The influence of vancomycin on extracellular matrix and pro-inflammatory cytokine expression in human articular chondrocytes

Accepted Manuscript Title: The influence of vancomycin on extracellular matrix and pro-inflammatory cytokine expressions to human articular chondrocytes Authors: Yi-Ru Chen, Jui-Hung Chang, Kai-Chiang Yang, Fedor Svyatoslavovich Senatov, Chang-Chin Wu, Mong-Hsun Tsai PII: DOI: Reference:

S1359-5113(17)31398-3 https://doi.org/10.1016/j.procbio.2017.11.007 PRBI 11200

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

28-8-2017 24-10-2017 13-11-2017

Please cite this article as: Chen Yi-Ru, Chang Jui-Hung, Yang Kai-Chiang, Senatov Fedor Svyatoslavovich, Wu Chang-Chin, Tsai Mong-Hsun.The influence of vancomycin on extracellular matrix and pro-inflammatory cytokine expressions to human articular chondrocytes.Process Biochemistry https://doi.org/10.1016/j.procbio.2017.11.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The influence of vancomycin on extracellular matrix and pro-inflammatory cytokine expressions to human articular chondrocytes

Short title: Influence of vancomycin on human articular chondrocytes

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Yi-Ru Chen a, Jui-Hung Chang b, Kai-Chiang Yang b, c, Fedor Svyatoslavovich

a.

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Senatov d, Chang-Chin Wu c, e, f, *, Mong-Hsun Tsai a, *

Institute of Biotechnology, College of Bio-Resources and Agriculture, National

School of Dental Technology, College of Oral Medicine, Taipei Medical

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b.

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Taiwan University, Taipei 106, Taiwan

Department of Orthopedics, National Taiwan University Hospital, College of

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c.

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University, Taipei 11031, Taiwan

Medicine, National Taiwan University, Taipei 10002, Taiwan Centre for Composite Materials, National University of Science and Technology

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d.

MISIS, Moscow 119991, Russia

Department of Biomedical Engineering, Yuanpei University of Medical

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e.

Technology, Hsinchu 30015, Taiwan Department of Orthopedics, En Chu Kong Hospital, New Taipei City 23702,

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f.

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Taiwan

*Correspondence to: Chang-Chin Wu MD, PhD., and Mon-Hsun Tsai PhD.

Yi-Ru Chen: [email protected] Jui-Hung Chang: [email protected] 1

Kai-Chiang Yang: [email protected] Fedor Svyatoslavovich Senatov: [email protected]

Chang-Chin Wu MD, PhD.

Hospital, College of Medicine, National Taiwan University No. 7, Chung Shan S. Rd., Taipei 10002, Taiwan

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Tel: +886-2-2312-3456 ex: 63982

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Associate Professor, Department of Orthopedics, National Taiwan University

Fax: +886-2-23224112

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E-mail: [email protected]

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Mon-Hsun Tsai PhD.

National Taiwan University

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Professor, Institute of biotechnology, College of Bio-Resources and Agriculture,

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No. 81, Chung-Xing Street, Taipei 106, Taiwan TEL: +886-2-3366-6009

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FAX: +886-2-33666001

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E-mail: [email protected]

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Graphical abstract

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Highlights  High dose of vancomycin (≥1000 μg/mL) has chondrotoxicity  Low dose of vancomycin (500 μg/mL) did not change the mRNA levels of ECMs and pro-inflammatory cytokines to chondrocytes  Vancomycin did not affect IκB to initiate the NF-κB pathway or COX-2 synthesis  Vancomycin at a low dosage did not have a deleterious effect on chondrocytes

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Abstract

Intravenous infusion or intra-articular administration of vancomycin is widely used to treat septic arthritis. Although vancomycin has been shown to cause cell death, its

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effects on chondrocytes have yet to be fully elucidated. Primary human chondrocytes

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were isolated and cultured in media supplemented with vancomycin at a series of

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concentrations (0, 100, 500, 1000, and 5000 μg/mL) to study the dose-toxicity. Since

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inflammatory chondrocytes are more catabolic than normal cells, we also evaluated the influence of vancomycin (500 μg/mL) on interleukin-1beta (IL-1β)-stimulated

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chondrocytes. Exposure of chondrocytes to a high dose of vancomycin (≥ 1000

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μg/mL) resulted in chondrotoxicity. Vancomycin treatment did not change the viability, morphology or glycosaminoglycan content of the normal or IL-1β-

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stimulated chondrocytes, or affect the mRNA levels of extracellular matrixes (aggrecan, collagen type I, II, and X) and pro-inflammatory cytokines (IL-1β, TNFα, IL-6, IL-10, MMP-3, MMP-9, and TIMP-1). ELISA revealed similar results in

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that vancomycin exposure did not affect the levels of PGE2, IL-1β, MMP-3, or MMP-9 in normal and IL-1β-stimulated cells. Western blotting further showed that vancomycin did not affect IκB and p50 to initiate the NF-κB pathway or COX-2 synthesis. Vancomycin at a low dosage (500 μg/mL) did not have a deleterious effect on chondrocytes. 3

Keywords: septic arthritis, antibiotic management, vancomycin, chondrocyte.

1. Introduction Septic arthritis is caused by direct inoculation or hematogenous spread of pathogens

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that can initiate an inflammatory process, cause rapid joint destruction, and even

result in morbidity and mortality [1]. The intravenous infusion of antibiotics such as

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vancomycin is a widely used strategy to treat septic arthritis, and even methicillin-

resistant Staphylococcus aureus has been shown to be susceptible to vancomycin [2].

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However, a high dose of vancomycin has been associated with nephrotoxicity which

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raises major concerns over its use [3]. Acute tubular necrosis has been shown to be

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caused by the intravenous infusion of vancomycin [4]. Vancomycin toxicity with

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continuous and intermittent infusion has been investigated, however the results have been inconsistent [5]. Some groups have tried delivering vancomycin through other

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routes, since intravenous infusion has also been reported to induce endothelial cell death, vein irritation and localized phlebitis [6]. Intraperitoneal vancomycin infusion

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has been proposed, however drug reactions with eosinophilia and systemic symptoms have still been reported [7].

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Local antibiotic delivery, such as antibiotic-laden cement beads and spacers,

may reduce the risk of systemic toxicity [8]. However, intravenous antibiotics and

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antibiotic-loaded cement carriers may not be able to achieve effective bactericidal levels [9]. Alternatively, the intra-articular administration of vancomycin has been reported to achieve high levels of local antibiotics. Roy et al. compared the joint and serum concentrations of vancomycin between intravenous and intra-articular administrations, and reported that intra-articular injections resulted in a relatively 4

higher peak concentration with bactericidal serum levels [10]. Unfortunately, local delivery systems for antibiotics have also been shown to cause skeletal cell toxicity, and exposure to high dosages of vancomycin has been shown to lead to changes in the spread, cell membrane, extension, and growth of osteoblasts and chondrocytes in vitro [11].

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Although concerns over antibiotic toxicity have been investigated, the cellular

and molecular mechanisms have yet to be fully elucidated. Therefore the aim of this

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study was to investigate the possible chondrotoxicity of vancomycin. Since

inflammatory chondrocytes are more catabolic than normal cells, we also evaluated

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the influence of vancomycin on interleukin-1beta (IL-1β)-stimulated chondrocytes

2. Materials and methods

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[12].

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2.1.Patient profile, chondrocyte isolation and cultivation Approval for the retrieval and use of human tissues in this study was granted by the

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Research Ethical Committee of En Chu Kong Hospital (ECKIRB1030502). Ten patients (four males and six females; average age 71.9 years; range 56 to 88 years)

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with knee osteoarthritis were enrolled, all of whom provided written informed consent. All of the patients received total knee replacement with preservation of the

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lateral compartments of the articular cartilage during surgery. The cartilaginous tissues were digested using 0.1% type II collagenase (9001-

12-1, Gibco, Life Technologies, NY) for cell isolation. The chondrocytes were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DMEM/F12) supplemented with 50 μg/mL L-ascorbic acid, 10% fetal bovine serum 5

(FBS, 10437-028, Gibco), and 1% antibiotics (P4083, Sigma-Aldrich, MO). The cells obtained from each donor were used independently. Proliferative chondrocytes (P3-P6) were used in this study.

2.2.Dose-toxicity study of vancomycin to chondrocytes

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Chondrocytes were seeded into culture plates and cultured in normal DMEM/F12

medium for 1 day to allow for cell adhesion. The culture medium was changed to a

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medium containing 1% FBS for serum starvation for 24 h. The low-serum medium was then removed and the cells were washed twice with PBS, and normal medium containing vancomycin (vancomycin hydrochloride, NDC 0409-4332-01, Hospira,

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IL) at different concentrations (0, 100, 500, 1000, and 5000 μg/mL) was added. The

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chondrocytes were cultured in the medium containing vancomycin for 1, 3, and 5

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days, and the morphology of the chondrocytes was recorded using an optical microscope. The cells were then detached and counted to identify cell proliferation.

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At pre-determined intervals, the culture medium was removed and preserved, and the viability of the chondrocytes was evaluated using a water-soluble

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tetrazolium salt-1 assay (Cell Proliferation Reagent WST-1, Roche, Mannheim, Germany). Serum free DMEM/F12 medium containing 10% WST-1 agent was

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added and incubated for 2 h. Cell activity was determined using a spectrophotometer (Multiskan™ GO Microplate Photometer, Thermo Fisher Scientific, Waltham, MA)

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at a wavelength of 460 nm. The supernatant of preserved medium was reacted with a lactate dehydrogenase assay (LDH, CytoTox 96® NonRadioactive Cytotoxicity Assay, G1780, Promega, WI). The results of the cytotoxicity assay were determined using the spectrophotometer at a wavelength of 490 nm.

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2.3.IL-1β stimulation and vancomycin supplementation To identify the effect of vancomycin on inflammatory chondrocytes, the cells were stimulated with IL-1β and then exposed to vancomycin. After culturing in 1% FBS medium for serum starvation for 24 h, the cells were stimulated with 10 ng/mL IL-1β (CYT-208, PorSpec, Israel) for an additional 24 h. The cells were then washed twice

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with PBS and then cultured in medium containing 500 μg/mL vancomycin (based on the results of dose-toxicity tests) for 1, 3, and 5 days. Un-stimulated chondrocytes

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were also cultured in regular medium or vancomycin-supplemented medium for the same periods. The cell morphology, proliferation, viability and cytotoxicity were

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determined as in the previous section.

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2.4.Toluidine blue staining and sulfated-glycosaminoglycan content

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After being cultured in the different culture media for 5 days, the chondrocytes were washed twice with PBS, fixed in neutral buffered 10% formalin solution, and stained

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with toluidine blue for 10 minutes to detect sulfated-glycosaminoglycan (sGAG). The amount of sGAG was quantified using a 1,9-dimethyl-methylene blue (DMMB,

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341088, Sigma-Aldrich) assay. In brief, the cells were digested in 0.1% papain

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solution (P4762, Sigma-Aldrich) at 60C for 16 h, and the digested sample was reacted with DMMB reagent with a standard curve of chondroitin sulfate sodium salt dilution (C4384, Sigma-Aldrich). Absorbance was detected at a wavelength of 530

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nm using a microplate reader. The sGAG content was normalized to the DNA content of each group.

2.5.RNA extraction and gene expression of the chondrocytes Quantitative real-time reverse transcription-polymerase chain reactions (qRT-PCRs) 7

were used to determine the relative gene expressions of the treated chondrocytes. Total RNA of the chondrocytes was extracted (NovelGene Biotech, Taiwan), and cDNA was synthesized (High-Capacity cDNA Reverse Transcription Kit, 4368814, ABI, MA) with a PCR system (T100TM Thermal Cycler, Bio-Rad, CA). FAM-labeled TaqMan gene expression kits were Hs00153936-m1 for aggrecan,

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Hs01028969-m1 for type I collagen, Hs00264051-m1 for type II collagen,

Hs00166657-m1 for type X collagen, Hs01555410-m1 for IL-1β, Hs01113624-m1

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for TNF-α, Hs00968305-m1 for metalloproteinase-3 (MMP-3), Hs00234579-m1 for MMP-9, and Hs00171558-m1 for tissue inhibitor of metalloproteinase-1 (TIMP-1).

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs02798991-g1) was used as the endogenous housekeeping gene with a qRT-PCR system (LightCycle 96,

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Roche). ΔCT was calculated by subtracting CT for GAPDH from CT for each target

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gene, where CT was the cycle threshold. The ΔCT for each group was further

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subtracted from the unstimulated chondrocytes group to obtain ΔΔCT. Relative

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expressions were calculated using the 2-ΔΔCT method.

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2.6.Quantification of pro-inflammatory cytokines After being treated with different media for 5 days, the culture supernatants were

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collected for the quantification of pro-inflammatory cytokines. The levels of PGE2 (514010, Cayman, MI), IL-1β (88-7010, eBioscience, CA), MMP-3 (DY513, R&D

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Systems, MN), and MMP-9 (DY911, R&D Systems, MN) were determined using a relevant enzyme-linked immunosorbent assay (ELISA) with a spectrophotometer.

2.7.Western blotting

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Cells were rinsed twice with PBS and lysed in RIPA buffer containing protease inhibitor cocktail tablets (Roche, Indianapolis, IN). Protein concentration was assessed using a BCA assay (Thermo Scientific, Rockford, IL), in which 20 μg of total cell extract protein per lane were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride

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membrane (Millipore, Bedford, MA). The membrane was incubated with primary

antibodies against NF-B (C-20), IB (C-21) (Santa Cruz Biotechnology, Santa Cruz,

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CA), COX-2 (Proteintech Group, Chicago, IL), p50 (Cell Signaling Technology,

Beverly, MA), and α-tubulin (CP06, Calbiochem, Bedford, MA), and then probed

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with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling

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Technology). Immune complexes were visualized using a SuperSignal West Pico

2.8.Statistical analysis

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Chemiluminescent Substrate (Thermo Scientific).

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The data obtained from each group were expressed as mean ± standard error. Statistical analyses of cell viability, cytotoxicity, qRT-PCR and ELISA were

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conducted using ANOVA with post-hoc Dunnett’s multiple comparison tests. A

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difference was considered to be significant when the p-value was less than 0.05.

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3. Results

3.1.Dose-toxicity study of vancomycin to chondrocytes Exposure of primary human chondrocytes to 1000 μg/mL and 5000 μg/mL vancomycin significantly decreased mitochondrial activity compared to the cells cultured in normal medium (p < 0.05 at day 1 and 3, p < 0.01 at day 5 with 1000 9

μg/mL; p < 0.01 at day 1, 3, and 5 with 5000 μg/mL). No significant differences were found in viability with 0, 100 and 500 μg/mL at day 1 and 3, but the cells treated with 100 μg/mL and 500 μg/mL had a significantly lower viability at day 5 (p < 0.05, Fig. 1A). There were also no significant differences in cytotoxicity among the cells treated with 0, 100, and 500 μg/mL at day 1, 3, and 5 (Fig. 1B). However,

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the cells treated with 1000 μg/mL and 5000 μg/mL had significantly higher LDH levels than those of the other three groups at day 1, 3, and 5 (all p < 0.01).

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Although the chondrocytes cultured in media containing 100 μg/mL and 500

μg/mL vancomycin showed similar growth kinetics to the cells cultured in regular

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medium, there were significantly fewer cells with 500 μg/mL treatment compared to

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those cultured in normal medium (p < 0.05 at day 1, and p < 0.01 at day 3 and 5; Fig. 1C). In addition, exposure of normal chondrocytes to high concentrations of

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vancomycin (1000 μg/mL and 5000 μg/mL) significantly decreased the number of cells (all p < 0.001). The cells isolated from the osteoarthritic patients had a slightly

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longer cell body but not an ideal cobblestone-like shape (Fig. 1D). The morphology of the chondrocytes treated with 100 μg/mL and 500 μg/mL vancomycin was not

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obviously changed. However, some of the cells treated with 1000 μg/mL vancomycin had fibrotic transformation, and the chondrocytes exposed to 5000

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μg/mL vancomycin developed apoptotic bodies after day 3.

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3.2.IL-1β stimulation and vancomycin supplementation IL-1β stimulation (10 ng/mL) significantly decreased the viability of the chondrocytes (p < 0.05 at day 1, and p < 0.0001 at day 3 and 5). However, vancomycin exposure did not further decrease the cell activity of the IL-1β-treated cells (Fig. 2A). IL-1β stimulation increased the LDH levels compared to normal 10

chondrocytes (all p < 0.001), however vancomycin treatment did not alter the cytotoxicity to the treated cells (Fig. 2B). IL-1β treatment also significantly decreased the number of cells (all p < 0.001), however the addition of vancomycin did not change the growth kinetics to IL-1β-treated chondrocytes (Fig. 2C). IL-1β stimulation resulted in chondrocyte hypertrophy, whereas vancomycin exposure did

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an irregular morphology in the IL-1β-stimulated-cells (Fig. 2D).

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not change the morphology of normal chondrocytes but caused cell transformation to

3.3.Toluidine blue staining for sulfated-glycosaminoglycan production

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All chondrocytes, including the IL-1β-treated cells and cells exposed to vancomycin,

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produced normal levels of sGAG in terms of positive toluidine blue staining (Fig.

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3A). Quantitative analysis by DMMB assay further revealed no significant

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differences in sGAG contents among the groups (Fig. 3B).

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3.4.Gene expression of the chondrocytes

In comparison with the normal chondrocytes, IL-1β stimulation caused significant

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increases in the mRNA levels of type I collagen (p < 0.01), IL-1β (p < 0.001), TNFα (p < 0.01), MMP-3 (p < 0.01) and MMP-9 (p < 0.05), but significant decreases in

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aggrecan (p < 0.01) and type II collagen (p < 0.01) expressions at day 1 (Fig. 4A). At days 3 and 5, the trends of mRNA expressions of IL-1β-stimulated chondrocytes

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were similar to those at day 1. There was no significant difference in type X collagen. Exposure of chondrocytes to 500 μg/mL vancomycin significantly increased the MMP-3 level (p < 0.05) at day 1 (Fig. 4A), however the expressions were decreased to the levels of normal cells at day 3 (Fig. 4B) and day 5 (Fig. 4C). Otherwise, vancomycin exposure did not result in any substantial changes in other 11

genes to unstimulated or IL-1β-stimulated chondrocytes.

3.5.Quantification of pro-inflammatory cytokines The results of ELISA revealed that IL-1β stimulation significantly increased the protein productions of PGE2 (p < 0.01, Fig. 5A), IL-1β (p < 0.001, Fig. 5A), and

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MMP-3 (p < 0.01, Fig. 5C) in the chondrocytes. There was no significant difference in MMP-9 production between the normal and IL-1β-stimulated chondrocytes (Fig.

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MMP-3 production in IL-1β-stimulated chondrocytes.

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5D). Likewise, exposure to 500 μg/mL vancomycin did not change PGE2, IL-1β, or

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3.6.Western blotting

Western blot analysis showed weak or no COX-2 protein expression in the

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chondrocytes under normal conditions. IL-1β stimulation decreased IκB but increased COX-2 and p50. However, no changes were observed in these protein

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expressions in the chondrocytes exposed to vancomycin. There was also no change in protein production when vancomycin was added to the IL-1β-treated

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chondrocytes (Fig. 6).

4. Discussion

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Although intra-articular administration can efficiently achieve high concentrations in local joints, a high dose of antibiotics has been reported to result in toxicity to skeletal cells. In addition to antibiotics, the topical application of antiseptics and anesthetics can also cause mitochondrial dysfunction and cell death in human chondrocytes [13, 14]. However, the toxic effects of antibiotics on chondrocytes are 12

unclear, which is why we investigated the possible cytotoxicity of vancomycin to normal and inflammatory human chondrocytes in this study. Dogan et al. reported that vancomycin did not exhibit chondrotoxicity in in vitro cultures, although they used a low dosage (16 μg/mL) [15]. In contrast, Antoci et al. identified a threshold of toxicity to mouse chondrocytic cell line N1511, and

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found that doses of vancomycin greater than 2000 μg/mL severely decreased cellular proliferation [11]. We further found that primary human chondrocytes were more

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susceptible to vancomycin toxicity than a chondrocytic cell line, and that exposure of primary chondrocytes to 1000 μg/mL vancomycin impaired mitochondrial

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activity, increased LDH release, and stopped cell proliferation. Similarly, Röhner et

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al. reported that antiseptic treatment increased LDH enzyme activity and decreased the number of cells in human chondrocytes [13]. Although exposure of chondrocytes

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to 500 μg/mL vancomycin did not alter the cytotoxicity, cell activity and proliferation were decreased. The long-term exposure of chondrocytes to antibiotics

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may therefore have impacts on cell growth. Likewise, quinolone antibiotics have been shown to have an inhibitory effect on osteoblast proliferation in vitro [16].

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Osteoarthritic chondrocytes are known to be more catabolic but less responsive to pro-inflammatory cytokine stimulation than normal cells [17]. Inflammatory

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chondrocytes have also been reported to show different responses to mechanical stimulation [18]. Thus, vancomycin may have different impacts on inflammatory

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chondrocytes. In this study, primary human chondrocytes were stimulated with IL1β and subsequently exposed to 500 μg/mL vancomycin. IL-1β treatment decreased mitochondrial activity and resulted in significant cell death, however vancomycin did not change cell viability or LDH level in IL-1β-stimulated chondrocytes. A previous porcine cartilage explant study found that loss of sGAG impaired 13

chondrocyte viability [19]. We found that sGAG contents were not affected despite changes in the activity and phenotype in the IL-1β-treated cells. Palmer et al. found that IL-1β stimulation induced the release of sGAG in cartilage [20]. In addition, antiseptics have been shown to decrease sGAG and other matrix synthesis of chondrocytes in vitro [21]. However, a previous study reported a quiescent state in

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sGAG synthesis during chondrocyte monolayer culture [22]. Therefore, further

studies are needed to clarify the effect of sGAG production in normal/stimulated

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chondrocytes with/without external treatment.

IL-1β treatment has been shown to induce hypertrophic transformation and

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evoke the synthesis of other inflammatory cytokines and cartilage-degrading

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proteases in chondrocytes [23]. In this study, the mRNA expressions of matrix

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proteins such as aggrecan and type II collagen were decreased while the expression of type I collagen was increased, indicating the presence of hypertrophy. The

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exogenous IL-1β further stimulated the endogenous expressions of IL-1β, TNF-α,

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and MMP-3 mRNA, and production of PGE2, IL-1β, and MMP-3 proteins. In addition, qRT-PCR and ELISA both showed that vancomycin exposure did not alter

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the synthesis of these cytokines in IL-1β-stimulated chondrocytes. Inflammatory mediators bind to their relevant membrane receptors to active the

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NEMO-IKK complex and ubiquitinated IκB to induce NF-κB activation in human chondrocytes [24]. The degradation of IκB allows for the translocation of the NF-κB

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dimer (p65-p50) to the nucleus, resulting in COX-2 overexpression, and eventually elevated PGE2 production [25]. Our Western blot analysis also showed that IL-1β stimulation led to a higher expression of NF-κB and COX-2 production. However, vancomycin did not activate the NF-κB pathway as evidenced by IκB and COX-2 production. Moreover, vancomycin did not increase the IL-1β-induced inflammatory 14

responses in human chondrocytes. In conclusion, exposure of human primary chondrocytes to a high dose of vancomycin (≥ 1000 μg/mL) resulted in chondrotoxicity and inhibited cell proliferation, whereas treatment with 500 μg/mL did not alter the cytotoxicity, morphology or sGAG contents of normal or IL-1β-stimulated chondrocytes. The

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mRNA levels of extracellular matrixes (aggrecan, collagen type I, II, and X) and pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-10, MMP-3, MMP-9, and

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TIMP-1) and protein levels (PGE2, IL-1β, MMP-3, and MMP-9) were not altered in normal or IL-1β-stimulated chondrocytes. The results of Western blotting further

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showed that vancomycin exposure did not activate IκB or p65-p50 in the NF-κB pathway or induce COX-2 synthesis. Therefore, vancomycin at a low dosage (500

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μg/mL) did not show a deleterious effect on chondrocytes. Intraarticular injection of

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proper concentration of vancomycin can be an alternative way to treat septic arthritis

Contributions

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with no direct harmful effect on chondrocytes.

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Yi-Ru Chen and Jui-Hung Chang participated in carrying out of the experiments, data analysis and writing of the manuscript. Kai-Chiang Yang and Chang-Chin Wu

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participated in research design, data analysis, writing of the manuscript and grant application. Fedor Svyatoslavovich Senatov and Mong-Hsun Tsai participated in

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data analysis and writing of the manuscript,

Ethical approval The study protocol was approved by the Institutional Review Board of En Chu Kong Hospital, and written informed consent was obtained from all patients. 15

Conflict of interest: The authors confirm that there are no conflicts of interest

Acknowledgment This study was supported by grants from the Ministry of Science and Technology

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(MOST103-2221-E-038-023) and En Chu Kong Hospital (ECK10506).

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References [1] Sharff KA, Richards EP, Townes JM. Clinical management of septic arthritis. Curr Rheumatol Rep 2013;15(6):332. [2] Frazee BW, Fee C, Lambert L. How common is MRSA in adult septic arthritis?

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Ann Emerg Med 2009;54(5):695-700.

[3] Hazlewood KA, Brouse SD, Pitcher WD, Hall RG. Vancomycin-associated

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nephrotoxicity: grave concern or death by character assassination? Am J Med 2010;123(2):182.e1-7

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[4] Shah-Khan F, Scheetz MH, Ghossein C. Biopsy-proven acute tubular necrosis due

N

to vancomycin toxicity. Int J Nephrol 2011;2011:436856.

M

Pharmacother 2013;47(2):219-27.

A

[5] DiMondi VP, Rafferty K. Review of continuous-infusion vancomycin. Ann

[6] Drouet M, Chai F, Barthélémy C, Lebuffe G, Debaene B, Décaudin B, Odou P.

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Influence of vancomycin infusion methods on endothelial cell toxicity. Antimicrob Agents Chemother 2015;59(2):930-4.

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[7] Kumar T, Teo I, McCormick BB. Systemic toxicity of intraperitoneal vancomycin. Case Rep Nephrol 2016;2016:3968690.

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[8] Hanssen AD. Local antibiotic delivery vehicles in the treatment of musculoskeletal infection. Clin Orthop Relat Res 2005;(437):91-6.

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[9] Whiteside LA, Roy ME, Nayfeh TA. Intra-articular infusion: a direct approach to treatment of infected total knee arthroplasty. Bone Joint J 2016;98-B(1 Suppl A):31-6.

17

[10] Roy ME, Peppers MP, Whiteside LA, Lazear RM. Vancomycin concentration in synovial fluid: direct injection into the knee vs. intravenous infusion. J Arthroplasty 2014;29(3):564-8. [11] Antoci V Jr, Adams CS, Hickok NJ, Shapiro IM, Parvizi J. Antibiotics for local delivery systems cause skeletal cell toxicity in vitro. Clin Orthop Relat Res.

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2007;462:200-6.

[12] Fan Z, Bau B, Yang H, Soeder S, Aigner T. Freshly isolated osteoarthritic

SC R

chondrocytes are catabolically more active than normal chondrocytes, but less responsive to catabolic stimulation with interleukin-1beta. Arthritis Rheum

U

2005;52(1):136-43.

N

[13] Röhner E, Kolar P, Seeger JB, Arnholdt J, Thiele K, Perka C, Matziolis G.

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Toxicity of antiseptics against chondrocytes: what is best for the cartilage in septic joint surgery? Int Orthop 2011;35(11):1719-23.

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[14] Grishko V, Xu M, Wilson G, Pearsall AW 4th. Apoptosis and mitochondrial

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dysfunction in human chondrocytes following exposure to lidocaine, bupivacaine, and ropivacaine. J Bone Joint Surg Am 2010;92(3):609-18.

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[15] Dogan M, Isyar M, Yilmaz I, Bilir B, Sirin DY, Cakmak S, Mahirogullari M. Are the leading drugs against Staphylococcus aureus really toxic to cartilage? J Infect

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Public Health 2016;9(3):251-8.

[16] Holtom PD, Pavkovic SA, Bravos PD, Patzakis MJ, Shepherd LE, Frenkel B.

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Inhibitory effects of the quinolone antibiotics trovafloxacin, ciprofloxacin, and levofloxacin on osteoblastic cells in vitro. J Orthop Res 2000;18(5):721-7.

[17] Fan Z, Bau B, Yang H, Soeder S, Aigner T. Freshly isolated osteoarthritic chondrocytes are catabolically more active than normal chondrocytes, but less

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responsive to catabolic stimulation with interleukin-1beta. Arthritis Rheum 2005;52(1):136-43. [18] Salter DM, Millward-Sadler SJ, Nuki G, Wright MO. Differential responses of chondrocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation. Biorheology 2002;39(1-2):97-108.

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[19] Otsuki S, Brinson DC, Creighton L, Kinoshita M, Sah RL, D'Lima D, Lotz M. The effect of glycosaminoglycan loss on chondrocyte viability: a study on

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porcine cartilage explants. Arthritis Rheum 2008;58(4):1076-85.

[20] Palmer AW, Wilson CG, Baum EJ, Levenston ME. Composition-function

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Osteoarthritis Cartilage 2009;17(8):1029-39.

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relationships during IL-1-induced cartilage degradation and recovery.

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[21] Röhner E, Hoff P, Winkler T, von Roth P, Seeger JB, Perka C, Matziolis G.

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Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis of human chondrocytes. J Histotechnol 2011;34(1):35-9.

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[22] Yang KC, Chen HT, Wu CC, Lian YJ, Chen LL, Sumi S, Huang TL. LGlutamine regulates the expression of matrix proteins, pro-inflammatory

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cytokines and catabolic enzymes in IL-1β-stimulated human chondrocytes. Process Biochemistry 2016;51(3):414-21.

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[23] Yang KC, Wu CC, Yu JS, Sumi S, Huang TL. L-Lysine regulates tumor necrosis factor-alpha and matrix metalloproteinase-3 expression in human osteoarthritic

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chondrocytes. Process Biochemistry 2016;51(7):904-11.

[24] Rahmati M, Mobasheri A, Mozafari M. Inflammatory mediators in osteoarthritis: A critical review of the state-of-the-art, current prospects, and future challenges. Bone 2016;85:81-90.

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[25] Marcu KB, Otero M, Olivotto E, Borzi RM, Goldring MB. NF-kappaB signaling:

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multiple angles to target OA. Curr Drug Targets 2010;11(5):599-613.

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Figure legends Fig. 1(A) Exposure of chondrocytes to 1000 μg/mL and 5000 μg/mL vancomycin significantly decreased cell activity. (B) There were also no significant differences in cytotoxicity among the cells treated with 0, 100, and 500 μg/mL at day 1, 3, and 5.

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However, the cells treated with 1000 μg/mL and 5000 μg/mL had significantly higher LDH levels than those of the other three groups at day 1, 3, and 5. (C)

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Chondrocytes cultured in media containing 100 μg/mL and 500 μg/mL vancomycin showed similar growth kinetics to cells cultured in regular medium, however

treatment with 500 μg/mL resulted in significantly fewer cells. Exposure of normal

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chondrocytes to high concentrations of vancomycin (1000 μg/mL and 5000 μg/mL)

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resulted in significantly fewer cells. (D) The chondrocytes isolated from osteoarthritic patients had a slightly longer cell body but not an ideal cobblestone-

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like shape. The morphology of the chondrocytes treated with 100 μg/mL and 500

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μg/mL vancomycin was not obviously changed. However, some chondrocytes treated with 1000 μg/mL had fibrotic transformation, and cells exposed to 5000

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μg/mL vancomycin developed apoptotic bodies.

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Fig. 2(A) IL-1β stimulation significantly decreased the viability of chondrocytes, but vancomycin exposure did not further decrease the cell activity of IL-1β-treated cells.

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(B) IL-1β stimulation increased the LDH levels compared to normal chondrocytes, and vancomycin did not change cytotoxicity to the treated cells. (C) IL-1β treatment also significantly decreased the number of cells, however the addition of vancomycin did not change the growth kinetics to IL-1β-treated chondrocytes. (D) IL-1β stimulation resulted in hypertrophy to chondrocytes; vancomycin exposure did 21

not change the morphology of normal chondrocytes but caused cell transformation to an irregular morphology in IL-1β-stimulated cells.

Fig. 3(A) All chondrocytes, including the IL-1β-treated cells and cells exposed to vancomycin produced normal expressions of sGAG and were positive for toluidine

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blue staining. (B) Quantitative analysis further revealed no significant differences in

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sGAG contents among the groups.

Fig. 4(A) IL-1β stimulation caused significant increases in the mRNA levels of type

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I collagen, IL-1β, TNF-α, MMP-3 and MMP-9, but significantly decreased

expressions of aggrecan and type II collagen at day 1. There was no significant

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difference in type X collagen. Exposure of chondrocytes to 500 μg/mL vancomycin

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increased MMP-3 level (p < 0.05) at day 1 but decreased the expression to that of normal cells at day 3 (B) and day 5 (C). Otherwise, vancomycin did not result in any

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substantial changes in other genes to unstimulated or IL-1β-stimulated chondrocytes.

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Fig. 5 IL-1β stimulation significantly increased the production of (A) PGE2, (B) IL1β, and (C) MMP-3 in chondrocytes. (D) There was no significant difference in

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MMP-9 production between normal and IL-1β-stimulated chondrocytes. Likewise, exposure to 500 μg/mL vancomycin did not change the production of PGE2, IL-1β,

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or MMP-3 in the IL-1β-stimulated chondrocytes.

Fig. 6 Weak or no COX-2 protein expression was found in the chondrocytes under normal conditions. IL-1β stimulation decreased IκB but significantly increased the expressions of COX-2 and p50. However, no changes were observed in these protein 22

expressions when the chondrocytes were exposed to vancomycin. There was also no change in protein production when vancomycin was added to IL-1β-treated

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chondrocytes.

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