Osteomyelitis, osteitis, spondylodiscitis, septic arthritis, and prosthetic joint infections still represent the worst complications of orthopedic surgery and traumatology. Successful treatment requires, besides surgical débridement, long-term systemic and high-concentration local antibiotic therapy, with possible local antibiotic concentrations of 100 g/ml and more. In this study, we investigated the effect of 20 different antibiotics on primary human osteoblasts (PHO), the osteosarcoma cell line MG63, and the epithelial cell line HeLa. High concentrations of fluoroquinolones, macrolides, clindamycin, chloramphenicol, rifampin, tetracycline, and linezolid during 48 h of incubation inhibited proliferation and metabolic activity, whereas aminoglycosides and inhibitors of bacterial cell wall synthesis did not. Twenty percent inhibitory concentrations for proliferation of PHO were determined as 20 to 40 g/ml for macrolides, clindamycin, and rifampin, 60 to 80 g/ml for chloramphenicol, tetracylin, and fluoroquinolones, and 240 g/ml for linezolid. The proliferation of the cell lines was always less inhibited. We established the measurement of extracellular lactate concentration as an indicator of glycolysis using inhibitors of the respiratory chain (antimycin A, rotenone, and sodium azide) and glycolysis (iodoacetic acid) as reference compounds, whereas inhibition of the respiratory chain increased and inhibition of glycolysis decreased lactate production. The measurement of extracellular lactate concentration revealed that fluoroquinolones, macrolides, clindamycin, rifampin, tetracycline, and especially chloramphenicol and linezolid impaired mitochondrial energetics in high concentrations. This explains partly the observed inhibition of metabolic activity and proliferation in our experiments. Because of differences in the energy metabolism, PHO provided a more sensitive model for orthopedic antibiotic usage than stable cell lines.
The aim of the study is to describe a model for testing biocompatibility of implant materials. Usually cells do not bind the biomaterial surface itself via integrins but adsorbed proteins of blood or interstitial fluids. To eliminate the influence of serum proteins on cell adhesion to the test materials we cultivated osteoprogenitor cells and osteoblasts with a serum replacement or with fetal calf serum, but seeded them likewise without serum or serum replacement on cell culture polystyrene, sandblasted titanium and titanium coated with the peptide c(RGDfK) or hydroxyapatite (Bonemaster) and determined cell adhesion. In addition, the surfaces were preincubated with the serum proteins albumin, fetuin, fibronectin and vitronectin to examine specifically their influence on cell adhesion. Clearly cell adhesion depended on cell culture conditions and state of differentiation, especially with prominent differences in adhesion to c(RGDfK). Precoating with serum proteins demonstrated that besides fibronectin and vitronectin fetuin can function as an adhesion protein, whereas albumin demonstrated an antiadhesive effect. Depending on the material they affected cell adhesion differently. Although osteoprogenitor cells and osteoblasts could bind to tissue culture polystyrene, titanium and especially hydroxyapatite without mediation of proteins, it has to be taken into consideration that cell spreading and proliferation of cells on a scaffold are more important than adhesion alone and may not be ensured in the absence of adhesion proteins.
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