/hydroxyapatite (CS/HA) bone graft substitutes are widely used being safe, osteoconductive and resorbable biomaterials that can be easily enriched with consistent amounts of antibiotics. In this in vitro study, the capability of the eluted antibiotics to select the tested bacterial strains for antibiotic resistance was evaluated to confirm the safe use of the product. Method. S. aureus, S. epidermidis and P. aeruginosa isolated in our Institute from bone and joint infection with different resistance phenotypes were used. 6 × 2.5 mm CS/HA discs were generated by pouring the antibiotic loaded formulations in a mold and were used as a modified disk diffusion test. The resistance selection was evaluated by subculturing cells growing on the edge of the zone of inhibition (ZOI) for seven days. Minimum inhibitory concentrations (MICs) of gentamicin and vancomycin were determined by broth microdilution method before and after the selection of resistance assay. In addition, MICs were assessed after seven day passage on antibiotic free agar plates to evaluate if eventual decrease of antibiotic susceptibility was stable or only transient. Results. Commonly, no adaptation in presence of both CS/HA formulations was observed by analysing ZOI on agar medium. The kinetic of decrease of the ZOI was similar between the strains, with the exception of gentamicin resistant staphylococci in presence of gentamicin loaded CS/HA, which was faster with respect to the susceptible strains. Conclusions. The present study shows that elution of gentamicin and vancomycin from CS/HA bone graft substitutes did not induce a decrease in susceptibility to these antibiotics in an in vitro setting, suggesting the safe use of the product

Aim. Chronic osteomyelitis often requires surgical debridement and local antibiotic treatment. Disadvantages of PMMA carriers include low dose release and the requirement of surgical removal in the case of PMMA-beads. Synthetic nanocrystalline calcium phosphate (nCP) materials, which mimic the chemical structure of the mineral composition of bone, have been well accepted as bone grafting materials due to their consistent osteoconductivity, ease of use, and mechanical properties. Such a material which remodels into native bone is a much more attractive option. The aim of this study is to investigate the release of gentamicin from CaP in vitro and in vivo when implanted in a rabbit femoral condyle defect. Method. Three formulations of nCP were evaluated in this study: putty, paste and porous. Four cylindrical dowels were made for each group with gentamicin sulphate at a concentration of 20mg/cc of paste. Material was eluted in PBS at 37C and pH 7.0 and elutions were tested every day up to 30 days. Eighteen New Zealand white rabbits will undergo surgeries. Briefly, a drill defect will be created in the metaphyseal bone of the lateral femoral condyle. The formulations will be implanted in the created defect at time of surgery and the wound will be closed. Blood will be collected regularly and analysed for gentamicin titers. Animals will be sacrificed at 6wk, 12wk or 24wk. Explanted femurs will be fixed, sectioned and stained. Results. At 7 days the in vitro elution, showed a continued release of gentamicin. A large amount of gentamicin is released within the first day followed by a slow controlled release. The nCP putty shows the slowest release, followed by the paste and porous formulations respectively. There is a significant increase in the elution with an increase in porosity of the material. We expect to observe a similar trend in the rabbit study with an improved healing. At 6wk we expect the implant material to be still present at the site of implantation, which would remodel by 12wk and 24wk to significant levels due to active ossification. Conclusions. nCP materials, which undergo remodelling, can be used an effective carrier for gentamicin or other antibiotic agents. Because of its potentially prolonged release of high levels of antimicrobial agents, this system could maintain long-term antibacterial effectiveness locally

Objectives. This study is to compare the elution characteristics, antimicrobial activity, and mechanical properties of antibiotic-loaded bone cement (ALBC) loaded with powder antibiotic, powder antibiotic with inert filler (xylitol), or liquid antibiotic, particularly focusing on vancomycin and amphotericin B. Methods. Cement specimens loaded with 2 g of vancomycin or amphotericin B powder (powder group), 2 g of antibiotic powder, and 2 g of xylitol (xylitol group) or 12 ml of antibiotic solution containing 2 g of antibiotic (liquid group) were tested. Results. Vancomycin elution was enhanced by 234% in the liquid group and by 12% in the xylitol group compared with the powder group. Amphotericin B elution was enhanced by 265% in the liquid group and by 65% in the xylitol group compared with the powder group. Based on the disk-diffusion assay, the eluate samples of vancomycin–loaded ALBC of the liquid group exhibited a significantly larger inhibitory zone than samples of the powder or xylitol group. Regarding the amphotericin B–loaded ALBCs, only the eluate samples of the liquid group exhibited a clear inhibitory zone which was not observed in either xylitol nor powder groups. The ultimate compressive strength was significantly reduced in specimens containing liquid antibiotics. Conclusions. Adding vancomycin or amphotericin B antibiotic powder in distilled water before mixing with bone cement can significantly improve the antibiotic-release efficacy than that by loading ALBC with the same dose of antibiotic powder. This simple, and effective method for preparation of ALBCs can significantly improve the antibiotic-release efficacy of ALBCs

The objective of this study was to compare the elution characteristics, antimicrobial activity and mechanical properties of antibiotic-loaded bone cement (ALBC) loaded with powdered antibiotic, powdered antibiotic with inert filler (xylitol), or liquid antibiotic, particularly focusing on vancomycin and amphotericin B. Cement specimens loaded with 2 g of vancomycin or amphotericin B powder (powder group), 2 g of antibiotic powder and 2 g of xylitol (xylitol group) or 12 ml of antibiotic solution containing 2 g of antibiotic (liquid group) were tested. Vancomycin elution was enhanced by 234% in the liquid group and by 12% in the xylitol group compared with the powder group. Amphotericin B elution was enhanced by 265% in the liquid group and by 65% in the xylitol group compared with the powder group. Based on the disk-diffusion assay, the eluate samples of vancomycin-loaded ALBC of the liquid group exhibited a significantly larger inhibitory zone than samples of the powder or the xylitol group. Regarding the ALBCs loaded with amphotericin B, only the eluate samples of the liquid group exhibited a clear inhibitory zone, which was not observed in either the xylitol or the powder groups. The ultimate compressive strength was significantly reduced in specimens containing liquid antibiotics. Adding vancomycin or amphotericin B antibiotic powder in distilled water before mixing with bone cement can significantly improve the efficiency of antibiotic release than can loading ALBC with the same dose of antibiotic powder. This simple and effective method for preparation of ALBCs can significantly improve the efficiency of antibiotic release in ALBCs. We thank H.Y. Hsu for performing the bioassay

Aim. To prevent infections after orthopaedic surgery, intravenous antibiotics are administered perioperatively. Cefazolin is widely used as the prophylactic antibiotic of choice. Systemic antibiotic therapy may however be less effective in longstanding surgery where bone allografts are used. Bone chips have been shown to be an effective carrier for certain types of antibiotics and may provide the necessary local antibiotic levels for prophylaxis. To be efficient a prolonged release is required. In contrast to vancomycin with proven efficient prolonged release from Osteomycin, this has not been described for cefazolin. We developed a protocol to bind cefazolin to bone chips by means of a hydrogel composed of proteins naturally present in the human body. Method. Three types of bone chips were evaluated: fresh frozen, decellularized frozen and decellularized lyophilized. Bone chips were incubated with 20 mg/ml cefazolin or treated with liquid hydrogel containing either 1 mg/ml fibrin or 1 mg/ml collagen and 20 mg/ml cefazolin. The cefazolin hydrogel was distributed in the porous structure by short vacuum treatment. Bone chips with cefazolin but without hydrogel were either incubated for 20 min- 4h or also treated with vacuum. Cefazolin elution of bone chips was carried out in fetal bovine serum and analysed by Ultra Performance Liquid Chromatography – Diode Array Detection. Results. Soaking of bone chips without hydrogel resulted in a quick release of cefazolin, which was limited to 4 hours. When vacuum was applied elution of >1 µg/ml cefazolin was measured for up to 36 hours. Combination with collagen hydrogel resulted in a higher cefazolin concentration released at 24 hours (3.9 vs 0.3 µg/ml), but not in a prolonged release. However, combination of decellularized frozen bone chips with fibrin hydrogel resulted in an initial release of 533 µg/ml followed by a gradual decline reaching the minimal inhibitory concentration for S. aureus at 72 hours (1.7 µg/ml), while not measurable anymore after 92 hours. Conclusions. Processed bone chips with hydrogel-cefazolin showed a markedly prolonged cefazolin release. When combined with a fibrin hydrogel, high initial peak levels of cefazolin were obtained, followed by a decreasing release over the following three days. This elution profile seems desirable, with high initial levels to maximize anti-bacterial action and low levels for a limited time to stimulate osteogenesis. Further preclinical studies are warranted to show effectiveness of hydrogel-cefazolin impregnated bone chips

This study was performed to investigate the concentration of silver ions release up to a time of 9 weeks as well as the antimicrobial activity of silver sulfate and Nano-silver mixed bone cement on Candida albicans, in expectation of a new way of therapy in manner of a time limited application – a silverions releasing bone cement spacer. Two different kinds of silver products were used and mixed with polymethylmetacrylate (PMMA, De Puy) bone cement:. Nano-silver with a particle size of 5–50 nm and active surface of 4 m2/ g. (Nanonet Styria, Austria). Silver sulfate in a finely powdered form (Fisher, GB). Concentrations of 0.1%, 0.5%, 1% and 5% of the Nano-silver and the silver-salt by weight were mixed with the dry powder portion of the cement. To test the silver-ions release from the silver-containing bone cement two models of elution, a static model and a dynamic model were created. To test the antifungal effectiveness of the various concentrations of Ag-PMMA the bone cement samples were tested by agar diffusion assay. With respect to minimal inhibition concentration (MIC) the sample containing 0.5 % silver sulfate showed required concentration at the dynamic elution model but none of the nano-silver samples did. In static elution model we measured the maximum concentration of 466.5 µg/l at the 0.5 % silver sulfate sample which is much below the toxic concentration. Agar diffusion assay showed no zone of inhibition from Nano-silver samples. In contrast, silver sulfate containing samples showed a zone of inhibition exactly growing, depending on the samples silver sulfate concentration. According to results, silver sulfate addition to PMMA might be another approach in treatment of candida associated periprosthetic joint infection

The rate of periprosthetic joint infections (PJI) after primary total hip arthroplasty (THA) is approximately 1%. As the number of THAs performed each year continue to increase (550,000 by 2030), a corresponding increase in the number of hip PJI cases is likely to occur. A chronic deep infection may be treated by either chronic suppression, irrigation and debridement, single-stage exchange, or two-stage exchange. In the United States, the gold standard for chronic PJI continues to be a two-stage exchange. The benefit of an antibiotic impregnated cement is that they produce higher local concentrations of antibiotics than systemic intravenous administration. Hip spacers may be either static or articulating. Static spacers are reserved for cases of massive acetabular bone loss in which an articulating spacer is not feasible. A static spacer consists of a block of antibiotic cement in the native acetabulum and antibiotic coated rod in the femoral canal. Limb shortening, loss of soft tissue planes, and disuse osteopenia and muscle atrophy are all limitations of static spacers. In contrast, articulating spacers fulfill the goals of the interim construct during two-stage exchange which is to enhance eradication of the infecting organism through drug elution, to maintain limb length, to facilitate exposure during revision surgery, and to improve functional mobilization. Articulating spacers may be divided into three general categories based on method of spacer creation: Handmade custom spacers, prefabricated spacers, custom molded spacers (hemiarthroplasty molds and molded stem with cemented all-polyethylene cup). Handmade custom spacers are usually created with K-wire or rush rods coated with antibiotic cement. Handmade spacers are relatively simple to make, they are economical, and the amount and type of antibiotics incorporated can be customised for the infecting organism. Commercially available hemiarthroplasty spacers can be either prefabricated (Spacer G, Exactech, Gainesville. FL) or made intraoperatively (Stage One, Zimmer Biomet, Warsaw, IN) are available in several head and stem sizes. The advantage of prefabricated spacers is that they do not require additional time to mold in the operating room. The downside of prefabricated spacers is that the antibiotic concentration and type is predetermined. A custom molded stem with cemented all-polyethylene cup can be made with off the shelf implants or used as part of a commercially available spacer (PROSTALAC, DePuy Synthes, Warsaw, IN). A common antibiotic/cement combination includes Tobramycin (3.6 g/40 g of cement) and vancomycin (1.5 g/40 g of cement). In all of these spacer constructs, the principles of using a high-elution cement mixed without a vacuum and with high doses of heat stable antibiotics are consistent. Tobramycin works synergistically to improve Vancomycin elution properties and is usually added in higher doses. Overall infection eradication is similar between all categories of spacers and range between 90–97%. Complications after placement of an articulating spacer are often specific to the type of spacer used. Handmade spaces that have K-wires for support are at risk for spacer cement fracture. Spacer dislocation is also a common complication in up to 15% of cases with all types of spacers. In addition, periprosthetic fractures can occur postoperatively in up to 10% of patients. Overall, despite this complication profile, articulating antibiotic spacers have excellent rates of infection eradication and offer improved mobilization in the interim two stage period and reduce operative time, complexity, and morbidity during reimplantation