Ro¨hner et al. Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis Two studies, which detected effects of antiseptics on bovine chondrocytes, were by Bates and co-workers and M u¨ller and colleagues.14,15 In the study of Bates and colleagues, bovine articular cartilage was treated with hydrogen peroxide and showed a signiﬁcant inhibition of proteoglycan synthesis. This result indicates that hydrogen peroxide is an active species of oxygen-derived reactive species, which is involved in the inhibition process.15 In the study of M u¨ller and Kramer, articular cartilage of bovine sesamoid bone was treated with betaisodona, polyhexanide, and octenidine in the presence of Escherichia coli and Staphylococcus aureus. One result of this study was that all tested antimicrobial irrigation ﬂuids inhibit proteoglycan synthesis. Additionally, M u¨ller and Kramer could show that betaisodona has the capacity to destroy microorganism in vitro in the presence of cartilage tissue without negative impact on chondro- cyte metabolism.14 In both studies, the mechanisms by which antiseptics cause an inhibition of proteo- glycan synthesis in cultured articular cartilage were not clear. In the present study, we evaluate the effects of polyhexanide and hydrogen peroxide on the extracellular cartilaginous matrix synthesis of pri- mary human chondrocytes. Materials and Methods Tissue culture plasticware were obtained from TPP (Trasadingen, Switzerland). Culture medium, phos- phate-buffered saline (PBS), trypsin, and fetal calf serum were purchased from Biochrom (Berlin, Germany). All other reagents were obtained from Sigma-Aldrich (Deisenhofen, Germany). Chondrocyte isolation and three-dimensional culture in alginate beads Chondrocyte isolation was performed as previously described.16,17 Cartilage was obtained from four human donors with knee osteoarthritis not presenting any kind of infectious signals. Experimental proto- cols were approved by the local ethics committee. Cartilage was minced and digested in medium containing 1 mg/ml pronase (Sigma-Aldrich, Deisen- hofen, Germany) for 30 minutes at 37uC. Next, digestion medium was discarded and the tissue was digested with medium containing 1 mg/ml clostridial collagenase (Sigma-Aldrich) for 18 hours at 37uC. Digested solution was ﬁltered (70 mm Nylon; BD Falcon, Bedford, Germany) and centrifuged at 1200 rev/min for 8 minutes. The supernatant was discarded and the cell pellet was washed three times with PBS. Then chondrocytes were suspended in DMEM/Ham’s F12 with 10% fetal bovine serum, 1% penicillin/streptomycine, and cultured at 37uC, 36 Journal of Histotechnology 2011 VOL. 34 NO. 1 95% air and 5% CO2 before use (Fig. 4). After conﬂuence, chondrocytes were detached with 2 ml trypsin EDTA solution, and were counted by using Casy Cell-Counter. Cells numbering 36106 was performed with 600 ml of 4.8% alginate gel (240 mg alginate powder with 5 ml NaCl). For production of the alginate beads, an 18-gauge needle and a 2 ml syringe were used. Each bead had the same volume of 0.1 ml with approximately 56105 cells and was dropped from a height of 10 cm into 10 ml of non- stirred calcium chloride solution with a molarity of 0.102. Then alginate beads were hardened for 10 min- utes and were suspended in six-well plates with 3 ml chondrogenic medium (DMEM/Ham’s F12 with 10% fetal bovine serum, 1% penicillin/streptomycine, 2% glutamine sulphate, ascorbic acid, TGF-beta, and dexamethasone) per well and cultured at 37uC, 95% air and 5% CO2 for 3 days before use. Treatment of alginate beads Alginate beads with chondrocytes were cultured in six-well plates with 3 ml of chondrogenic medium. Two times (10 and 30 minutes), three antiseptic treatments (polyhexanide, hydrogen peroxide, or PBS control), and four replicates for each condition were assessed. After removal of the medium, 3 ml of 100% of concentrated 0.04% polyhexanide (Cha- rite´, Berlin, Germany) or 3% hydrogen peroxide (Charite´), or PBS (control) were added for 10 and 30 minutes. After treatment with antiseptics, all alginate beads were washed three times with PBS and were re-suspended in chondrogenic medium for 3 weeks. Alcian blue staining After 3 weeks of cultivation in chondrogenic medium at 37uC, 95% air, and 5% CO2, alginate beads were washed twice with PBS, ﬁxed with 4% formalin, and rinsed again for 30 minutes. After cell staining with haematoxylin and eosin (Chroma, Brattleboro, VT, USA), the ﬁxed alginate beads were dehydrated with a rising series of graded ethanol (10 minutes with 70%, 25 minutes with 80%, 25 minutes with 90%, 30 minutes with 96%, 1 hour with 100%) and soaked in xylene for 30 minutes. Then alginate beads were inﬁltrated with 60uC parafﬁn for 60 minutes and were sectioned into 4 mm sections. Sections were ﬂoated in a 56uC distilled water bath and collected on slides. These slides were soaked in xylene for 10 minutes and dipped in a declining series of graded ethanol (2 minutes in 100%, 2 minutes in 96%, 2 minutes in 90%, 2 minutes in 80%, 2 min- utes in 70%, and 2 minutes in distilled water). Next,