In vitro response of THP-1 derived macrophages to antimicrobially effective PHMB-coated Ti6Al4V alloy implant material with and without contamination with S. epidermidis and P. aeruginosa.

AIM: Periprosthetic joint infections are a devastating complication after arthroplasty, leading to rejection of the prosthesis. The prevention of septic loosening may be possible by an antimicrobial coating of the implant surface. Poly (hexamethylene) biguanide hydrochloride [PHMB] seems to be a suitable antiseptic agent for this purpose since previous studies revealed a low cytotoxicity and a long-lasting microbicidal effect of Ti6Al4V alloy coated with PHMB. To preclude an excessive activation of the immune system, possible inflammatory effects on macrophages upon contact with PHMB-coated surfaces alone and after killing of S. epidermidis and P. aeruginosa are analyzed. METHODS: THP-1 monocytes were differentiated to M0 macrophages by phorbol 12-myristate 13-acetate and seeded onto Ti6Al4V surfaces coated with various amounts of PHMB. Next to microscopic immunofluorescence analysis of labeled macrophages after adhesion on the coated surface, measurement of intracellular reactive oxygen species and analysis of cytokine secretion at different time points without and with previous bacterial contamination were conducted. RESULTS: No influence on morphology of macrophages and only slight increases in iROS generation were detected. The cytokine secretion pattern depends on the surface treatment procedure and the amount of adsorbed PHMB. The PHMB coating resulted in a high reduction of viable bacteria, resulting in no significant differences in cytokine secretion as reaction to coated surfaces with and without bacterial burden. CONCLUSION: Ti6Al4V specimens after alkaline treatment followed by coating with 5-7 µg PHMB and specimens treated with H2O2 before PHMB-coating (4 µg) had the smallest influence on the macrophage phienotype and thus are considered as the surface with the best cytocompatibility to macrophages tested in the present study.

Negative Magnetic Sorting Preserves the Functionality of Ex Vivo Cultivated Non-Adherent Human Monocytes.

BACKGROUND: Monocyte-derived macrophages or dendritic cells are of increasing interest for cellular therapeutic products to treat inflammation-related diseases and cancer. However, the isolation method and the culture conditions applied influence the functionality of cells. For some approaches, the adhesion-induced differentiation into macrophages must be prevented to maintain functions attributed to circulating monocytes. The effects of the isolation method on the functionality of non-adherent peripheral monocytes have not yet been investigated. METHODS: The present study examines the impact of the isolation method on cell viability, growth, metabolism, inflammation-induced cytokine response, migratory capacity, and adherence of non-adherent human peripheral monocytes. The monocytes were isolated by magnetic sorting using either positive or negative selection and cultured in cell-repellent plates. RESULTS: The purity and yield of monocytes were higher after positive selection. However, the adherence and migratory capacity, cytokine response, and metabolic activity were decreased compared to negatively selected monocytes. The impaired functionality presented in combination with cell shrinking, thus, indicates the start of cell viability loss. Negatively selected non-adherent monocytes showed no impairment in functionality, and the viability remained high. In conclusion, this approach is better suited for conducting ex vivo modifications of monocytes prior to the intended experimental setup or therapeutic application.

Mimicking of Blood Flow Results in a Distinct Functional Phenotype in Human Non-Adherent Classical Monocytes.

Ex vivo culture conditions during the manufacturing process impact the therapeutic effect of cell-based products. Mimicking blood flow during ex vivo culture of monocytes has beneficial effects by preserving their migratory ability. However, the effects of shear flow on the inflammatory response have not been studied so far. Hence, the present study investigates the effects of shear flow on both blood-derived naïve and activated monocytes. The activation of monocytes was experimentally induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which acts as a pro-survival and growth factor on monocytes with a potential role in inflammation. Monocytes were cultured under dynamic (=shear flow) or static conditions while preventing monocytes' adherence by using cell-repellent surfaces to avoid adhesion-induced differentiation. After cultivation (40 h), cell size, viability, and cytokine secretion were evaluated, and the cells were further applied to functional tests on their migratory capacity, adherence, and metabolic activity. Our results demonstrate that the application of shear flow resulted in a decreased pro-inflammatory signaling concurrent with increased secretion of the anti-inflammatory cytokine IL-10 and increased migratory capacity. These features may improve the efficacy of monocyte-based therapeutic products as both the unwanted inflammatory signaling in blood circulation and the loss of migratory ability will be prevented.

In vitro evaluation of contact-active antibacterial efficacy of Ti-Al-V alloys coated with the antimicrobial agent PHMB.

Immobilized polycationic substances on biomaterial surfaces kill adhering bacteria upon contact and are considered a promising non-antibiotic alternative. Unfortunately, there is no generally accepted in vitro method for quantitatively evaluating the antibacterial efficacy of contact-active non-leachable antimicrobial surfaces. Moreover, guidelines of generally accepted international industrial standards do not reflect the basic principle of bacterial contamination and/or are performed in the presence of a solid covering material. Therefore, in the present study, six bacterial adherence tests on non-porous surfaces with no covering material were compared with respect to their efficacy and reproducibility, as well as to evaluate the bactericidal contact-killing of relevant device-associated slime-producing bacteria using antimicrobially coated Ti6Al4V surfaces with positively-charged poly(hexamethylene biguanide) hydrochloride (PHMB). After direct bacterial inoculation to simulate a perioperative infection, non-leaching PHMB reacts bactericidally against the slime-producing bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa after surface contact. The 6-h drop technique was found to be a suitable method to quantitatively evaluate contact-active antibacterial surfaces. Adjunctively, however, damage of bacterial membrane integrity should be confirmed by LIVE/DEAD staining and the presence of non-leaching agents. STATEMENT OF SIGNIFICANCE: Unintentional perioperative bacterial adhesion to implant surfaces can generate biomaterial-associated infections. Adhered bacteria produce biofilms that protect them from antibiotic attack, which may be complicated by possible antibiotic resistance. Polycationic surfaces can prevent such unwanted biofilm formation by killing bacteria upon initial contact. Unfortunately, no reliable in vitro methods exist to evaluate the efficacy of contact-active antimicrobial surfaces. In this study, we show that the 6-h drop technique may be a suitable method to evaluate positively-charged contact-killing surfaces. Identification of suitable screening assays for evaluating the bactericidal efficacy of non-leachable antimicrobial agents will greatly improve this newly developing field as a prophylactic alternative to postoperative treatment of implant-associated infections by antibiotics.

Poly (hexamethylene biguanide), adsorbed onto Ti-Al-V alloys, kills slime-producing Staphylococci and Pseudomonas aeruginosa without inhibiting SaOs-2 cell differentiation.

Antimicrobial coating of implant material with poly(hexamethylene biguanide) hydrochloride (PHMB) may be an eligible method for preventing implant-associated infections. In the present study, an antibacterial effective amount of PHMB is adsorbed on the surface of titanium alloy after simple chemical pretreatment. Either oxidation with 5% H2 O2 for 24 hr or processing for 2 hr in 5 M NaOH provides the base for the subsequent formation of a relatively stable self-assembled PHMB layer. Compared with an untreated control group, adsorbed PHMB produces no adverse effects on SaOs-2 cells within 48 hr cell culture, but promotes the initial attachment and spreading of the osteoblasts within 15 min. Specimens were inoculated with slime-producing bacteria to simulate a perioperative infection. Adsorbed PHMB reacts bactericidally against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa after surface contact. Adhered SaOs-2 cells differentiate and produce alkaline phosphatase and deposit calcium within 4 days in a mineralization medium on PHMB-coated Ti6Al4V surfaces, which have been precontaminated with S. epidermidis. The presented procedures provide a simple method for generating biocompatibly and antimicrobially effective implant surfaces that may be clinically important.