Ro¨hner et al. Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis Figure 3 Cell number of chondrocytes per view field after treatment with PBS, polyhexanide, or hydrogen peroxide. Chondrocytes treated with antiseptics were compared with PBS-treated chondrocytes (control) after (A) 10 and (B) 30 minutes. n54, mean¡SEM. versus the control group (Figs. 1 and 2). The positive Alcian blue staining of the control group indicates a high concentration of proteoglycans (58.92¡1.96). In contrast, a decrease of Alcian blue staining suggests a lower concentration of proteoglycans seen in chondrocytes incubated in the indicated antiseptics (Figs. 1 and 2). The comparison between polyhexanide- and hy- drogen peroxide-incubated chondrocytes shows that human chondrocytes have a higher intensity of Alcian blue staining after treatment with hydrogen peroxide than after treatment with polyhexanide. This was detected for each treatment time point (10 minutes: 15.49¡1.29 versus 12.11¡4.13; 30 minutes: 12.31¡0.56 versus 10.27¡1.03; P,0.05) (Fig. 2). Analysis of the cell number per view ﬁeld and morphological characteristics showed no sig- niﬁcant differences between control (PBS), and between hydrogen peroxide or polyhexanide (Figs. 2 and 3). Discussion The optimal therapy of joint infections is currently still an unsolved problem.18 In contrast to treatment using antibiotics which has brought a therapeutic window based on different mechanisms of action, antiseptics act through their more or less undiffer- entiated cell toxicity. Despite their importance in eradication of bacteria, the damage of antiseptic application to cartilage tissue is still investigated insufﬁciently.19 Our results show that both polyhex- anide and hydrogen peroxide inhibit proteoglycan synthesis of human chondrocytes, which could po- ssibly result in cartilage degradation. Additionally, we could show that polyhexanide inhibits proteogly- can synthesis more than hydrogen peroxide. Several studies indicated that a dysbalance in chondrocyte homeostasis, induced by antiseptics or antibiotics, results in a degradation of cartilage tissue.14,15,19 38 Journal of Histotechnology 2011 VOL. 34 NO. 1 Taking other studies into account, the aim of the present study was to compare the effects of polyhex- anide and hydrogen peroxide, representing two of the most frequently used antiseptics, on the extracellular cartilaginous matrix synthesis of human chondrocytes. Resulting from the fact that in monolayer culture the diffusion barrier is minimal, in vitro results show higher cell toxicity than in vivo examinations. A chondrocyte culture with a stable phenotype and a cartilage-like barrier for antiseptics inﬂuence was achieved by using an alginate matrix.12 The incubation times (10 and 30 minutes) represent a clinically relevant dimension in septic joint surgery and should therefore allow conclusions from the in vitro data. In addition to our analysis of cartilaginous matrix production, we detected the cell number per view ﬁeld and cell morphology of the treated articular chon- drocytes. Unexpectedly, the cell number and morph- ology did not signiﬁcantly differ between control group and antiseptic treated chondrocytes embedded Figure 4 Experimental protocol for chondrocyte isolation from cartilage tissue.