Treatment of vascular graft infections: gentamicin-coated ePTFE grafts reveals strong antibacterial properties in vitro.

Vascular graft infections (VGI) are severe complications in prosthetic vascular surgery with an incidence ranging from 1 to 6%. In these cases, synthetic grafts are commonly used in combination with antimicrobial agents. Expanded polytetrafluoroethylene (ePTFE) is in clinical use as a synthetic graft material and shows promising results by influencing bacterial adhesion. However, the literature on antibiotic-bound ePTFE grafts is scarce. Gentamicin is a frequently used antibiotic for local treatment of surgical site infections, but has not been evaluated as antimicrobial agent on ePTFE grafts. In this study, we examine the antimicrobial efficacy and biocompatibility of novel types of gentamicin-coated ePTFE grafts in vitro. ePTFE grafts coated with gentamicin salt formulations with covalently-bound palmitate were evaluated in two drug concentrations (GP1.75% and GP3.5%). To investigate effects from types of formulations, also suspensions of gentamicin in palmitate as well as polylactide were used at comparable levels (GS + PA and GS + R203). Antibacterial efficacies were estimated by employing a zone of inhibition, growth inhibition and bacterial adhesion assay against Staphylococcus aureus (SA). Cytotoxicity was determined with murine fibroblasts according to the ISO standard 10993-5. Gentamicin-coated ePTFE grafts show low bacterial adherence and strong antibacterial properties in vitro against SA. Bactericidal inhibition lasted until day 11. Highest biocompatibility was achieved using gentamicin palmitate GP1.75% coated ePTFE grafts. ePTFE grafts with gentamicin-coating are effective in vitro against SA growth and adherence. Most promising results regarding antimicrobial properties and biocompatibility were shown with chemically bounded gentamicin palmitate GP1.75% coatings. Graphical abstract.

Novel antimicrobial coatings based on polylactide for plastic biliary stents to prevent post-endoscopic retrograde cholangiography cholangitis.

BACKGROUND: Cholangitis is a common complication after endoscopic retrograde cholangiography (ERC). OBJECTIVES: To evaluate antimicrobial coatings for biliary plastic stents in relation to efficacy against biliary pathogens, drug release and toxicity. METHODS: Biliary plastic stents were prepared by coating using a polylactide drug carrier. Stent coatings contained 4% (w/w) drug content of Resomer-octenidine (RO), Resomer-octenidine and citrate (ROC), Resomer-triclosan (RT) or Resomer-gentamicin (RG). Drug-release kinetics, antimicrobial efficacies of coated biliary stents against biliary pathogens and biocompatibilities were tested. Antimicrobial efficacy measurements included MIC testing, zone of inhibition (ZOI) assays and log reduction in bacterial suspensions. RESULTS: Continuous drug release was observed in all antimicrobial stent coatings for at least 168 h with an initial peak within the first 24 h. RT-, ROC- and RG-coated stents resulted in the following log reductions in suspensions: Escherichia coli (-0.3, -7.4 and -6.6, respectively); Enterococcus faecalis (-0.05, -6.3 and -3.9, respectively); and Candida albicans (-0.04, -1.5 and -0.2, respectively). ROC had the highest log reduction in suspension and the most favourable time course of ZOI (≥2 mm, over 72 h) against all tested pathogens. Although RT coatings showed the lowest MICs, they had the lowest ZOIs after 24 h. Concerning RO, acceptable biocompatibility could only be reached by adding a citrate component. RG showed the largest ZOI after 24 h against E. coli (19.3 mm) and E. faecalis (5.1 mm), whereas the ZOI was lower against C. albicans (1.3 mm) compared with ROC (3.7 mm). CONCLUSIONS: ROC corresponds most closely to the requirements of an ideal antimicrobial stent coating to prevent post-ERC cholangitis, showing the highest log reduction in pathogen counts, the most favourable time course of ZOI and high biocompatibility.

In vitro Growth Pattern of Primary Human Osteoblasts on Calcium Phosphate- and Polymethylmethacrylate-Based Bone Cement.

BACKGROUND/PURPOSE: Polymethylmethacrylate (PMMA) and calcium phosphate (Ca-P) cements are widely used for arthroplasty surgery and augmentation of bone defects. However, aseptic implant loosening in absence of wear-induced osteolysis indicates an unfavourable interaction between the cement surface and human osteoblasts. Our underlying hypothesis is that cement surfaces directly modify cell viability, proliferation rate, and cell differentiation. METHODS: To test this hypothesis, we examined primary human osteoblasts harvested from six individuals. These cells were pooled and subsequently seeded directly on cement pellets prepared from Palacos® R, Palacos® R+G, and Norian® Drillable cements. After incubation for 24 and 72 h, cell viability, proliferation rate, apoptosis rate, and cell differentiation were analysed. RESULTS: Upon cultivation of human osteoblasts on cement surfaces, we observed a significantly reduced cell viability and DNA content compared to the control. Analysis of the apoptosis rate revealed an increase for cells on Palacos R and Norian Drillable, but a significant decrease on Palacos R+G compared to the control. Regarding osteogenic differentiation, significantly lower values of alkaline phosphatase enzyme activity were identified for all cement surfaces after 24 and 72 h compared to cultivation on tissue culture plastic, serving as control. CONCLUSIONS: In summary, these data suggest a limited biocompatibility of both PMMA and Ca-P cements, necessitating further research to reduce unfavourable cell-cement interactions and consequently extend implant survival.

Patient-specific effects of soluble factors from Staphylococcus aureus and Staphylococcus epidermidis biofilms on osteogenic differentiation of primary human osteoblasts.

Due to the frequency of biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis in orthopedics, it is crucial to understand the interaction between the soluble factors produced by prokaryotes and their effects on eukaryotes. Our knowledge concerning the effect of soluble biofilm factors (SBF) and their virulence potential on osteogenic differentiation is limited to few studies, particularly when there is no direct contact between prokaryotic and eukaryotic cells. SBF were produced by incubating biofilm from S. aureus and S. epidermidis in osteogenic media. Osteoblasts of seven donors were included in this study. Our results demonstrate that the detrimental effects of these pathogens do not require direct contact between prokaryotic and eukaryotic cells. SBF produced by S. aureus and S. epidermidis affect the metabolic activity of osteoblasts. However, the effect of SBF derived from S. aureus seems to be more pronounced compared to that of S. epidermidis. The influence of SBF of S. aureus and S. epidermidis on gene expression of COL1A1, ALPL, BGLAP, SPP1, RUNX2 is bacteria-, patient-, concentration-, and incubation time dependent. Mineralization was monitored by staining the calcium and phosphate deposition and revealed that the SBF of S. epidermidis markedly inhibits calcium deposition; however, S. aureus shows a less inhibitory effect. Therefore, these new findings support the hypotheses that soluble biofilm factors affect the osteogenic processes substantially, particularly when there is no direct interaction between bacteria and osteoblast.

Antimicrobial peptides in human synovial membrane as (low-grade) periprosthetic joint infection biomarkers.

BACKGROUND: Safe diagnosis of periprosthetic joint infection (PJI) is of utmost importance for successful exchange arthroplasty. However, current diagnostic tools show insufficient accuracy in the clinically common and challenging chronic low-grade infections. To close this diagnostic gap, reliable (bio)markers display the most promising candidates. Antimicrobial peptides (AMPs) are part of the innate immune response towards microbial growth. Recently we could show significant intraarticular levels of human cathelicidin LL-37 and ß-defensin-3 (HBD-3) with high diagnostic accuracy in PJI synovial fluid. Consequently, these promising biomarkers were evaluated in PJI synovial membrane and synoviocytes, which may significantly facilitate histological diagnosis of PJI to improve outcome of septic joint replacement. METHODS: In this prospective single-center controlled clinical study (diagnostic level II), consecutive patients with total hip (THR) and knee (TKR) replacements were included undergoing primary arthroplasty (n = 8), surgical revision due to aseptic loosening (n = 9) and septic arthroplasty with coagulase-negative staphylococci (n = 8) according to the criteria of the Musculoskeletal Infection Society (MSIS). Semiquantitative immunohistochemical (IHC) analysis of LL-37, HBD-3 and HBD-2 in synovial membrane and isolated synoviocytes based on Total Allred Score (TS) and Immunoreactive Remmele and Stegner score (IRS) was performed. For statistical analysis, SPSS 26.0/R3.6.3 (p < 0.05) was used. RESULTS: The AMPs LL-37 and HBD-3 were significantly elevated (up to 20×) in synovial membranes from PJI compared to aseptic loosening or primary arthroplasty. The area under the curve (AUC) in a receiver operating characteristic curve analysis was equal to 1.0 for both scores revealing excellent diagnostic accuracy. Isolated synoviocytes as cellular AMP source showed comparable results with a significant LL-37/HBD-3-increase up to 3 × in PJI. In contrast, local HBD-2 levels were negligible (p > 0.23) upon PJI with a lower diagnostic accuracy (AUC = 0.65) in analogy to our previous findings with synovial fluid. CONCLUSIONS: Our results implicate AMPs as promising and specific biomarkers for the histological diagnosis of PJI.

Full biomechanical mapping of the ovine knee joint to determine creep-recovery, stiffness and thickness variation.

BACKGROUND: Clinical cartilage repair strategies can be tested using the sheep model as suggest by the European Medicines Agency. To characterize variation within the joint a full biomechanical mapping is necessary. The aim of this study is to establish a loading model, to map regional differences within the knee and determine reference areas for area specific replacement techniques. METHODS: A porous indenter was selected to evaluate 22 defined test locations (femoral condyles, tibia plateau, patella, femoral groove) on ovine knees (n = 7). A high-dynamic force-controlled micro creep and creep-recovery indentation test system applied five loading (0.11 MPa) and unloading (5.6 kPa) cycles for 60 s each and recorded creep-recovery. Needle indentation was used to measure cartilage thickness and calculate total strain. FINDINGS: Steady state behaviour was observed from the third cycle and further evaluated. Little variation of stiffness in N/mm was found within the patella (4.3SD0.5) and femoral groove (8.1SD0.7) compared to larger variations in the femur (7.9SD2.0) and tibia (7.5SD3.2). Creep indentation showed values of 14.5%(SD2.7%) for the patella and 17.4%(SD3%) for the femoral grove opposed to 13.4%(SD4.3%) for the femoral condyles and 21.8%(SD6.6%) for the tibia plateau. Similar trends were observed analysing creep-recovery. Values were normalized to cartilage thickness which ranged between 0.36 mm and 1.14 mm. INTERPRETATION: Our setup allows a reliable evaluation of zonal differences. Homogenous biomechanical behaviour is found within the patella and femoral groove whereas significant biomechanical variation within the femoral condyles and tibia plateau indicates the need for site-specific cartilage repair products.

Correction: Viable adhered Staphylococcus aureus highly reduced on novel antimicrobial sutures using chlorhexidine and octenidine to avoid surgical site infection (SSI).

[This corrects the article DOI: 10.1371/journal.pone.0190912.].

Viable adhered Staphylococcus aureus highly reduced on novel antimicrobial sutures using chlorhexidine and octenidine to avoid surgical site infection (SSI).

BACKGROUND: Surgical sutures can promote migration of bacteria and thus start infections. Antiseptic coating of sutures may inhibit proliferation of adhered bacteria and avoid such complications. OBJECTIVES: This study investigated the inhibition of viable adhering bacteria on novel antimicrobially coated surgical sutures using chlorhexidine or octenidine, a critical factor for proliferation at the onset of local infections. The medical need, a rapid eradication of bacteria in wounds, can be fulfilled by a high antimicrobial efficacy during the first days after wound closure. METHODS: As a pretesting on antibacterial efficacy against relevant bacterial pathogens a zone of inhibition assay was conducted with middle ranged concentrated suture coatings (22 µg/cm). For further investigation of adhering bacteria in detail the most clinically relevant Staphylococcus aureus (ATCC®49230™) was used. Absorbable braided sutures were coated with chlorhexidine-laurate, chlorhexidine-palmitate, octenidine-laurate, and octenidine-palmitate. Each coating type resulted in 11, 22, or 33 µg/cm drug content on sutures. Scanning electron microscopy (SEM) was performed once to inspect the coating quality and twice to investigate if bacteria have colonized on sutures. Adhesion experiments were assessed by exposing coated sutures to S. aureus suspensions for 3 h at 37°C. Subsequently, sutures were sonicated and the number of viable bacteria released from the suture surface was determined. Furthermore, the number of viable planktonic bacteria was measured in suspensions containing antimicrobial sutures. Commercially available sutures without drugs (Vicryl®, PGA Resorba®, and Gunze PGA), as well as triclosan-containing Vicryl® Plus were used as control groups. RESULTS: Zone of inhibition assay documented a multispecies efficacy of novel coated sutures against tested bacterial strains, comparable to most relevant S. aureus over 48 hours. SEM pictures demonstrated uniform layers on coated sutures with higher roughness for palmitate coatings and sustaining integrity of coated sutures. Adherent S. aureus were found via SEM on all types of investigated sutures. The novel antimicrobial sutures showed significantly less viable adhered S. aureus bacteria (up to 6.1 log) compared to Vicryl® Plus (0.5 log). Within 11 µg/cm drug-containing sutures, octenidine-palmitate (OL11) showed the highest number of viable adhered S. aureus (0.5 log), similar to Vicryl® Plus. Chlorhexidine-laurate (CL11) showed the lowest number of S. aureus on sutures (1.7 log), a 1.2 log greater reduction. In addition, planktonic S. aureus in suspensions were highly inhibited by CL11 (0.9 log) represents a 0.6 log greater reduction compared to Vicryl® Plus (0.3 log). CONCLUSIONS: Novel antimicrobial sutures can potentially limit surgical site infections caused by multiple pathogenic bacterial species. Therefore, a potential inhibition of multispecies biofilm formation is assumed. In detail tested with S. aureus, the chlorhexidine-laurate coating (CL11) best meets the medical requirements for a fast bacterial eradication. This suture coating shows the lowest survival rate of adhering as well as planktonic bacteria, a high drug release during the first-clinically most relevant- 48 hours, as well as biocompatibility. Thus, CL11 coatings should be recommended for prophylactic antimicrobial sutures as an optimal surgical supplement to reduce wound infections. However, animal and clinical investigations are important to prove safety and efficacy for future applications.

Growth promoting in vitro effect of synthetic cyclic RGD-peptides on human osteoblast-like cells attached to cancellous bone.

In tissue engineering, the application of biofunctional compounds on biomaterials such as integrin binding RGD-peptides has gained growing interest. Anchorage-dependent cells like osteoblasts bind to these peptides thus ameliorating the integration of a synthetic implant. In case sterilized bone grafts are used as substitutes for reconstruction of bone defects, the ingrowth of the implanted bone is often disturbed because of severe pretreatment such as irradiation or autoclaving, impairing the biological and mechanical properties of the bone. We report for the first time on the in vitro coating of the surface of freshly resected, cleaned bone discs with synthetic, cyclic RGD-peptides. For this approach, two different RGD-peptides were used, one containing two phosphonate anchors, the other peptide four of these binding moieties to allow efficient association of these reactive RGD-peptides to the inorganic bone matrix. Human osteoblast-like cells were cultured on RGD-coated bone discs and the adherence and growth of the cells were analyzed. Coating of bone discs with RGD-peptides did not improve the adhesion rate of osteoblast-like cells to the discs but significantly (up to 40%) accelerated growth of these cells within 8 days after attachment. This effect points to pretreatment of bone implants, especially at the critical interface area between the implanted bone and the non-resected residual bone structure, before re-implantation in order to stimulate and enhance osteointegration of a bone implant.

Expression of the Tumor-associated Mucin 1 Epitope Analyzed with the Humanized PankoMab-GEX™ Antibody in Malignant and Normal Tissues of the Head and Neck.

BACKGROUND: During neoplasia, glycosylation changes. In this setting, mucins, especially mucin 1 (MUC1), become carriers for oncofetal carbohydrates and relieve invasive growth. The recently described tumor-associated MUC1 epitope TA-MUC1 is primarily restricted to malignancies and is overexpressed in these tissues. The humanized monoclonal antibody PankoMab-GEX specifically recognizes TA-MUC1. MATERIALS AND METHODS: Laryngeal cancer specimens (n=125) and normal tissue of head and neck (n=7) were used in this study. Paraffin-embedded sections were incubated with PankoMab-GEX. Staining reaction was carried out using peroxidase (POD) labeling and diaminobenzidine (DAB). Breast cancer tissue was used as positive control and negative control used non-specific mouse IgM. Semi-quantitative evaluation by two independent double-blinded investigators, including a pathologist, used the immunoreactive score (IRS) of Remmele and Stegner. RESULTS: A total of 31 out of 125 laryngeal cancer specimens were classified as G1. Of these, 22 (71%) were completely negative for TA-MUC1, the remaining 9 showed very weak staining, with an IRS of 2. A total of 94 cases of cancer specimens were classified as G2 and G3; 34 of them were also negative, but 60 had an IRS of up to 9. All investigated normal tissue of the upper aerodigestive tract was completely negative for TA-MUC1. CONCLUSION: G1 tumors are completely negative or do not reach an IRS relevant range. The finding that G1 tumors are completely negative for TA-MUC1 or have IRS≤2 can be helpful for histopathological examination, especially concerning tumor grading. Therefore, this antibody holds great potential for use as a therapeutic antibody in laryngeal cancer.

Methylation of the ER-alpha Promoter Is Influenced by its Ligand Estrogen in Osteosarcoma Cells SAOS-2 In Vitro.

BACKGROUND/AIM: The aggressive fast-growing osteosarcoma is the most common primary malignant bone tumor. The relevance of estrogen as a key player in bone metabolism and bone tumor is well-known. At the molecular level, estrogen activates the estrogen receptor α (ERα) as a natural ligand of this receptor. ERα acts as a transcription factor by binding to the "estrogen response element" (ERE) and regulates the expression of a various number of genes. Epigenetic processes, e.g. the methylation of the "cytosine-phosphatidyl-guanine (CpG) islands" can change the transcription of target genes and subsequently the protein expression. As DNA methylation is generally associated with gene transcription repression, up until now little is known about the ERα methylation in osteosarcoma cells. The aim of the present pilot study was to evaluate the methylation status of ERα in osteosarcoma cells SAOS-2 and MG 63 after stimulation with estrogen. MATERIALS AND METHODS: SAOS-2 and MG 63 cells were cultured in DMEM. After treatment with 10 nmol estrogen (E2) for 24 h, the expression of ERα was detected by immunocytochemistry (ICC). As controls we used untreated cells. Staining was evaluated semi-quantitatively by the immunoreactive score of Remmele and Stegner (IRS). To determine mRNA gene expression, extracted RNA was transcribed into c-DNA and a quantitative real-time-PCR (qRT-PCR) was carried out. The semi quantitative evaluation of the ERα mRNA was based on the 2(-ΔΔct) method using untreated cells as reference control. One microgram of each extracted genomic DNA sample was converted with bisulfite and a real-time methylation-specific PCR (rt-MSP) was performed. RESULTS: The estrogen-stimulated SAOS-2 cells showed a significant increase of ERα expression. A 7-fold up-regulation of ERα mRNA confirmed the results of immunocytochemistry. Methylation of the ERα promoter was not detected in treated cells. In contrast, we identified methylation of the ERα promoters in untreated cells. The staining of MG 63 cells showed a weak gain of ERα expression in the stimulated cells, as well as a weak increase of the ER-α mRNA (2-fold). Methylation of the ERα promoters was not detectable in either treated or untreated cells. CONCLUSION: The methylation status of ERα in osteosarcoma cells is affected by estrogen. These findings indicate that epigenetic changes of genomic DNA regulate ERα synthesis. Taken together, our results suggest that SAOS-2 cells can be an interesting model for further investigating ERα synthesis. In addition, the evaluation of ERα methylation in osteosarcoma specimens is in progress.

Spontaneous in vitro transformation of primary human osteoblast-like cells.

BACKGROUND: Two new tumor-like cell lines were established which developed spontaneously in vitro from normal human primary osteoblast-like cells originating from non-oncogenic bone surgery. MATERIALS AND METHODS: The tumor cell properties studied included morphology, proliferation characteristics in normal and low-serum media, and anchorage-independent growth in soft agarose. RESULTS AND CONCLUSION: Karyotyping of the cells showed numerous rearrangements and abnormalities. These results pointed to the tumorigenic potential of the cells and demonstrate the importance of biosafety in tissue engineering and therapeutic cell applications when prolonged culture conditions are required.