Orthopedics

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Toxicity of Polyhexanide and Hydrogen Peroxide on Human Chondrocytes In Vitro

Eric Röhner, MD; Joern B. Seeger, MD; Paula Hoff, MD; Stephanie Dähn-Wollenberg; Carsten Perka, MD; Georg Matziolis, MD, PhD

Abstract

The treatment of acute joint infections has an important impact on long-term outcome and remains an unsolved problem. The most frequent bacteria are staphylococci, streptococci, and gram-negative bacteria. In septic surgery, polyhexanide and hydrogen peroxide are the most frequently used local antiseptics. The aim of this study was to examine the hypothesis that antiseptics induce cell death of human chondrocytes after a short incubation time.

Human chondrocytes were treated with different concentrations of polyhexanide and hydrogen peroxide. Toxicity analysis was determined by visualization of cell structure using light microscopy, lactate dehydrogenase release, and determination of living and total cell numbers after addition of polyhexanide and hydrogen peroxide. Light microscopic data revealed a defect cell structure after addition of both antiseptics. Lactate dehydrogenase activity showed a significant increase of enzyme expression after a short incubation with polyhexanide. The determination of vital chondrocytes showed a significant decrease of vital and total cell numbers after addition with polyhexanide and hydrogen peroxide.

Both antiseptic solutions induce significant cell death of human chondrocytes after a short incubation time. Polyhexanide possibly has more toxic potential than hydrogen peroxide against human chondrocytes after an application >15 minutes. Therefore, both substances should only be applied for a short time (<15 minutes) and the joint irrigated to wash out the antiseptic substance prior to wound closure.

Drs Röhner, Seeger, Perka, and Matziolis and Ms Dähn-Wollenberg are from the Department of Traumatology and Orthopedics, and Dr Hoff is from the Department of Rheumatology and Clinical Immunology, Charité – Universitätsmedizin, Berlin, Germany.

Drs Röhner, Seeger, Hoff, Perka, and Matziolis and Ms Dähn-Wollenberg have no relevant financial relationships to disclose.

The treatment of acute joint infections has an important impact on long-term outcome and remains an unsolved problem. 1 The most frequent bacteria are staphylococci, streptococci, and gram-negative bacteria. 2–5

In contrast to the hip joint, which is rarely infiltrated for diagnostic or therapeutic reasons, shoulder and knee joint infections often result from infiltrations with local anesthetics, glucocorticoids, or hyaluronic acid. 3,6,7 While there is consensus on a staged operative treatment, the intra-articular application of antiseptic substances remains controversial. 8–10 It is evident that mechanical elimination of bacteria through lavage and surgical debridement can be supported by antiseptic substances; however, all of these have considerable tissue toxicity. This results from the fact that in contrast to antibiotic substances, there is no difference between eukaryontic and procariontic cells. Therefore, the tissue toxicity of antiseptic substances must be determined for every tissue coming into contact. There is ongoing discussion regarding elimination of bacteria versus toxicity against chondrocytes. Studies demonstrate polyhexanide being less toxic than hydrogen peroxide, Dibromol, Kodan, and chlorhexidingluconate. 11,12

In surgical practice, 2 of the most frequently used antiseptics are polyhexanide and hydrogen peroxide. Both antiseptics are used for local wound debridement and joint lavage in septic surgery. Literature reviews revealed insufficient data about the effects of these antiseptics on human cartilage. We therefore hypothesized that polyhexanide and hydrogen peroxide both have a toxic effect on human chondrocytes in vitro, even after short-term incubation, and that hydrogen peroxide is less toxic than polyhexanide.

Tissue culture plasticware was obtained from TPP (Trasadingen, Switzerland). Culture medium, phosphate buffer saline, trypsin, and fetal calf serum were purchased from Biochrom (Berlin, Germany). All other reagents were obtained from Sigma-Aldrich (Deisenhofen, Germany).

Chondrocyte isolation was performed as previously described. 13 Cartilage was obtained from 6 human donors with knee osteoarthritis not presenting with any infectious signals. Experimental protocols were approved by the local ethics committee. Cartilage was minced and digested in medium containing 1 mg/mL pronase (Sigma-Aldrich) for 30 minutes at 37°C. Next, digestion medium was discarded…

Abstract

The treatment of acute joint infections has an important impact on long-term outcome and remains an unsolved problem. The most frequent bacteria are staphylococci, streptococci, and gram-negative bacteria. In septic surgery, polyhexanide and hydrogen peroxide are the most frequently used local antiseptics. The aim of this study was to examine the hypothesis that antiseptics induce cell death of human chondrocytes after a short incubation time.

Human chondrocytes were treated with different concentrations of polyhexanide and hydrogen peroxide. Toxicity analysis was determined by visualization of cell structure using light microscopy, lactate dehydrogenase release, and determination of living and total cell numbers after addition of polyhexanide and hydrogen peroxide. Light microscopic data revealed a defect cell structure after addition of both antiseptics. Lactate dehydrogenase activity showed a significant increase of enzyme expression after a short incubation with polyhexanide. The determination of vital chondrocytes showed a significant decrease of vital and total cell numbers after addition with polyhexanide and hydrogen peroxide.

Both antiseptic solutions induce significant cell death of human chondrocytes after a short incubation time. Polyhexanide possibly has more toxic potential than hydrogen peroxide against human chondrocytes after an application >15 minutes. Therefore, both substances should only be applied for a short time (<15 minutes) and the joint irrigated to wash out the antiseptic substance prior to wound closure.

Drs Röhner, Seeger, Perka, and Matziolis and Ms Dähn-Wollenberg are from the Department of Traumatology and Orthopedics, and Dr Hoff is from the Department of Rheumatology and Clinical Immunology, Charité – Universitätsmedizin, Berlin, Germany.

Drs Röhner, Seeger, Hoff, Perka, and Matziolis and Ms Dähn-Wollenberg have no relevant financial relationships to disclose.

Correspondence should be addressed to: Eric Röhner, MD, Department of Traumatology and Orthopedics, Charité – Universitätsmedizin, Charitéplatz 1, 10117, Berlin, Germany (eric.roehner@charite.de).
Posted Online: July 07, 2011

The treatment of acute joint infections has an important impact on long-term outcome and remains an unsolved problem. 1 The most frequent bacteria are staphylococci, streptococci, and gram-negative bacteria. 2–5

In contrast to the hip joint, which is rarely infiltrated for diagnostic or therapeutic reasons, shoulder and knee joint infections often result from infiltrations with local anesthetics, glucocorticoids, or hyaluronic acid. 3,6,7 While there is consensus on a staged operative treatment, the intra-articular application of antiseptic substances remains controversial. 8–10 It is evident that mechanical elimination of bacteria through lavage and surgical debridement can be supported by antiseptic substances; however, all of these have considerable tissue toxicity. This results from the fact that in contrast to antibiotic substances, there is no difference between eukaryontic and procariontic cells. Therefore, the tissue toxicity of antiseptic substances must be determined for every tissue coming into contact. There is ongoing discussion regarding elimination of bacteria versus toxicity against chondrocytes. Studies demonstrate polyhexanide being less toxic than hydrogen peroxide, Dibromol, Kodan, and chlorhexidingluconate. 11,12

In surgical practice, 2 of the most frequently used antiseptics are polyhexanide and hydrogen peroxide. Both antiseptics are used for local wound debridement and joint lavage in septic surgery. Literature reviews revealed insufficient data about the effects of these antiseptics on human cartilage. We therefore hypothesized that polyhexanide and hydrogen peroxide both have a toxic effect on human chondrocytes in vitro, even after short-term incubation, and that hydrogen peroxide is less toxic than polyhexanide.

Materials and Methods

Tissue culture plasticware was obtained from TPP (Trasadingen, Switzerland). Culture medium, phosphate buffer saline, trypsin, and fetal calf serum were purchased from Biochrom (Berlin, Germany). All other reagents were obtained from Sigma-Aldrich (Deisenhofen, Germany).

Chondrocyte Isolation and Culture

Chondrocyte isolation was performed as previously described. 13 Cartilage was obtained from 6 human donors with knee osteoarthritis not presenting with any infectious signals. Experimental protocols were approved by the local ethics committee. Cartilage was minced and digested in medium containing 1 mg/mL pronase (Sigma-Aldrich) for 30 minutes at 37°C. Next, digestion medium was discarded and the tissue was digested with medium containing 1 mg/mL clostridial collagenase (Sigma-Aldrich) at 37°C overnight. Digested solution was filtered (70 µm Nylon; BD Falcon, Bedford, Germany) and centrifuged at 1200 rpm for 8 minutes. The supernatant was discarded and the cell pellet was washed 3 times with phosphate buffer saline. Chondrocytes were suspended in Dulbecco’s Modification of Eagle’s Medium/Hams-F12 with 10% fetal bovine serum and 1% penicillin/streptomycine and cultured at 37°C, 95% air and 5% CO 2. Experiments were performed immediately.

Chondrocytes Treatment and Detection of Cell Structure

Human chondrocytes were cultured and grown on 24-well plates at a density of subconfluence and were added to 100 µL of 10% and 100% solutions of concentrated 0.04% polyhexanide (Charité, Berlin, Germany) and 3% hydrogen peroxide (Charité) for 5, 15, and 30 minutes. Phosphate buffer saline-treated chondrocytes were used as control. Immediately after incubation time, the results were interpreted with light microscopy analysis (Axiovert 40 C Light Microscope, lens 10×0.25, ocular 10×18; Zeiss, Göttingen, Germany). The view fields were then digitized by a digital camera (Canon EOS 500D, 15.1 megapixels; Lake Success, New York).

Activity of Lactate Dehydrogenase

Chondrocyte monolayers were challenged with different concentrations (10%, 100%) of polyhexanide and hydrogen peroxide, phosphate buffer saline (negative control), and 2% Triton X-100 (Sigma-Aldrich) (positive control) for 5, 15, and 30 minutes. Lactate dehydrogenase activity in the supernatant was determined by the colorimetric measurement of the reduction of sodium pyruvate in the presence of nicotinamide adenine dinucleotide plus hydrogen and expressed as the percentage of total enzyme activity liberated from chondrocytes in the presence of the antiseptics.

Determination of Total Cell Number and Vital Cells

Isolated human chondrocytes were counted by using the Casy Cell-Counter and Analyser System (Schärfe-System, Reutlingen, Germany), and grown on 24-well plates at a density of 2×104 cells per well, and were incubated with different concentrations (10%, 100%) of polyhexanide and hydrogen peroxide, phosphate buffer saline (negative control) and 2% Triton X-100 (positive control) for 5, 15, and 30 minutes. After removing of growth medium, all cells were detached with 100 µL trypsin. The detection of living and total cell numbers was determined using Casy Cell-Counter and Analyser System.

Statistical Analysis

A nonparametric Wilcoxon matched-pairs test was used. A P value <.05 was considered significant, and a P value <.001 was considered highly significant.

Results

Chondrocytes incubated with polyhexanide and hydrogen peroxide showed an increased number of cells with defect cell structure after an incubation time of 30 minutes as revealed by light microscopy. There were no defective chondrocytes when cultured without antiseptic solutions (Figure ). The incubation with Triton X-100 as a known mediator of necrosis revealed clear induction of necrosis in human chondrocytes (Figure ). Hydrogen peroxide-incubated chondrocytes were shrunken and globular and showed losses of cell contacts (Figure ). In contrast, polyhexanide-incubated chondrocytes were swollen and showed a defective cell structure (Figure ).

Light microscopy detection showed variation of cell structure after incubation with antiseptics. Normal cell structure when cultured without antiseptics (A). Totally destroyed cell structure found after incubation with Triton X-100 as positive control (B). Hydrogen peroxide-incubated chondrocytes showed losses of cell contact and were globular and partly shrunken (C). Polyhexanide-incubated chondrocytes were partly swollen and showed a defective cell structure (D). One representative result of at least 3 independently performed experiments is shown.

Figure 1:. Light microscopy detection showed variation of cell structure after incubation with antiseptics. Normal cell structure when cultured without antiseptics (A). Totally destroyed cell structure found after incubation with Triton X-100 as positive control (B). Hydrogen peroxide-incubated chondrocytes showed losses of cell contact and were globular and partly shrunken (C). Polyhexanide-incubated chondrocytes were partly swollen and showed a defective cell structure (D). One representative result of at least 3 independently performed experiments is shown.

Lactate dehydrogenase activity was analyzed in the supernatant of each cell culture. Compared to the control, we noted significant lactate dehydrogenase release after 5 minutes of chondrocyte incubation with polyhexanide, indicating beginning necrotic cell death at that stage (Figures ). Triton X-100 was used as a known mediator of cell necrosis. We detected no lactate dehydrogenase release at each time after incubation with hydrogen peroxide. The determination of vital and total cell numbers of antiseptics-incubated chondrocytes showed a significant decrease of vital and total cell numbers after a short incubation time of 5 minutes ( P<.05) (Figures , ). In comparing both antiseptics (100%), we detected significant differences of living cells after an incubation time of 30 minutes (Figure ). There were no significant differences in the comparison of antiseptics (10%) incubation (Figure ). The detection of total cell numbers showed a significant decrease of total cell numbers after 5, 15, and 30 minutes after incubation with hydrogen peroxide and polyhexanide (10% and 100%), P<.05. In comparing both antiseptics (10% and 100%), we detected a significant decrease of total cell numbers of hydrogen peroxide-incubated chondrocytes ( P<.05) (Figures ).

Lactate dehydrogenase activity was analyzed in the supernatant of each cell culture. Significant lactate dehydrogenase release detected after 5 minutes’ incubation with 100% polyhexanide (A) and after 10 minutes’ incubation with 10% polyhexanide versus control (B). There was no lactate dehydrogenase activity after incubation with 10% and 100% hydrogen peroxide. In the comparison of both antiseptics (10% and 100%), significant differences of lactate dehydrogenase activity were detected after an incubation time of 5, 15, and 30 minutes. Nonparametric Wilcoxon matched-pairs test; n=6; mean±standard error of mean. P<.05 was considered significant. Abbreviations: ctr, control; LDH, lactate dehydrogenase; OD, optical density.

Figure 2:. Lactate dehydrogenase activity was analyzed in the supernatant of each cell culture. Significant lactate dehydrogenase release detected after 5 minutes’ incubation with 100% polyhexanide (A) and after 10 minutes’ incubation with 10% polyhexanide versus control (B). There was no lactate dehydrogenase activity after incubation with 10% and 100% hydrogen peroxide. In the comparison of both antiseptics (10% and 100%), significant differences of lactate dehydrogenase activity were detected after an incubation time of 5, 15, and 30 minutes. Nonparametric Wilcoxon matched-pairs test; n=6; mean±standard error of mean. P<.05 was considered significant. Abbreviations: ctr, control; LDH, lactate dehydrogenase; OD, optical density.

Antiseptic-incubated chondrocytes showed a significant decrease of vital cells after 5, 15, and 30 minutes versus negative control (A). In comparing both antiseptics (100%), significant differences of vital cell numbers were detected after an incubation time of 30 minutes. There were no significant differences after incubation with 10% antiseptics (B). Nonparametric Wilcoxon matched-pairs test; n=6; mean±standard error of mean. P<.05 was considered significant.

Figure 3:. Antiseptic-incubated chondrocytes showed a significant decrease of vital cells after 5, 15, and 30 minutes versus negative control (A). In comparing both antiseptics (100%), significant differences of vital cell numbers were detected after an incubation time of 30 minutes. There were no significant differences after incubation with 10% antiseptics (B). Nonparametric Wilcoxon matched-pairs test; n=6; mean±standard error of mean. P<.05 was considered significant.

Antiseptic-incubated chondrocytes showed a significant decrease of total cell numbers after 5, 15, and 30 minutes versus negative control (A). In the comparison of both antiseptics (10% and 100%), a significant decrease of total cell numbers was detected after incubation with hydrogen peroxide (B). Non-parametric Wilcoxon matched-pairs test; n=6; mean±standard error of mean. P<.05 was considered significant.

Figure 4:. Antiseptic-incubated chondrocytes showed a significant decrease of total cell numbers after 5, 15, and 30 minutes versus negative control (A). In the comparison of both antiseptics (10% and 100%), a significant decrease of total cell numbers was detected after incubation with hydrogen peroxide (B). Non-parametric Wilcoxon matched-pairs test; n=6; mean±standard error of mean. P<.05 was considered significant.

Discussion

In contrast to antibiotics, which have a broad therapeutic window based on their differential mechanisms of action, antiseptics function through their more or less undifferentiated cell toxicity. Despite their importance in eradication of bacteria, the application of antiseptics to cartilage tissue is insufficiently investigated. We investigated 2 of the most frequently used antiseptics in orthopedic surgery. We analyzed and compared 3 different points of time of antiseptic incubation (5, 15, and 30 minutes). Despite that, we observed that the results in vitro are based on direct chondrocyte incubation. Therefore, in vitro results show higher cell toxicity than in vivo examinations. In vitro, antiseptic solutions do not have to pass different barriers like chondrocytes matrix.

In the present study, the first point of investigation was the detection of cell structure using light microscopy. After incubation with polyhexanide, the qualitative analysis showed some chondrocytes with a defective cell structure that were swollen. This could result from cell damage seen at the beginning of cell necrosis. 14 In contrast, hydrogen peroxide-incubated chondrocytes were shrunken and globular and showed losses of cell contact. This could result from cell damage seen by apoptotic cell death. 14,15 In addition, the second point of investigation was the detection of lactate dehydrogenase activity as a marker of advanced cell death. 15,16 Our data showed significant lactate dehydrogenase activity after a short incubation time of 5 minutes with both concentrations of polyhexanide. There was no lactate dehydrogenase activity after incubation with hydrogen peroxide. Lactate dehydrogenase activity after polyhexanide incubation indicated the loss of membrane plasma integrity as possible marker of cell necrosis. 15,16 Absence of lactate dehydrogenase activity after incubation with hydrogen peroxide could support the hypothesis that hydrogen peroxide induced less necrotic cell death and has less toxic potential than polyhexanide.

In the present study, the detection of total cell number and cell viability showed that human chondrocytes were affected negatively after a short incubation time of 5 minutes with both antiseptics in comparison to the control. Similar results were shown by Ince et al. 17 In their study, human osteoblasts and endothelial cells were incubated with different concentrations of polyhexanide for 6 hours. A low concentration of 0.0006% polyhexanide demonstrated a significant decrease of cell number and viability. Compared to both antiseptics, we showed that cell viability of hydrogen peroxide-incubated chondrocytes was significantly higher than viability of polyhexanide-incubated chondrocytes after 30 minutes. In contrast, total cell numbers of hydrogen peroxide-incubated chondrocytes were significantly lower in each concentration and point of incubation time in comparison to polyhexanide-incubated chondrocytes.

In in vitro studies with human cells, uncontrollable influences may play an important role, such as contamination or deviations in temperature or barometric pressure. Therefore, our cell model may have limitations. The results of our study do not represent an in vivo situation, as in vitro results show higher cell toxicity than in vivo examinations based on the direct chondrocyte incubation. Antiseptic solutions in vitro do not have to pass different barriers like chondrocytes matrix.

Conclusion

The present study shows that both antiseptics induce cell death of human chondrocytes after a short incubation time. In consideration of our results and the results of studies found in the literature, polyhexanide has more toxic potential than hydrogen peroxide against human chondrocytes after an application >15 minutes. Therefore, both substances should only be applied for a short time (<15 minutes) and the joint irrigated to wash out the antiseptic substance prior to wound closure.

References

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10.3928/01477447-20110526-02

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