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Orthopaedic Proceedings
Vol. 97-B, Issue SUPP_16 | Pages 145 - 145
1 Dec 2015
Putzeys G Croes K Boudewijns M Lambrecht S Voet P
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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


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_19 | Pages 66 - 66
22 Nov 2024
Ye Z van der Wildt B Vogely C Weinans H Poot A van der Wal B
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Aim. Prosthetic joint infections (PJI) remain a great challenge in orthopedic surgery with a high mortality rate. It is particularly complicated by biofilms and infections caused by Methicillin-resistant Staphylococcus aureus (MRSA). It concurrently shields bacteria from host immune responses and confers resistance to antibiotics. This study aims to investigate the efficacy of radioimmunotherapy as an innovative therapeutic modality to address the challenges posed by MRSA and its biofilm. Method. We induced specific monoclonal antibodies 4497-IgG1 as carriers, which target wall teichoic acids (WTA) existing on MRSA and its biofilm. Radionuclides actiniumr-225 (. 225. Ac, α-emitter) and lutetium-177 (. 177. Lu, β-emitter) were conjugated with mAbs using DOTA as chelator. Quality control was assessed using thin layer chromatography and immunoreactivity assays. . 225. Ac- and . 177. Lu-labelled 4497-IgG1 were employed to evaluate the susceptibility of MRSA and its biofilm to the radioimmunotherapy in vitro. Planktonic MRSA and biofilms, at concentrations of 10. 8. and 10. 7. CFU/mL, were incubated at 37°C for 60 minutes in PBS containing either . 225. Ac-mAb (0 - 14.8 kBq) or . 177. Lu-mAb (0 - 14.8 MBq). Radiolabelled dunituximab and free radionuclides serve as isotype-matched negative control. The bacterial viability and metabolic activity were subsequently quantified using CFU and XTT assays. Results. The radiochemical purity of the . 225. Ac-mAbs and . 177. Lu-mAbs complex were determined to be 95.4% and 96.16%. Immunoreactivity fractions of them were measured at 81.8% and 80.8%. . 225. Ac-mAbs and . 177. Lu-mAbs exhibited significant and dose-dependent antimicrobial effects on both planktonic MRSA and biofilm. . 225. Ac- and . 177. Lu-4497IgG1 at doses of 7.4 kBq and 7.4 MBq resulted in more than 4-log reduction in bacterial counts. In biofilms, 2-log reduction at the highest . 225. Ac radioactivity of 14,8kBq. The . 177. Lu complex showed a strong dose-dependent effect, with a reduction of up to 4-log. The XTT assay confirmed these findings, showing a decrease in metabolic activity corresponding to a decrease in bacterial counts, and a slight increase in metabolic activity at the lower dose. Conclusions. Our study demonstrates the efficacy of . 225. Ac and . 177. Lu-labelled 4497-IgG1 antibodies in mediating dose-dependent bactericidal effects against planktonic MRSA and biofilms in vitro. This indicates that radioimmunotherapy could be a potential targeted therapeutic strategy against MRSA and its biofilm. Further research in preclinical and clinical settings is warranted to validate and refine these findings on biofilm-associated implant infections


Orthopaedic Proceedings
Vol. 106-B, Issue SUPP_19 | Pages 22 - 22
22 Nov 2024
Söderquist B Möller M Salihovic S
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Aim. Dalbavancin is a lipoglycopeptide with a broad antimicrobial spectrum against Gram-positive bacteria and effect against microorganisms in biofilm in vitro. Its pharmacokinetic properties, with an exceptionally long half-life of approximately 300 hours, allow for simplified administration that may be of value in the long-term treatment of bone and joint infections, such as prosthetic joint infections (PJIs). Several case reports and case series with “off-lable” treatment with dalbavancin of PJIs exist, but the optimal dosing regimen remains to be defined. Therapeutic drug monitoring (TDM) is recommended for treatment with >2 doses of dalbavancin. In the absence of TDM, the Swedish national guidelines for bone and joint infections (2023, . www.infektion.net. ) recommends a loading dose of dalbavancin 1,500 mg on day 1 and 1,500 mg on days 8 – 14, after which from day 28 1,000 mg is given biweekly or 500 mg every week. The aim of the present study was to determine trough levels of dalbavancin in patients with long-term treatment of PJIs according to the national guidelines. Method. Twelve patients with PJI were treated with at least 6 doses of dalbavancin, of which the first two doses were 1500 mg and the following doses were 1000 every second week, and prospectively sampled biweekly for determination of serum concentrations (trough levels) of dalbavancin which was measured by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS/MS). The renal function was also examined. Results. The median serum concentration 14 days after the first dose of dalbavancin 1500 mg was 36.3 mg/L (range 6.6 – 62.4 mg/L). The median value 14 days after the second dose of 1500 mg (day 27 – 28) was 48.2 mg/L (range 12.2 – 77.3 mg/L). The trough value after the last dose of a total of 6 – 7 doses was as median 43.1 mg/L (range 26.2 – 97.5 mg/L). Three patients showed a tendency towards successive accumulation of dalbavancin during treatment. None of the patients, including those three with increasing through levels during treatment, showed any significant alteration in creatinine nor glomerular filtration rate. Conclusions. TDM during long-term treatment with dalbavancin is recommended to avoid the risk of accumulation and unnecessarily high trough values. With TDM, the dosing interval can be extended in several cases. In addition, with the support of TDM, subtherapeutic serum concentrations, with the risk of developing resistance, can be avoided


Orthopaedic Proceedings
Vol. 104-B, Issue SUPP_10 | Pages 81 - 81
1 Oct 2022
Hvistendahl MA Bue M Hanberg P Kaspersen AE Schmedes AV Stilling M Høy K
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Background. Surgical site infection following spine surgery is associated with increased morbidity, mortality and increased cost for the health care system. The reported pooled incidence is 3%. Perioperative antibiotic prophylaxis is a key factor in lowering the risk of acquiring an infection. Previous studies have assessed perioperative cefuroxime concentrations in the anterior column of the cervical spine with an anterior surgical approach. However, the majority of surgeries are performed in the posterior column and often involve the lumbar spine. Accordingly, the objective was to compare the perioperative tissue concentrations of cefuroxime in the anterior and posterior column of the same lumbar vertebra using microdialysis in an experimental porcine model. Method. The lumbar vertebral column was exposed in 8 female pigs. Microdialysis catheters were placed for sampling in the anterior column (vertebral body) and posterior column (posterior arch) within the same vertebra (L5). Cefuroxime (1.5 g) was administered intravenously over 10 min. Microdialysates and plasma samples were continuously obtained over 8 hours. Cefuroxime concentrations were quantified by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry. Microdialysis is a catheter-based pharmacokinetic tool, that allows dynamic sampling of unbound and pharmacologic active fraction of drugs e.g., cefuroxime. The primary endpoint was the time with cefuroxime above the clinical breakpoint minimal inhibitory concentration (T>MIC) for Staphylococcus aureus of 4 µg/mL as this has been suggested as the best predictor of efficacy for cefuroxime. The secondary endpoint was tissue penetration (AUC. tissue. /AUC. plasma. ). Results. Mean T>MIC 4 µg/mL (95% confidence interval) was 123 min (105–141) in plasma, 97 min (79–115) in the anterior column and 93 min (75–111) in the posterior column. Tissue penetration (95% confidence interval) was incomplete for both the anterior column 0.48 (0.40–0.56) and posterior column 0.40 (0.33–0.48). Conclusions. Open lumbar spine surgery often involves extensive soft tissue dissection, stripping and retraction of the paraspinal muscles which may impair the local blood flow exposing the lumbar vertebra to postoperative infections. A single intravenous administration of 1.5 g cefuroxime resulted in comparable T>MIC between the anterior and posterior column of the lumbar spine. Mean cefuroxime concentrations decreased below the clinical breakpoint MIC for S. aureus of 4 µg/mL after 123 min (plasma), 97 min (anterior column) and 93 min (posterior column). This is shorter than the duration of most lumbar spine surgeries, and therefore alternative dosing regimens should be considered in posterior open lumbar spine surgeries lasting more than 1.5 hours


Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_7 | Pages 59 - 59
1 Jul 2020
Qiu H Cheng T Chim SM Zhu S Xu H Qin A Wang C Teguh D Zhang G Tickner J Yao F Vrielink A Smithers L Pavlos N Xu J
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Bone is a connective tissue that undergoes constant remodeling. Any disturbances during this process may result in undesired pathological conditions. A single nucleotide substitution (596T-A) in exon eight which leads to a M199K mutation in human RANKL was found to cause osteoclast-poor autosomal recessive osteopetrosis (ARO). Patients with ARO cannot be cured by hematopoietic stem cell transplantation and, without proper treatments, will die in their early age. To date, how this mutation alters RANKL function has not been characterized. We thus hypothesized that hRANKL M199 residue is a structural determinant for normal RANKL-RANK interaction and osteoclast differentiation. By sharing our findings, we aim to achieve an improved clinical outcome in treating bone-related diseases such as osteoporosis, ARO and osteoarthritis. Site-directed mutagenesis was employed to create three rat RANKL mutants, replacing the methionine 200 (human M199 equivalent residue) with either lysine (M200K), alanine (M200A) or glutamic acid (M200E). Recombinant proteins were subsequently purified through affinity chromatography and visualized by Coomassie blue staining and western blot. MTS was carried out before osteoclastogenesis assay in vitro to measure the cellular toxicity. Bone resorption pit assay, immuno-fluorescent staining, luciferase reporter assay, RT-PCR, western blot and calcium oscillation detection were also conducted to explore the biological effect of rRANKL mutants. Computational modeling, thermal Shift Assay, western blot and protein binding affinity experiments were later carried out for structural analyses. rRANKL mutants M200K/A/E showed a drastically reduced ability to induce osteoclast formation and did not demonstrate features of competitive inhibition against wild-type rRANKL. These mutants are all incapable of supporting osteoclastic polarization and bone resorption or activating RANKL-induced osteoclast marker gene transcription. Consistently, they were unable to induce calcium flux, and also showed a diminished induction of IκBa degradation and activation of NF-kB and NFATc1 transcriptional activity. Furthermore, the transcriptional activation of the antioxidant response element (ARE) crucial in modulating oxidative stress and providing cytoprotection was also unresponsive to stimulation with rM200s. Structural analyses showed that rM200 is located in a hydrophobic pocket critical for protein folding. Thermal shift and western blot assays suggested that rM200 mutants formed unstructured proteins, with disturbed trimerisation and the loss of affinity to its intrinsic receptors RANK and OPG. Taken together, we first demonstrates the underlying cause of M199-meidated ARO in a cellular and molecular level by establishing a phenotype in BMMs similar to observed in human samples. Further investigation hints the structural significance of a hydrophobic pocket within the TNF-like region. Combined with pharmaceutical studies on small-molecule drugs, this finding may represent a therapeutic target motif for future development of anti-resorptive treatments


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_14 | Pages 19 - 19
1 Dec 2019
Duportail C Gerard M Kathleen C Putzeys G Thorrez L
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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


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_14 | Pages 20 - 20
1 Dec 2019
Hanberg PE J⊘rgensen AR Stilling M Thomassen M Bue M
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Aim. Cefuroxime is a time-dependent antibiotic widely used as intravenous perioperative prophylaxis in spine surgery. A previous study has indicated that a single dose of cefuroxime provided insufficient spine tissue concentrations for spine procedures lasting more than 2–3 hours. Due to the fact that postoperative pyogenic spondylodiscitis is associated with prolonged antimicrobial therapy and high relapse rates, we aimed to evaluate if a twofold increase of standard dosage of 1.5g cefuroxime given as one double dose or two single doses with 4-hours intervals will lead to sufficient cefuroxime spine tissue concentrations throughout the dosing interval. Method. This is preliminary data for 8 out of 16 female pigs. Data from all 16 pigs will be included for the conference. Eight pigs were randomized into two groups: Group A received one double dose of cefuroxime (3g) as a bolus, and Group B received two single doses of cefuroxime (2×1.5g) with 4-hours intervals. Measurements were obtained from plasma, subcutaneous tissue (SCT), vertebral cancellous bone and the intervertebral disc (IVD) for 8-hours thereafter. Microdialysis was applied for sampling in solid tissues. The cefuroxime concentrations were determined using ultra-high performance liquid chromatography. Results. The time with concentrations above the minimal inhibitory concentration (T>MIC) for the clinical breakpoint MIC for Staphylococcus aureus of 4 μg/ml, was superior in all compartments when administering cefuroxime as two single doses with 4-hours intervals. For the target MIC of 4 μg/ml, the mean T>MIC in all compartments ranged between 53–73% and 85–95% for Group A and B, respectively. For both groups the area under the concentration-curve (AUC) was higher for plasma compared to the remaining compartments, and the lowest AUCs were found in the vertebral cancellous bone and the IVD. There were no differences in AUC between the two groups. Furthermore, the maximal concentrations were lower for both vertebral cancellous bone and IVD compared to both SCT and plasma. When comparing the two groups, higher maximal concentrations were found in all compartments for Group A. Tissue penetration was incomplete and delayed for all compartments and comparable between the two groups. Conclusions. Despite comparable pharmacokinetic results between the two groups, Group B exhibited superior T>MIC in all compartments for the clinical breakpoint MIC for Staphylococcus aureus of 4 μg/ml. As such administration of cefuroxime as two single doses with 4-hours intervals provided sufficient cefuroxime spine tissue concentrations for a minimum of 85% of an 8-hour dosing interval, which may be acceptable for most spine procedures


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_4 | Pages 43 - 43
1 Apr 2019
Friedrich CR Baker E Bhosle S Justin D
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Periprosthetic infection remains a clinical challenge that may lead to revision surgeries, increased spending, disability, and mortality. The cost for treating hip and knee total joint infections is anticipated to be $1.62 billion by 2020. There is a need for implant surface modifications that simultaneously resist bacterial biofilm formation and adhesion, while promoting periprosthetic bone formation and osseointegration. In vitro research has shown that nanotextured titanium promotes osteoblast differentiation, and upregulates metabolic markers of osteoblast activity and osteoblast proliferation. In vivo rat studies confirmed increased bone-implant contact area, enhanced de novo bone formation on and adjacent to the implant, and higher pull-out forces compared to non-textured titanium. The authors have advanced a benign electrochemical anodization process based on ammonium fluoride that creates a nanotube surface in as little as 10 minutes (Fig. 1), which can also integrate antibacterial nanosilver (Fig. 2). The work reported here summarizes in vitro post-inoculation and in vivo post-implantation studies, showing inherent inhibition of methicillin-resistant Staphylococcus aureus (MRSA) by titanium surfaces with nanotubes (TiNT), nanotubes with nanosilver (TiNT+Ag), plain (Ti), and thermal plasma sprayed (TPS) titanium. Ti6Al4V was the base material for all surfaces. In vitro studies evaluated Ti, TPS, four TiNT groups with varying nanotube diameters (60nm, 80nm, 110nm, 150nm), and TiNT+Ag. After seeding with MRSA (10. 5. , 10. 6. , and 10. 8. CFU/mL), the 110nm diameter nanotubes showed MRSA inhibition up to three-orders of magnitude lower than the Ti and TPS surfaces at 2, 6, and 48 hours. Following on the in vitro results, New Zealand White rabbits underwent a bilateral implantation of intramedullary tibial implants of the four material groups (4 mm outside diameter; 110nm NT diameter on TiNT and TiNT+Ag implants). One intramedullary canal was inoculated with clinically-derived MRSA (10. 5. CFU in broth) at the time of implantation; one canal had only culture media introduced (control). At a 2-week endpoint, limbs were harvested for analysis, including implant sonication with sonicant bacterial cultured, histology, and microcomputed chromatography. In the sonicant analysis cohort, TPS showed the lowest average MRSA count, while TiNT and TiNT+Ag were the highest. There was one sample each of TPS, TiNT and TiNT+Ag that showed no MRSA. After an additional 24-hour implant incubation, the TiNT and TiNT+Ag samples had no bacteria, but the TPS grew bacteria; therefore, the authors hypothesize that MRSA more readily releases from the TiNT and TiNT+Ag implants during sonication, indicating weaker biofilm adhesion and development. Histologic analysis is currently underway. In a therapeutic experiment, rabbits underwent bilateral implantation, followed by 1 week of infection development, and then 1 week of vancomycin treatment. At the endpoint, implants were sonicated and bacteria was quantified from the sonicant. TiNT showed viable MRSA at only 30% that of TPS-coated levels, while TiNT+Ag implants showed viable MRSA at only 5% that of TPS-coated levels (Fig. 3). These early results indicate that the TiNT and TiNT+Ag surfaces have some inherent antibacterial activity against MRSA, which may increase the efficacy of systemic antibiotic treatments in the setting of periprosthetic joint infections


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_22 | Pages 62 - 62
1 Dec 2017
Hanberg PE Bue M Sørensen HB Søballe K Tøttrup M
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Aim. Pyogenic spondylodiscitis is associated with prolonged antimicrobial therapy and high relapse rates. Nevertheless, tissue pharmacokinetic studies of relevant antimicrobials in both prophylactic and therapeutic situations are still sparse. Previous approaches based on bone biopsy and discectomy exhibit important methodological limitations. The objective of this study was therefore to assess the concentration of cefuroxime in intervertebral disc (IVD), vertebral body cancellous bone, subcutaneous adipose tissue (SCT) and plasma pharmacokinetics after single dose administration by use of microdialysis (MD) in a large animal model. Method. Ten female pigs were assigned to receive 1,500 mg of cefuroxime intravenously over 15 min. Measurements of cefuroxime were obtained from plasma, SCT, the vertebral cancellous bone and the IVD for 8 hours thereafter. MD was applied for sampling in solid tissues. The cefuroxime concentration in both the MD and plasma samples was determined using ultra-high performance liquid chromatography. Results. For both the IVD and the vertebral cancellous bone, the area under the concentration-curve from zero to the last measured value was significantly lower than that of free plasma. Tissue penetration of cefuroxime was incomplete for the IVD, whereas for vertebral cancellous bone and SCT it was not. Furthermore, the penetration of cefuroxime from plasma to IVD was delayed. Additionally, a noticeable prolonged elimination rate of cefuroxime in the IVD was found. The maximal concentration and the elimination of cefuroxime were reduced in IVD compared to both SCT and vertebral cancellous bone. Due to this delay in elimination of cefuroxime, the time with concentrations above the minimal inhibitory concentration (T>MIC) was significantly higher in IVD than in SCT, vertebral cancellous bone and free plasma for MICs up to 6 μg/ml. Conclusions. MD was successfully applied for serial assessment of the concentration of cefuroxime in the IVD and the vertebral cancellous bone. Penetration of cefuroxime from plasma to IVD was found to be incomplete and delayed, but due to a prolonged elimination, the best results regarding T>MIC was found in IVD


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_22 | Pages 74 - 74
1 Dec 2017
Bue M Tøttrup M Hanberg P Langhoff O Sorensen HB Thillemann TM Andersson TL Søballe K
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Aim. The incidence of orthopaedic methicillin-resistant staphylococcus aureus infections is increasing. Vancomycin may therefore play an increasingly important role in orthopaedic perioperative antimicrobial prophylaxis. Adequate antimicrobial concentrations at target site is essential for prevention of orthopaedic infections. Current studies investigating perioperative bone and soft tissue concentrations of vancomycin are sparse and challenged by a lack of appropriate methods. The aim of this study was therefore to assess the concentration of vancomycin in plasma, subcutaneous tissue and bone after single dose administration using microdialysis (MD) in patients undergoing total knee replacement. Method. 1,000 mg of vancomycin was postoperatively administered intravenously over 100 minutes to 10 male patients undergoing primary total knee replacement. Vancomycin concentrations in plasma, subcutaneous tissue (SCT), cancellous and cortical bone were measured the following 8 hours. MD was applied for sampling in solid tissues. The vancomycin concentration in MD-samples was determined using ultra-high performance liquid chromatography, whilst the free plasma concentration was determined using a chemistry analyzer*. Results. For all extravascular tissue, an impaired penetration was demonstrated, with lower area under the concentration-time curve (AUC) compared to free plasma. The lowest AUC was found in cortical bone. For all tissues, tissue penetration expressed as the ratio of the area under the concentration–time curve from 0 to the last measured value (AUC0-last tissue/AUC0-last plasma) were below 0.5. The time to a mean clinically relevant minimal inhibitory concentration (MIC) of 2 mg/L were 3, 36, 27 and 110 min for plasma, SCT, cancellous and cortical bone, respectively. As opposed to the other compartments, a mean MIC of 4 mg/L was not reached in cortical bone. The AUC0-last and peak drug concentrations (Cmax) for SCT, cancellous and cortical bone were lower than those of free plasma. The time to Cmax was higher for all tissues compared with free plasma. Conclusions. Penetration of vancomycin to bone and SCT was found to be impaired and delayed in male patients undergoing total knee replacement surgery. Adequate perioperative vancomycin concentrations may not be reached at target site using standard prophylactic dosage. *Cobas c501


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_23 | Pages 66 - 66
1 Dec 2016
Samara E Moriarty F Decosterd LA Richards G Gautier E Wahl P
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Aim. Thermal stability is a key property determining the suitability of an antibiotic agent for local application. Long-term data describing thermal stability without interference from carrier materials are scarce. Method. In this study, a total of 38 common antibiotic agents have been maintained at 37 °C in saline solution, and degradation and antibacterial activity assessed over 6 weeks. The impact of an initial supplementary heat exposure mimicking exothermically-curing bone cement has also been tested. Antibiotic degradation was assessed by chromatography coupled to mass spectrometry or immunoassays, as appropriate. Antibacterial activity was determined by Kirby-Bauer disk diffusion assay. Results. The heat exposure mimicking curing bone cement had minimal effect on stability for most antibiotics, except for gentamicin, which experienced approximately 25% degradation as measured by immunoassay. Beta-lactam antibiotics were found to degrade quite rapidly at 37°C regardless of whether there was an initial heat exposure or not. However some of them maintained relevant concentrations and activity for 2–3 weeks, particularly aztreonam. Excellent long-term stability was observed for aminoglycosides, glycopeptides, tetracyclines and quinolones under both conditions. Conclusions. This study provides a valuable dataset for orthopaedic surgeons considering local application of antibiotics. For example, tobramycin would be more suitable for application with bone cement than gentamicin, as it was found to be resistant to heat exposure mimicking curing bone cement


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_23 | Pages 67 - 67
1 Dec 2016
Thorrez L Putzeys G Kathleen C Boudewijns M Christiane D
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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


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_6 | Pages 101 - 101
1 Mar 2017
Wimmer M Pacione C Yuh C Laurent M Chubinskaya S
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Introduction. There is interest in minimally invasive solutions that reduce osteoarthritic symptoms and restore joint mobility in the early stages of cartilage degeneration or damage. The aim of the present study was to evaluate the Biolox®delta alumina-zirconia composite as a counterface for articulation against live cartilage in comparison to the clinically relevant CoCrMo alloy using a highly controlled in vitro ball-on-flat articulation bioreactor that has been shown to rank materials in accord with clinical experience. Methods. The four-station bioreactor was housed in an incubator. The dual axis concept of this simulator approximates the rolling-gliding kinematics of the joint. Twelve 32 mm alumina-zirconia composite femoral heads (Biolox®delta, CeramTec GmbH, Germany) and twelve 32 mm CoCrMo femoral heads (Peter Brehm GmbH, Germany) made up the testing groups. Each head articulated against a cartilage disk of 14 mm diam., harvested from six months old steers. Free-swelling control disks were obtained as well. Testing was conducted in Mini ITS medium for three hours daily over 10 days applying a load of 40 N (∼2 MPa). PG/GAG was determined using the dimethylmethylene blue (DMMB) assay. Hydroxyproline was analyzed by high performance liquid chromatography coupled to a mass spectrometer. Additionally, at test conclusion, chondrocyte survival was determined using Live/Dead assay. Histological analysis was performed using a modified Mankin score. The effect of articulating material (ceramic, CoCrMo) on the various outputs of interest was evaluated using ANOVA. Blocking was performed with respect to the animals. The Mankin scores were compared using the Kruskal–Wallis test. Results. Cells stayed alive during the course of the 3-week experiment with cell survival values close to or at 80% at test completion. There was no difference between ceramic and free swelling control tissue. However, cell count values were inferior for CoCrMo in the superficial zone (p= 0.003). Tested tissue suffered mostly structural abnormalities. In many samples, the superficial layer was disturbed (and sometimes absent), but deeper layers were little affected. The average Mankin scores were in the range of 2 (out of 14) for both materials (p=0.772; Fig. 1). PG/GAG content in medium was highest for CoCrMo (Fig. 2). Though despite a 10% difference between CoCrMo and ceramic, this did not manifest in statistical significance (p=0.315). Similarly, hydroxyproline release into medium was higher for CoCrMo than ceramic (Fig. 3). This difference (28%) was statistically significant (p=0.024). Discussion. Overall, the results indicate that ceramic-on-cartilage induces less tissue and cell damage than metal-on-cartilage. However, only the hydroxyproline measurements reached statistical significance, partially due to a large variation within both material groups. Current understanding of cartilage wear is still incomplete. While studies have utilized the coefficient of friction against artificial materials as a surrogate wear marker, the best way to determine wear in in-vitro experiments is not well-established. Here, we used the matrix components proteoglycan and hydroxyproline to predict cartilage damage, but further work is necessary to elucidate the mechanobiological reasons for damage. In summary, from this study, Biolox®delta ceramic is generally superior to CoCrMo in the articulation against hyaline cartilage. For any figures or tables, please contact authors directly (see Info & Metrics tab above).


Orthopaedic Proceedings
Vol. 97-B, Issue SUPP_15 | Pages 43 - 43
1 Dec 2015
Hansen K Rasmussen L Skov O Nielsen F Stage T Jørgensen U
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In Denmark the most common postoperative pathogen is S. aureus (1), sensitive to dicloxacillin. These bacteria can cause a postoperative infection despite using prophylactic antibiotics. Whether the tissue concentration reached is above the minimal inhibitory concentration (MIC) for the pathogens is unknown, and if lower than expected could result in a postoperative infection. Thus a trial was conducted, measuring the actual tissue concentration of dicloxacillin in human muscle and adipose tissue and compared these to the plasma concentration. MIC for dicloxacillin against S. aureus was determined using the broth macrodilution method. Six healthy male volunteers aging 25 to 27 years (body-mass-index; 20–28), were recruited. A CMA63 (Mdialysis, Stockholm, Sweden) catheter was placed in the subcutaneous tissue of the abdomen and in the rectus muscle of the thigh and the volunteers given 2 g dicloxacillin intravenously over 5 minutes. In 10 min intervals for the following 6 hours, samples from blood and Microdialysis fluid (flowrate 5 ml/min) were collected. Recovery was determined in vitro. Plasma was isolated from blood samples. The unbound dicloxacillin was isolated from plasma using filter plates (AcroPrep 30K Omega, Pall Corporation, US) centrifuged for 30 minutes at 1000 × g and 37°C. All samples were analyzed with High Performance Liquid Chromatography. MIC was determined to be 0.125 µg/ml. Average recovery was 73,7 % Maximum concentrations were reached in muscle tissue after a median of 0.5 hours and adipose tissue after 0.8 hours. The geometric mean ration (GMR) of AUC0-6h for adipose tissue compared to plasma was 0.32 [0.15–0.71]. GMR of AUC0-6h for muscle tissue compared to plasma and adipose tissue compared to muscle showed no statistically significant differences. The tissue concentrations were above MIC for 3.4 hours for adipose tissue and 4.1 hours for muscle tissue. The administration of prophylactic dicloxacillin should be given at least 30 minutes prior to incision to ensure maximum tissue concentrations at the onset of surgery. A second dose should be given after 3.4 hours in case of long surgery time. Since the dicloxacillin concentration reached in the adipose tissue is lower than in plasma, it should be investigated whether this difference is more prominent in adipose patients or patients with impaired peripheral circulation, since these patients are at a greater risk of postoperative infections


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_9 | Pages 78 - 78
1 May 2016
Narayan V
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The ATTUNE™ Knee System (DePuy Synthes) comprises of a tibial insert that is made from AOX™, an antioxidant-stabilized polyethylene. The antioxidant used in AOX is pentaerythritol tetrakis [3-(3, 5-di-tertiary butyl-4-hydroxyphenyl)] propionate (PBHP). A biological risk assessment of the degradation products arising from PBHP has been performed. This assessment focuses on the requirements of ISO 10993–1:2009, ISO 14971:2007, and the Medical Device Directive 93/42/EEC. Because the orthopedic implant is a permanent implant, consideration has been given to all relevant endpoints defined by ISO 10993–1 Biological evaluation of medical devices – Part 1: Evaluation and testing within a risk management process. Comprehensive biocompatibility testing including long-term (26 weeks) subcutaneous implantation has been conducted which confirms the biosafety of the polyethylene compound[1]. In addition to the biological safety testing completed, the overall safety and the associated toxicological risk of exposure to degradation products of PBHP has been given due consideration. The guidelines for the Threshold for Toxicological Concern (TTC) provided by The Product Quality Research Institute (PQRI) Leachables and Extractables Working Group were used in the assessment[2]. This working group is a collaboration of chemists and toxicologists from the U.S. Food and Drug Administration (FDA), industry, and academia. The TTC principle allows safety assessment in the absence of substance-specific hazard data, based on very low levels of exposure to that substance. A Margin of Safety (MOS) is calculated as the ratio of the threshold safety value to the actual exposure quantities determined and used in the assessment. A MOS value greater than 1 is typically judged by risk assessors and regulatory bodies to be unlikely to cause harm and the risk may be considered low. The identity of the degradation products as well as the corresponding 30-day leachable quantities from a water:acetone extraction media has been previously reported [3] and provided here (Table 1). The amount of leachables determined from Table 1 for all products were well below the TTC of 150 ng/device and hence no toxicological risks were identified for these compounds. In order to further examine the toxicological risk assessment, aggressive extraction using Dynamic Head Space (DHS) extraction was done and analytical testing was performed on the degradation products of PBHP using gas chromatography/mass spectrometry (GC /MS). These estimated quantities along with literature information from biological safety studies of the chemicals that were identified from the quantitative GC/MS analysis of degradation products of PBHP were used in the review and toxicological assessment per the methodology described in ISO 14971 and ISO 10993–18. The extraction and analysis confirmed the same sixteen compounds previously identified. The quantities and the calculated margins of safety are summarized (Table 2). In conclusion, upon review of actual test results of PBHP degradation products (Table 1), there is little probability that these organic degradation products would cause a systemic reaction and not be safe. Thus, the potential biological hazards identified in ISO 10993–1:2009 due to the quantified leachables have been verified to be minimal with a high Margin of Safety relative to the Threshold of Toxicological Concern


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXIII | Pages 125 - 125
1 May 2012
Nguyen H Gineyts E Wu A Cassady A Bennett M Morgan D Delmas P Forwood M
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It is not known if the radiation sterilisation dose (RSD) of 25 kGy affects mechanical properties and biocompability of allograft bone by alteration of collagen triple helix or cross-links. Our aim was to investigate the mechanical and biological performance, cross-links and degraded collagen content of irradiated bone allografts. Human femoral shafts were sectioned into cortical bone beams (40 × 4 × 2 mm) and irradiated at 0, 5, 10, 15, 20, and 25 kGy for three-point bending tests. Corresponding cortical bone slices were used for in vitro determination of macrophage activation, osteoblast proliferation and attachment, and osteoclast formation and fusion. Subsequently, irradiated cortical bone samples were hydrolised for determination of pyridinoline (PYD), deoxypyridinoline (DPD), and pentosidine (PEN) by high performance liquid chromatography (HPLC) and collagen degradation by the alpha chymotrypsin (ï. j. CT) method. Irradiation up to 25 kGy did not affect the elastic properties of cortical bone, but the modulus of toughness was decreased from 87% to 74% of controls when the gamma dose increased from 15 to 25 kGy. Macrophages activation, the proliferation and attachment of osteoblasts on irradiated bone was not affected. Osteoclast formation and fusion were less than 40% of controls when cultured on bone irradiated at 25 kGy, and 80% at 15 kGy. Increasing radiation dose did not significantly alter the content of PYR, DPD or PEN but increased the content of denatured collagen. Cortical allografts fragility increases at doses above 15 kGy. Decreased osteoclast viability at these doses suggests a reduction in the capacity for bone remodelling. These changes were not correlated with alterations in collagen cross-links but in degradation to the collagen secondary structure as evidenced by increased content of denatured collagen


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XLI | Pages 58 - 58
1 Sep 2012
Young S Vince K Coleman B
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Despite modern surgical techniques, reported rates of deep infection following Total Knee Replacement (TKR) persist between 1–2.5%. Coagulase-negative staphylococcus (CNS) has become the most common causative organism, and while growth of CNS is more indolent thanstaphylococcus aureus, it has a relatively higher minimum inhibitory concentration (MIC) against cephalosporins. Tissue concentrations of prophylactic antibiotics may fall below this level during TKR with conventional ‘systemic’ dosing. Regional administration of prophylactic antibiotics via a foot vein following tourniquet inflation has been shown to provide tissue concentrations approximately 10 times higher than systemic dosing, however cannulation of a foot vein is difficult, time consuming, and may compromise sterility. Intraosseous cannulation offers an alternative method of accessing the vascular system, and the aim of this study was to assess its effectiveness in administration of prophylactic antibiotics. 22 patients undergoing primary total knee arthroplasty were randomised into two groups. Group 1 received 1g of cephazolin systemically 10 minutes prior to tourniquet inflation. In Group 2 the EZ-IO tibial cannulation system was used, and 1g of cephazolin was administered intraosseously in 200ml of normal saline following tourniquet inflation and prior to skin incision. Subcutaneous fat and femoral bone samples were taken at set intervals during the procedure, and antibiotic concentrations measured using High Performance Liquid Chromatography (HPLC). There were no significant differences in patient demographics, comorbidities, or physical parameters between groups. The overall mean tissue concentration of cephazolin in subcutaneous fat was 185.9μg/g in the intraosseous group and 10.6μg/g in the systemic group (p<0.01). The mean tissue concentration in bone was 129.9 μg/g in the intraosseous group and 11.4μg/g in the systemic group (p<0.01). These differences were consistent across all sample time points throughout the procedure. No complications occurred in either group. Intraosseous regional administration can achieve tissue levels of antibiotic over an order of magnitude higher than systemic administration. Further work is required to determine if there is clinical benefit in preventing infection, particularly against CNS. This novel mode of drug administration may also have other applications, allowing ‘surgical site delivery’ of medication while minimising systemic side effects