A Novel In Vitro System for Comparative Analyses of Bone Cells and Bacteria under Electrical Stimulation

Electrical stimulation is a promising approach to enhance bone regeneration while having potential to inhibit bacterial growth. To investigate effects of alternating electric field stimulation on both human osteoblasts and bacteria, a novel in vitro system was designed. Electric field distribution was simulated numerically and proved by experimental validation. Cells were stimulated on Ti6Al4V electrodes and in short distance to electrodes. Bacterial growth was enumerated in supernatant and on the electrode surface and biofilm formation was quantified. Electrical stimulation modulated gene expression of osteoblastic differentiation markers in a voltage-dependent manner, resulting in significantly enhanced osteocalcin mRNA synthesis rate on electrodes after stimulation with 1.4VRMS. While collagen type I synthesis increased when stimulated with 0.2VRMS, it decreased after stimulation with 1.4VRMS. Only slight and infrequent influence on bacterial growth was observed following stimulations with 0.2VRMS and 1.4VRMS after 48 and 72 h, respectively. In summary this novel test system is applicable for extended in vitro studies concerning definition of appropriate stimulation parameters for bone cell growth and differentiation, bacterial growth suppression, and investigation of general effects of electrical stimulation

Prospective data collection and analysis of perforations and tears of latex surgical gloves during primary endoprosthetic surgeries

Introduction: Surgical gloves are used to prevent contamination of the patient and the hospital staff with pathogens. The aim of this study was to examine the actual effectiveness of gloves by examining the damage (perforations, tears) to latex gloves during surgery in the case of primary hip and knee prosthesis implantation. Materials and methods: Latex surgical gloves used by surgeons for primary hip and knee replacement surgeries were collected directly after the surgery and tested using the watertightness test according to ISO EN 455-1:2000.Results: 540 gloves were collected from 104 surgeries. In 32.7% of surgeries at least one glove was damaged. Of all the gloves collected, 10.9% were damaged, mainly on the index finger. The size of the perforations ranged from ≤1 mm to over 5 mm. The surgeon’s glove size was the only factor that significantly influenced the occurrence of glove damage. Surgeon training level, procedure duration, and the use of bone cement had no significant influence.Conclusions: Our results highlight the high failure rate of surgical gloves. This has acute implications for glove production, surgical practice, and hygiene guidelines. Further studies are needed to detect the surgical steps, surface structures, and instruments that pose an increased risk for glove damage

Co-Culture of S. epidermidis and Human Osteoblasts on Implant Surfaces: An Advanced In Vitro Model for Implant-Associated Infections.

OBJECTIVES:Total joint arthroplasty is one of the most frequent and effective surgeries today. However, despite improved surgical techniques, a significant number of implant-associated infections still occur. Suitable in vitro models are needed to test potential approaches to prevent infection. In the present study, we aimed to establish an in vitro co-culture setup of human primary osteoblasts and S. epidermidis to model the onset of implant-associated infections, and to analyze antimicrobial implant surfaces and coatings. MATERIALS AND METHODS:For initial surface adhesion, human primary osteoblasts (hOB) were grown for 24 hours on test sample discs made of polystyrene, titanium alloy Ti6Al4V, bone cement PALACOS R®, and PALACOS R® loaded with antibiotics. Co-cultures were performed as a single-species infection on the osteoblasts with S. epidermidis (multiplicity of infection of 0.04), and were incubated for 2 and 7 days under aerobic conditions. Planktonic S. epidermidis was quantified by centrifugation and determination of colony-forming units (CFU). The quantification of biofilm-bound S. epidermidis on the test samples was performed by sonication and CFU counting. Quantification of adherent and vital primary osteoblasts on the test samples was performed by trypan-blue staining and counting. Scanning electron microscopy was used for evaluation of topography and composition of the species on the sample surfaces. RESULTS:After 2 days, we observed approximately 104 CFU/ml biofilm-bound S. epidermidis (103 CFU/ml initial population) on the antibiotics-loaded bone cement samples in the presence of hOB, while no bacteria were detected without hOB. No biofilm-bound bacteria were detectable after 7 days in either case. Similar levels of planktonic bacteria were observed on day 2 with and without hOB. After 7 days, about 105 CFU/ml planktonic bacteria were present, but only in the absence of hOB. Further, no bacteria were observed within the biofilm, while the number of hOB was decreased to 10% of its initial value compared to 150% in the mono-culture of hOB. CONCLUSION:We developed a co-culture setup that serves as a more comprehensive in vitro model for the onset of implant-associated infections and provides a test method for antimicrobial implant materials and coatings. We demonstrate that observations can be made that are unavailable from mono-culture experiments

Cell viability evaluation of human primary osteoblasts in mono-culture.

<p><b>A)</b> Metabolic activity of human primary osteoblasts (hOB) incubated for 2 and 7 days on Ti6Al4V, PALACOS^® R, and PALACOS^® R+G+V, and measured using the WST-1 assay. Values are given as percentage of the polystyrene control. PALACOS^® R+G+V displayed lower cell activity than the other materials on both days. (*) Denotes significance with respect to the polystyrene control at each time point, and (+) significance with respect to the same material on day 2 (n = 4, mean ± SEM). <b>B)</b> Live/dead staining of the hOB cultured in a monolayer on the test samples after 2 and 7 days of incubation. All images were taken at 40x magnification. Scale bars are 200 μm.</p

Evaluation of metabolic activity of biofilm-bound <i>S</i>. <i>epidermidis</i> in mono-culture.

<p><b>A)</b> Metabolic activity of biofilm-bound <i>S</i>. <i>epidermidis</i> incubated for 2 and 7 days on Ti6Al4V, PALACOS^® R, and PALACOS^® R+G+V, and measured using the WST-1 assay. Values are given as a percentage of the polystyrene control. PALACOS^® R+G+V displayed approximately 25% and 0% activity after 2 and 7 days respectively. (*) Denotes significance with respect to the polystyrene control at each time point, and (+) significance with respect to the same material on day 2 (n = 4, mean ± SEM). <b>B)</b> Live/dead staining of the biofilm-bound <i>S</i>. <i>epidermidis</i> on the test samples after 2 and 7 days of incubation. All images were made at 400x magnification. Scale bars are 20 μm.</p

Quantification of viable planktonic <i>S</i>. <i>epidermidis</i> by CFU counting.

<p>Viable planktonic <i>S</i>. <i>epidermidis</i> were quantified in the medium after 2 and 7 days of cultivation with and without hOB on the 4 test samples (polystyrene control, TI6Al4V, PALACOS^® R, PALACOS^® R+G+V). Qualitatively, we observed the same results as for biofilm-bound <i>S</i>. <i>epidermidis</i>, except for PALACOS^® R+G+V where viable cells remain after 2 days in both experiments and after 7 days in the co-culture. (*) Denotes significance with respect to the polystyrene control, and (#) significance with respect to the mono-culture (n = 4, mean ± SEM).</p

SEM images of different samples.

<p>Row 1: Test samples in medium. Rows 2 and 3: hOB mono-culture on test samples. Rows 4 and 5: <i>S</i>. <i>epidermidis</i> mono-culture on test samples. Rows 6 and 6: Co-culture of hOB and <i>S</i>. <i>epidermidis</i> on test samples. Images were taken at magnification 500x, 2000x or 2500x.</p

Quantification of viable biofilm-bound <i>S</i>. <i>epidermidis</i> by CFU counting.

<p><i>S</i>. <i>epidermidis</i> mono-culture and co-culture with hOB in the biofilm on the 4 test samples (polystyrene control, TI6Al4V, PALACOS^® R, PALACOS^® R+G+V) after 2 and 7 days. PALACOS^® R+G+V test samples showed minimal viable bacteria except in the co-culture after 2 days. (*) Denotes significance with respect to the polystyrene control, and (#) significance with respect to the mono-culture (n = 4, mean ± SEM).</p