BAG-S53P4 as an Additive to Bone Allografts: A Laboratory Study Using an Uniaxial Compression Test

ABSTRACT: We want to address the clinical issue of too sparse supply of allograft in total hip replacement and ambitions of controlling the grain size distribution. Bioglass BAG-S53P4 was evaluated as a bone graft additive to chemically treated allografts with controlled grain size distribution. Allografts were chemically cleaned (CG) and mixed with BAG-S53P4 additive (BG) for comparison. All samples were compacted with a dropped weight apparatus and then underwent a uniaxial compression test. The yield limit was determined by a uniaxial compression test and density was recorded while flowability was calculated. There was no difference between the yield stress limit of BG and CG after compaction (p¼0.432). Adding BAG-S53P4 reduced flowability and could indicate better interlocking mechanism between particles. Adding BAG-S53P4 seems to have no impact on the yield stress limit. The extended allografts withstand the compaction equally good which makes it a valid bone substitute in total hip replacement. An in vivo loaded study is needed before clinical use can be recommended

Efficacy of Antibacterial Bioactive Glass S53P4 Against S. aureus Biofilms Grown on Titanium Discs In Vitro

ABSTRACT: We evaluated the effectiveness of different sizes of bioactive glass S53P4 against Staphylococcus aureus biofilms grown on metal discs in vitro. S. aureus biofilms were cultivated on titanium discs. BAG-S53P4 (0.5–0.8mm and <45 mm) were placed in contact with the discs containing biofilms. Glass beads (0.5mm) were used as a control. After each interval, the pH from each sample was measured. Colony forming units were counted for the biofilm recovery verification. In parallel, we tested the activity of bioactive glass against S. aureus planktonic cells. We found that BAG-S53P4 can suppress S. aureus biofilm formation on titanium discs in vitro. The suppression rate of biofilm cells by BAG-S53P4 <45mm was significantly higher than by BAG-S53P4 0.5–0.8mm. BAG-S53P4 has a clear growth-inhibitory effect on S. aureus biofilms. BAG-S53P4 <45mm is more efficient against biofilm growth in vitro comparing with BAG-S53P4 0.5–0.8mm. Bioactive glass S53P4 has potential to be used as bone substitute for the resolution of infection complications in joint replacement surgeries and treatment of chronic osteomyelitis. 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:175–177, 2014

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

Bactericidal Activity of N-Chlorotaurine against Biofilm-Forming Bacteria Grown on Metal Disks

Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies

Influence of poly-N-acetylglucosamine in the extracellular matrix on N-chlorotaurine mediated killing of Staphylococcus epidermidis

N–chlorotaurine (NCT) has recently been shown to have bactericidal activity against bacterial biofilm on metal discs (Coraca-Huber et al., 2014). In a biofilm, Staphylococcus epidermidis polymerizes poly-N-acetylglucosamine (PNAG) to form an extracellular matrix (ECM). Pseudomonas aeruginosa does not express this PNAG and has been shown to be highly susceptible to NCT. We compared the action of NCT on S. epidermidis 1457, a PNAG positive strain (SE1457) and S. epidermidis 1457- M10 an isogenic PNAG negative mutant (SE1457 M10). NCT-mediated killing was more effective and quicker on the PNAG negative strain SE1457 M10. Bacteria hidden in biofilms for prolonged periods of time were generally more susceptible than freshly formed biofilms. The differences in NCT-mediated killing might not be direct effects since NCT did not react with the monomeric N-acetylglucosamine, but might be explained by denser growth in the PNAG-containing biofilm produced by the wild type strain, which results in delayed penetration of NCT. The higher susceptibility of older biofilms to NCTmediated killing could be explained by more pronounced 3D architecture and subsequent larger surface area for interactions with NCT

From Mendel’s discovery on pea to today’s plant genetics and breeding

In 2015, we celebrated the 150th anniversary of the presentation of the seminal work of Gregor Johann Mendel. While Darwin’s theory of evolution was based on differential survival and differential reproductive success, Mendel’s theory of heredity relies on equality and stability throughout all stages of the life cycle. Darwin’s concepts were continuous variation and “soft” heredity; Mendel espoused discontinuous variation and “hard” heredity. Thus, the combination of Mendelian genetics with Darwin’s theory of natural selection was the process that resulted in the modern synthesis of evolutionary biology. Although biology, genetics, and genomics have been revolutionized in recent years, modern genetics will forever rely on simple principles founded on pea breeding using seven single gene characters. Purposeful use of mutants to study gene function is one of the essential tools of modern genetics. Today, over 100 plant species genomes have been sequenced. Mapping populations and their use in segregation of molecular markers and marker–trait association to map and isolate genes, were developed on the basis of Mendel's work. Genome-wide or genomic selection is a recent approach for the development of improved breeding lines. The analysis of complex traits has been enhanced by high-throughput phenotyping and developments in statistical and modeling methods for the analysis of phenotypic data. Introgression of novel alleles from landraces and wild relatives widens genetic diversity and improves traits; transgenic methodologies allow for the introduction of novel genes from diverse sources, and gene editing approaches offer possibilities to manipulate gene in a precise manner

Sterilization Procedures for Titanium Alloy Surfaces Leads to Higher Expression of Biofilm-Related Staphylococcus aureus Genes

Background: Around 1&ndash;2% of all implantation surgeries lead to implant-related infections, incurring costs of 40,000–160,000 per total hip PJI. The 5-year mortality rate of prosthetic joint infections is up to 21%. To prevent infections during surgery, sterile surgery rooms and procedures have been developed and certified standards have been established. To guarantee the sterility, implants can be acquired already sterile from manufacturers. Some titanium implants can be delivered unsterilized with a manual for sterilization procedure in compliance with ISO 17664. The aim of this study is to evaluate if the most used sterilization methods (steam sterilization in an autoclave and UV light sterilization) of titanium alloys, can influence the biofilm forming capacity of Staphylococcus aureus. In this study, we examined the influence of sterilization methods on the gene expression of biofilm-associated genes and regulators. Methods: We compared gene expression of icaADBC, SarA, SigB, and SodA on titanium CP4 and Ti6Al4V alloys sterilized by UV-light and pressurized saturated steam sterilization. We performed RT-qPCR after RNA extraction of Staphylococcus aureus ATCC 29213. In addition, bacterial cell growth on the sterilized titanium surfaces was examined by colony forming unit counting on agar plates after 24 h of incubation. Results: Colony forming units of S. aureus on titanium CP4 samples showed a higher tendency in colony counts when sterilized with UV light than with pressurized saturated steam (autoclaved). Similarly, colony forming unit counts on Ti6Al4V samples showed tendencies of higher numbers on UV light sterilized samples than on autoclaved samples. Gene expression of icaADBC, SarA and SodA between steamed samples and UV light sterilized samples showed no difference on titanium CP4 samples, whereas SigB showed higher gene expression on titanium CP4 samples when sterilized with UV light than in an autoclave. On autoclaved Ti6Al4V samples, all examined genes showed 4 to 9 times higher fold changes in gene expression than on UV light sterilized samples. Conclusions: This study indicates that steam sterilization of Ti6Al4V can increase biofilm formation of S. aureus on its surface. The significantly increased gene expression of biofilm responsible genes may indicate a modification of titanium surfaces on alloy components. This may promote biofilm formation that can lead to implant-infections in vivo