Raman microscopic spectroscopy as a diagnostic tool to detect Staphylococcus epidermidis in bone grafts.

INTRODUCTION: Raman microscopic spectroscopyis a new approach for further characterization and detection of molecular features in many pathological processes. This technique has been successfully applied to scrutinize the spatial distribution of small molecules and proteins within biological systems by in situ analysis. This study uses Raman microscopic spectroscopyto identify any in-depth benefits and drawbacks in diagnosing Staphylococcus epidermidis in human bone grafts. MATERIAL AND METHODS: 40 non-infected human bone samples and 10 human bone samples infected with Staphylococcus epidermidis were analyzed using Raman microscopic spectroscopy. Reflectance data were collected between 200 cm-1 and 3600 cm-1 with a spectral resolution of 4 cm-1 using a Senterra II microscope (Bruker, Ettlingen, Germany). The acquired spectral information was used for spectral and unsupervised classification, such as principal component analysis. RESULTS: Raman measurements produced distinct diagnostic spectra that were used to distinguish between non-infected human bone samples and Staphylococcus epidermidis infected human bone samples by spectral and principal component analyses. A substantial loss in bone quality and protein conformation was detected by human bone samples co-cultured with Staphylococcus epidermidis. The mineral-to-matrix ratio using the phosphate/Amide I ratio (p = 0.030) and carbonate/phosphate ratio (p = 0.001) indicates that the loss of relative mineral content in bones upon bacterial infection is higher than in non-infected human bones. Also, an increase of alterations in the collagen network (p = 0.048) and a decrease in the structural organization and relative collagen in infected human bone could be detected. Subsequent principal component analyses identified Staphylococcus epidermidis in different spectral regions, respectively, originating mainly from CH2 deformation (wagging) of protein (at 1450 cm-1) and bending and stretching modes of C-H groups (∼2800-3000 cm-1). CONCLUSION: Raman microscopic spectroscopyis presented as a promising diagnostic tool to detect Staphylococcus epidermidis in human bone grafts. Further studies in human tissues are warranted.

Material modifications enhancing the antibacterial properties of two biodegradable poly(3-hydroxybutyrate) implants.

The aim of this study was to evaluate the antimicrobial efficacy of adding a gentamicin palmitate (GP) coating and zirconium dioxide (ZrO2) to biodegradable poly(3-hydroxybutyrate) (PHB) to reduce biofilm formation. Cylindrical pins with and without a coating were incubated in Müller-Hinton broth inoculated with 2 × 105 colony-forming units (CFU) ml-1 of Staphylococcus aureus for 2 d or 7 d, then sonicated to disrupt biofilms. Pure PHB (PHB + GP) and PHB pins with ZrO2 added (PHBzr + GP) were coated with GP and compared with PHB pins lacking a coating (PHB). Cells (CFU) were counted to quantify the number of bacteria in the biofilm and a cell proliferation assay was employed to evaluate metabolic activity, and scanning electron microscopy (SEM) was performed to visualize the structure of the biofilm. After 2 d of incubation there were significantly more cells in biofilms on PHB pins than PHB + GP and PHBzr + GP pins (p < 0.0001), and cells in the sonication fluid obtained from GP-coated pins exhibited significantly lower metabolic activity than cells from uncoated PHB pins (p < 0.0001). After 7 d of incubation metabolic activity was lowest for PHBzr + GP, with significant differences between PHB and PHBzr + GP (p = 0.001). SEM revealed more cells attached to the surface, and more structured biofilms, on pins without a coating. Coating pins with GP significantly reduced early biofilm formation on PHB implants. This could lower the potential risk of surgical site infections when using PHB implants. Addition of ZrO2 might further enhance the antibacterial properties. Such modification of the implant material should therefore be considered when developing new biodegradable PHB implants.

[Potential of allogeneic bone grafts as antibiotic carriers : Effect of different preparation processes on efficacy]. / Allogene spongiöse Knochenpräparate als Antibiotikaträger : Einfluss verschiedener Aufbereitungsverfahren auf die Wirksamkeit.

BACKGROUND: The rising number of primary joint replacements worldwide is causing an increase of endoprosthetic revision surgery due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome, and the use of antibiotic-impregnated bone grafts might control the infection and provide a good support for the implant. In this study, we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. METHODS: Heat-treated, lyophilized and fresh frozen cryopreserved bone chips were impregnated with gentamicin sulphate, gentamicin palmitate and vancomycin, and calcium carbonate/calcium sulphate treated with antibiotics. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. RESULTS: The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during the entire experiment. This might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high on the first and second days, then decreased and stayed at a low rate until the end of the second week. CONCLUSION: Depending on the surgical strategy, either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within 2 weeks after surgery. In the case of uncemented revision of joint replacements, allogeneic bone can deliver therapeutic doses of gentamicin and peak levels immediately and a fortnight after implantation.

Mini-midvastus total knee arthroplasty does not result in superior gait pattern.

PURPOSE: Previous studies dealing with gait after minimally invasive surgery (MIS) total knee arthroplasty (TKA) are rare and insufficient. It was the purpose of the study to determine in a prospective, comparative setting whether MIS influences the outcome of TKA in terms of typical 3D gait parameters. METHODS: Patients scheduled for TKA or MIS TKA were invited to participate. MIS TKA was defined as TKA with shorter skin incision, mini-midvastus arthrotomy, special instruments, and avoidance of tibiofemoral dislocation and patella eversion. All other intra- and perioperative aspects were identical for both groups. A 3D gait analysis was performed with a VICON system 1 month preoperative and 8 weeks post-operative. A multivariate analysis of variance was conducted including the main effects time (pre- and post-surgery) and surgical group and the group-by-time interaction effect. RESULTS: Seventeen MIS TKA patients and 20 TKA patients were eligible for the final analysis. We determined neither inter-group differences nor time × group interactions for any gait variables (temporospatial, ground reaction forces, joint angles and joint moments)­except for the varus-valgus knee kinematics. In pre- to post-operative comparison, the maximum valgus sway increased in the MIS group, whereas it decreased in the conventional group (p = 0.001). CONCLUSION: From our findings, it was concluded that MIS TKA does not result in a superior walking pattern 8 weeks post-operative. Because we previously also observed mini-midvastus MIS TKA to have equal or slightly inferior results with regard to knee scores, knee torque, radiographic outcome and tourniquet/operating time, we discontinued the procedure. LEVEL OF EVIDENCE: Prospective comparative study, Therapy, Level II.

Effect of storage temperature on gentamicin release from antibiotic-coated bone chips.

Freezing is the most common method for storing bones until use in skeletal reconstruction. However, the effect of freezing on antibiotic delivery from antibiotic-coated bone has not been evaluated. In this study, we compared antibiotic delivery in vitro from gentamicin-coated human bone stored at different temperatures. Bone chips obtained from human femur heads were chemically cleaned and mixed with gentamicin sulfate. Samples were stored for 4 months at -20 °C, 4 months at -80 °C, or evaluated immediately without freezing. Antibiotic release from the bone chips was measured using Bacillus subtilis as an indicator strain. Zones of inhibition and rates of gentamicin release were similar in all three groups. Storage at -20 and -80 °C for bone allografts has no effect on gentamicin release from chemically cleaned bone chips.

Effect of two cleaning processes for bone allografts on gentamicin impregnation and in vitro antibiotic release.

Bone allografts are a useful and sometimes indispensable tool for the surgeon to repair bone defects. Microbial contamination is a major reason for discarding allografts from bone banks. To improve the number of safe allografts, we suggest chemical cleaning of the grafts followed by antibiotic impregnation. Comparison of two chemical cleaning processes for bone allografts aiming for antibiotic impregnation and consequently delivery rates in vitro. Bone chips of 5-10 mm were prepared from human femoral heads. Two cleaning methods (cleaning A and cleaning B) based on solutions containing hydrogen peroxide, paracetic acid, ethanol and biological detergent were carried out and compared. After the cleaning processes, the bone chips were impregnated with gentamicin. Bacillus subtilis bioassay was used to determine the gentamicin release after intervals of 1-7 days. Differences were compared with non-parametric Mann-Whitney U tests. The zones of inhibition obtained from the bone grafts cleaned with both cleaning processes were similar between the groups. The concentration of the released antibiotic was decreasing gradually over time, following a similar pattern for both groups. The cleaning procedure A as well as the cleaning procedure B for bone allografts allowed the impregnation with gentamicin powder in the same concentrations in both groups. The delivery of gentamicin was similar for both groups. Both cleaning procedures were easy to be carried out, making them suitable for routine use at the bone banks.

Staphylococcus aureus biofilm formation and antibiotic susceptibility tests on polystyrene and metal surfaces.

AIM: We compared the MBEC™-HTP assay plates made of polystyrene with metal discs composed of TMZF(®) and CrCo as substrates for biofilm formation. METHODS AND RESULTS: Staphylococcus aureus was grown on polystyrene and on metal discs made of titanium and chrome-cobalt. Antibiotic susceptibility was assessed by examining the recovery of cells after antibiotic exposure and by measuring the biofilm inhibitory concentration (BIC). The minimal inhibitory concentration (MIC) was assessed with planktonic cells. Bacterial growth was examined by scanning electron microscopy. The antibiotic concentration for biofilm inhibition (BIC) was higher than the MIC for all antibiotics. Microscopic images showed the biofilm structure characterized by groups of cells covered by a film. CONCLUSIONS: All models allowed biofilm formation and testing with several antibiotics in vitro. Gentamicin and rifampicin are the most effective inhibitors of Staph. aureus biofilm-related infections. We recommend MBEC™-HTP assay for rapid testing of multiple substances and TMZF(®) and CrCo discs for low-throughput testing of antibiotic susceptibility and for microscopic analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: In vitro assays can improve the understanding of biofilms and help developing methods to eliminate biofilms from implant surfaces. One advantage of the TMZF(®) and CrCo discs as biofilm in vitro assay is that these metals are commonly used for orthopaedic implants. These models are usable for future periprosthetic joint infection studies.