Viability Of Human Chondrocytes Research Articles

Dihydrotanshinone I protects human chondrocytes and alleviates damage from spontaneous osteoarthritis in a guinea pig model

Osteoarthritis (OA) is the most common degenerative joint disease. Currently, no satisfactory pharmacological treatment exists for OA. The potential anti-inflammatory properties of Dihydrotanshinone I (DHT) have been reported, but its effects on OA are unclear. In this study, we assess the impact of DHT on the viability of human chondrocytes in vitro. We then use a guinea pig model to investigate the effects of DHT on knee osteoarthritis progression. Twelve-week-old Dunkin Hartley guinea pigs spontaneously developing OA were intraperitoneally injected with different doses of DHT for eight weeks. Micro-CT analysis was performed on the subchondral bone in the knee, and histological assessment of the knee joint was done using stained sections, the ratio of hyaline to calcified cartilage, and Mankin scores. DHT successfully restored IL-1β-induced decreases in cell viability in human primary chondrocytes. In the guinea pig model, intraperitoneal injections of DHT ameliorated age-induced OA, effectively reduced the expression level of two cartilage metabolism-related genes (ADAMTS4 and MMP13) and decreased the inflammatory biomarker IL-6 in the serum of guinea pigs developing spontaneous osteoarthritis. These findings demonstrate DHT’s protective effects on chondrocytes and suggest that it alleviates cartilage degradation and proteoglycan loss in OA.

Upregulating miR-181b promotes ferroptosis in osteoarthritic chondrocytes by inhibiting SLC7A11

BackgroundOsteoarthritis (OA) is a common disease with a complex pathology. This study aimed to investigate the correlation between the aberrant upregulation of miR-181b and ferroptosis in chondrocytes during the progression of OA.MethodsAn OA cell model was constructed with erastin. Ferrostatin-1 (Fer), bioinformatics, and dual-luciferase activity reports were used to investigate the effect of miR-181b on OA. Finally, a rat model of OA was induced by monosodium iodoacetate to verify that miR-181b inhibits SLC7A11 gene expression and increases ferroptosis.ResultsThe results showed that Fer could effectively reverse the erastin-induced inhibition of human chondrocyte viability, increase the level of collagenous proteins in human chondrocytes, and inhibit oxidative stress and ferroptosis. MiR-181b is abnormally elevated in OA cell models. Transfection of a miR-181b inhibitor could increase the expression levels of the ferroptosis-related proteins solute carrier family 7 members 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), thereby inhibiting the occurrence of ferroptosis in chondrocytes. In addition, hsa-miR-181b-5p and SLC7A11 have a targeted regulatory effect. Transfection of SLC7A11 siRNA effectively abrogated the increase in chondrocyte viability induced by the miR-181 inhibitor and increased ferroptosis. Finally, miR-181b was shown to exacerbate OA disease progression by inhibiting SLC7A11 gene expression and increasing ferroptosis in a rat OA model.ConclusionsElevating miR-181b may mediate chondrocyte ferroptosis by targeting SLC7A11 in OA.

Evaluation of the anti-inflammatory activity of fisetin-loaded nanoparticles in an in vitro model of osteoarthritis

Cartilage lesions, especially osteoarthritis (OA), are a common health problem, causing pain and disability in various age groups, principally in older adults and athletes. One of the main challenges to be considered in cartilage tissue repair is the regeneration of cartilage tissue in an active inflammatory environment. Fisetin has various biological effects including anti-inflammatory, antioxidant, apoptotic, and antiproliferative activities. The only disadvantages of fisetin in the pharmaceutical field are its instability and low solubility in aqueous media. This study is aimed at preparing chitosan (CS)-based nanoparticles to yield fisetin with improved bioavailability features. Then, the effect of fisetin-loaded nanoparticles (FNPs) on inflammatory responses in interleukin-1β (IL-1β) pretreated human chondrocytes has also been investigated. FNPs presented an average size of 363.1 ± 17.2 nm and a zeta potential of + 17.7 ± 0.1 mV with encapsulation efficiency (EE) and loading capacity (LC) of 78.79 ± 7.7% and 37.46 ± 6.6%, respectively. The viability of human chondrocytes was not affected by blank nanoparticles (BNPs) up to a concentration of 2000 μg/mL. In addition, the hemolysis results clearly showed that FNPs did not damage the red blood cells (RBCs) and had good hemocompatibility within the range investigated. FNPs, similar to fisetin, were able to inhibit the inflammatory responses induced by IL-1β such as the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) while increasing the production of an anti-inflammatory cytokine such as interleukin-10 (IL-10). Overall, the in vitro evaluation results of the anti-inflammatory activity showed that FNPs can serve as delivery systems to transfer fisetin to treat inflammation in OA.

Ex Vivo Toxicity of Commonly Used Topical Antiseptics and Antibiotics on Human Chondrocytes.

Topical povidone-iodine, chlorhexidine, bacitracin, and vancomycin are commonly used antiseptic and antimicrobial agents to reduce risk and treat surgical site infections in numerous orthopedic procedures. Chondrocytes potentially may be exposed to these agents during operative procedures. The impact of these topical agents on chondrocyte viability is unclear. The goal of this study is to determine human chondrocyte viability ex vivo after exposure to commonly used concentrations of these topical antiseptic and antimicrobial agents. Human osteochondral plugs were harvested from the knee joint of a human decedent within 36 hours of death. Individual human osteochondral plugs were exposed to normal saline as a control; a range of concentrations of povidone-iodine (0.25%, 0.5%, and 1%), chlorhexidine (0.01% and 0.5%), and bacitracin (10,000 units/L, 50,000 units/L, and 100,000 units/L) for 1-minute lavage; or a 48-hour soak in vancomycin (0.16 mg/mL, 0.4 mg/mL, and 1.0 mg/mL) with nutrient media. Chondrocyte viability was evaluated with a live/dead viability assay at 0, 2, 4, and 6 days after exposure to bacitracin at 0, 3, and 6 days). Control subjects showed greater than 70% viability at all time points. Povidone-iodine, 0.5% chlorhexidine, and vancomycin showed significant cytotoxicity, with viability dropping to less than 40% by day 6. Chondrocytes exposed to 0.01% chlorhexidine maintained viability. Chondrocytes exposed to bacitracin showed viability until day 3, when there was a large drop in viability. Commonly used topical concentrations of povidone-iodine, vancomycin, and bacitracin are toxic to human chondrocytes ex vivo. A low concentration of chlorhexidine appears safe. Caution should be used when articular cartilage may be exposed to these agents during surgery. [Orthopedics. 2022;45(5):e263-e268.].

EtoGel for Intra-Articular Drug Delivery: A New Challenge for Joint Diseases Treatment.

Ethosomes® have been proposed as potential intra-articular drug delivery devices, in order to obtain a longer residence time of the delivered drug in the knee joint. To this aim, the conventional composition and preparation method were modified. Ethosomes® were prepared by using a low ethanol concentration and carrying out a vesicle extrusion during the preparation. The modified composition did not affect the deformability of ethosomes®, a typical feature of this colloidal vesicular topical carrier. The maintenance of sufficient deformability bodes well for an effective ethosome® application in the treatment of joint pathologies because they should be able to go beyond the pores of the dense collagen II network. The investigated ethosomes® were inserted in a three-dimensional network of thermo-sensitive poloxamer gel (EtoGel) to improve the residence time in the joint. Rheological experiments evidenced that EtoGel could allow an easy intra-articular injection at room temperature and hence transform itself in gel form at body temperature into the joint. Furthermore, EtoGel seemed to be able to support the knee joint during walking and running. In vitro studies demonstrated that the amount of used ethanol did not affect the viability of human chondrocytes and nanocarriers were also able to suitably interact with cells.

In Vitro Effects of Low Doses of β-Caryophyllene, Ascorbic Acid and d-Glucosamine on Human Chondrocyte Viability and Inflammation

β-caryophyllene (BCP), a plant-derived sesquiterpene, has been reported to have anti-inflammatory and antioxidant effects. The purpose of this study is to evaluate the effects of BCP in combination with ascorbic acid (AA) and d-glucosamine (GlcN) against macrophage-mediated inflammation on in vitro primary human chondrocytes. Changes in cell viability, intracellular ROS generation, gene expression of pro-inflammatory mediators, metalloproteinases (MMPs), collagen type II and aggrecan were analyzed in primary human chondrocytes exposed to the conditioned medium (CM) of activated U937 monocytes and subsequently treated with BCP alone or in combination with AA and GlcN. The CM-induced chondrocyte cytotoxicity was reduced by the presence of low doses of BCP alone or in combination with AA and GlcN. The exposure of cells to CM significantly increased IL-1β, NF-κB1 and MMP-13 expression, but when BCP was added to the inflamed cells, alone or in combination with AA and GlcN, gene transcription for all these molecules was restored to near baseline values. Moreover, chondrocytes increased the expression of collagen type II and aggrecan when stimulated with AA and GlcN alone or in combination with BCP. This study showed the synergistic anti-inflammatory and antioxidative effects of BCP, AA and GlcN at low doses on human chondrocyte cultures treated with the CM of activated U937 cells. Moreover, the combination of the three molecules was able to promote the expression of collagen type II and aggrecan. All together, these data could suggest that BCP, AA and GlcN exert a chondro-protective action.

Modulation of the Inflammatory Process by Hypercholesterolemia in Osteoarthritis.

Objective: Several studies have linked metabolic syndrome to the development of osteoarthritis (OA) through hypercholesterolemia, one of its components. However, epidemiological studies showed contradictory results, and it is not clear how hypercholesterolemia itself, or oxidized LDL (oxLDL)—a pathological molecule potentially involved in this relationship—could be affecting OA. The objectives of this study were to investigate the effect of hypercholesterolemia induced by high-fat diet (HFD) in cartilage from OA rabbits, and how oxLDL affect human chondrocyte inflammatory and catabolic responses.Design: New Zealand rabbits were fed with HFD for 18 weeks. On week 6, OA was surgically induced. At the end of the study, cartilage damage and IL-1β, IL-6, MCP-1, MMP-13, and COX-2 expression in articular cartilage were evaluated. In addition, cultured human OA articular chondrocytes were treated with oxLDL at concentrations equivalent to those expected in synovial fluid from HFD rabbits, in the presence of IL-1β and TNFα. The effect of oxLDL on cell viability, nitric oxide production and catabolic and pro-inflammatory gene expression was evaluated.Results: HFD intake did not modify cartilage structure or pro-inflammatory and catabolic gene expression and protein presence, both in healthy and OA animals. OxLDL did not affect human chondrocyte viability, ADAMTS5 and liver X receptor (LXR) α gene expression, but decreased the induction of IL-1β, IL-6, MCP-1, MMP-13, iNOS, and COX-2 gene expression and MMP-13 and COX-2 protein presence, evoked by cytokines.Conclusions: Our data suggest that cholesterol intake per se may not be deleterious for articular cartilage. Instead, cholesterol de novo synthesis and altered cholesterol metabolism could be involved in the associations observed in human disease.

Vancomycin is toxic to human chondrocytes in vitro.

Vancomycin powder (VP) is a well-established topical antibiotic used in spinal surgery to prevent surgical site infections. More recently its extension to hip and knee arthroplasty was introduced. The aim of this study was to examine toxic effects of VP on the viability of human chondrocytes. Our hypothesis was that VP damages human chondrocytes in vitro with increasing concentration and length of exposure. Primary human chondrocytes were isolated and cultured from donated human knee joints. VP was added to these cultures with increasing concentrations (0-50mg/ml) and length of exposure (0-336h). Toxicity and viability were analyzed using LDH und XTT Elisa assays. Cell structure and determination of vital versus dead cells were visualized using light microscopy and fluorescence microscopy. Light microscopy and fluorescence microscopy visualized defect cell structures and cell death proportional to increasing dose and length of exposure to VP. The analysis of LDH activity data showed toxic effects on chondrocytes as early as 2,5min after exposure to VP. XTT activity data revealed a significant toxic threshold of a VP concentration above 12.5mg/ml. These results show that exposure to high VP concentrations yields to a damage of human chondrocytes in vitro. Chondrotoxicity is an immediate effect that is proportional to VP concentration. Therefore, the intraarticular use of high concentrations of vancomycin powder in the presence of native cartilage tissue must be considered critically.

Tranexamic acid is toxic on human chondrocytes, in vitro

The aim of this study was to examine toxic effects of tranexamic acid (TXA) on the viability of human chondrocytes. Our hypothesis was that TXA damages human chondrocytes. Chondrocytes were cultured from donated human knee joints. TXA was added to these cultures. Toxicity were analyzed by using LDH und XTT Elisa assays, light microscopy and fluorescence microscopy. The results show that TXA damages human chondrocytes in vitro. We cannot recommend the use of TXA in hemiarthroplasty of the hip or unicompartmental knee arthroplasty in higher concentrations.

Short exposure to tranexamic acid does not affect, in vitro, the viability of human chondrocytes

BackgroundOnly few studies have investigated the effect of topical application of tranexamic acid (TXA) on “minimally” invasive joint surgical procedures in which articular cartilage is preserved; for this reason, actually many surgeons avoid the use of topical TXA even if the disadvantage related to a blood loss can occur. The aim of this study was to evaluate the cytotoxicity, on human chondrocytes, of TXA at different concentrations and times of exposure and the mechanisms of cell death.MethodsExperiments were carried out on isolated human chondrocytes harvested from eight patients who underwent total knee replacement. Cell viability was determined using XTT assay and was assessed at 0, 24 and 48 h intervals after a 10-min-long treatment, followed by thorough washes, or at 24 and 48 h of treatment at TXA concentrations of 20, 50, 70 and 100 mg/ml. Cell cycle alterations and occurrence of cell death for apoptosis or necrosis were assessed by cytofluorimetry. Data were analyzed using Proc Mixed Procedure; LSMEANS was used to compare multiple group means with Tukey’s honestly significant difference test.ResultsA significant correlation between the controlled for factors (type of treatment, time and concentration) was found in the performed experiment. No significant effect on cell viability was observed when the TXA exposure was limited to 10 min, while for increased exposure, 24 and 48 h, a remarkable reduction was found; cell death occurred by apoptosis and was already appreciable after 24 h, reaching a statistical significance after the 48-h-long treatment.ConclusionA prolonged exposure to TXA may cause cartilage damage, thus its topical application can be expanded also to clinical scenarios that include retention of native cartilage chondrocytes, only if it is limited to few minutes and used at concentrations of 70 mg/ml or less.

Is tranexamic acid toxic to articular cartilage when administered topically? What is the safe dose?

The intra-articular administration of tranexamic acid (TXA) has been shown to be effective in reducing blood loss in unicompartmental knee arthroplasty and anterior cruciate reconstruction. The effects on human articular cartilage, however, remains unknown. Our aim, in this study, was to investigate any detrimental effect of TXA on chondrocytes, and to establish if there was a safe dose for its use in clinical practice. The hypothesis was that TXA would cause a dose-dependent damage to human articular cartilage. The cellular morphology, adhesion, metabolic activity, and viability of human chondrocytes when increasing the concentration (0 mg/ml to 40 mg/ml) and length of exposure to TXA (0 to 12 hours) were analyzed in a 2D model. This was then repeated, excluding cellular adhesion, in a 3D model and confirmed in viable samples of articular cartilage. Increasing concentrations above 20 mg/ml resulted in atypical morphology, reduced cellular adhesion and metabolic activity associated with increased chondrocyte death. However, the cell matrix was not affected by the concentration of TXA or the length of exposure, and offered cellular protection for concentrations below 20 mg/ml. These results show that when in vitro chondrocytes are exposed to higher concentrations of TXA, such as that expected following recommended intra-articular administration, cytotoxicity is observed. This effect is dose-dependent, such that a tissue concentration of 10 mg/ml to 20 mg/ml could be expected to be safe. Cite this article: Bone Joint J 2018;100-B:404-12.

Catalase Enhances Viability of Human Chondrocytes in Culture by Reducing Reactive Oxygen Species and Counteracting Tumor Necrosis Factor-α-Induced Apoptosis

Background/Aims: Both physiologic remodeling and pathologic regeneration of cartilage tissue rely upon chondrocyte functions and are benefited from factors that promote viability and inhibit apoptosis of the cell, and associated mechanisms. High level of reactive oxygen species (ROS) and proinflammatory cytokines activate apoptosis signaling and initiate cell death, which can be attenuated by antioxidants. This study examined the effect of catalase (CAT) on ROS and tumor necrosis factor-α (TNF-α)-induced apoptosis in human C28/I2 chondrocytes cultured in monolayer. Methods: Chondrocytes were treated with diluted CAT in the presence or absence of TNF-α and compared to untreated cells. Levels of hydrogen peroxide (H2O2) and mitochondrial membrane potential (Δψm) were measured using fluorescent labeling, cell apoptosis was assayed by flow cytometry using Annexin V/propidium iodide (PI) staining, gene expression was detected by quantitative real time polymerase chain reaction (qRT-PCR) and the proteins were investigated by Western blotting. Results: CAT effectively reduced the intracellular ROS caused by the monolayer culture system, enhanced the Δψm depending on the presence of TNF-α and promoted morphological features at sub-cellular level. CAT also attenuated the TNF-α-upregulated expression of factors/mediators of extrinsic cell death cascade and apoptotic caspases, ultimately resulted in promoted cellular viability. Conclusion: The anti-apoptotic effect of CAT on chondrocytes via scavenging ROS and suppressing TNF-α-induced cell apoptosis by TNF/TNF receptor (TNFR) mediated death signaling pathway and potentiate CAT as a complementary agent beneficial to cartilage remodeling and regeneration in vivo, and cell-based therapies of cartilage repair demanding viable cells expanded ex vivo.

Protective Effect of Ginger (Zingiber officinale Roscoe) Extract against Oxidative Stress and Mitochondrial Apoptosis Induced by Interleukin-1β in Cultured Chondrocytes

Aims: The protective effects of ginger (Zingiber officinale Roscoe) extract on IL-1β-mediated oxidative stress and mitochondrial apoptosis were investigated in C28I2 human chondrocytes. Methods: The effects of various concentrations of ginger extract on C28I2 human chondrocyte viability were evaluated in order to obtain noncytotoxic concentrations of the drug by methylthiotetrazole assay. The cells were pretreated with 5 and 25 μg/mL ginger extract for 24 h, followed by incubation with IL-1β (10 ng/mL) for 24 h. The effects of ginger extract on IL-1β-induced intracellular reactive oxygen species (ROS) production and lipid peroxidation were examined. The mRNA expressions of antioxidant enzymes including catalase, superoxide dismutase-1, glutathione peroxidase-1, glutathione peroxidase-3, and glutathione peroxidase-4 were evaluated by reverse transcription polymerase chain reaction. The protein expressions of Bax, Bcl-2, and caspase-3 were analyzed by Western blotting. Results: No cytotoxicity was observed at any concentration of ginger extract in C28I2 cells. Ginger extract pretreatment remarkably increased the gene expression of antioxidant enzymes and reduced the IL-1β-induced elevation of ROS, lipid peroxidation, the Bax/Bcl-2 ratio, and caspase-3 activity. Conclusions: Ginger extract could considerably reduce IL-1β-induced oxidative stress and consequent mitochondrial apoptosis as the major mechanisms of chondrocyte cell death. These beneficial effects of ginger extract may be due to its antioxidant properties. It may be considered as a natural herbal product to prevent OA-induced cartilage destruction in the clinical setting.

Cell-laden biphasic scaffolds with anisotropic structure for the regeneration of osteochondral tissue

Sufficient treatment of chondral and osteochondral defects to restore function of the respective tissue remains challenging in regenerative medicine. Biphasic scaffolds that mimic properties of bone and cartilage are appropriate to regenerate both tissues at the same time. The present study describes the development of biphasic, but monolithic scaffolds based on alginate, which are suitable for embedding of living cells in the chondral part. Scaffolds are fabricated under sterile and cell-compatible conditions according to the principle of diffusion-controlled, directed ionotropic gelation, which leads to the formation of channel-like, parallel aligned pores, running through the whole length of the biphasic constructs. The synthesis process leads to an anisotropic structure, as it is found in many natural tissues. The two different layers of the scaffolds are characterized by different microstructure and mechanical properties which provide a suitable environment for cells to form the respective tissue. Human chondrocytes and human mesenchymal stem cells were embedded within the chondral layer of the biphasic scaffolds during hydrogel formation and their chondrogenic (re)differentiation was successfully induced. Whereas viability of non-induced human mesenchymal stem cells decreased during culture, cell viability of human chondrocytes and chondrogenically induced human mesenchymal stem cells remained high within the scaffolds over the whole culture period of 3 weeks, demonstrating successful fabrication of cell-laden centimetre-scaled constructs for potential application in regenerative treatment of osteochondral defects. Copyright © 2014 John Wiley & Sons, Ltd.

The mechanical microenvironment of high concentration agarose for applying deformation to primary chondrocytes

Cartilage and chondrocytes experience loading that causes alterations in chondrocyte biological activity. In vivo chondrocytes are surrounded by a pericellular matrix with a stiffness of ~25–200kPa. Understanding the mechanical loading environment of the chondrocyte is of substantial interest for understanding chondrocyte mechanotransduction. The first objective of this study was to analyze the spatial variability of applied mechanical deformations in physiologically stiff agarose on cellular and sub-cellular length scales. Fluorescent microspheres were embedded in physiologically stiff agarose hydrogels. Microsphere positions were measured via confocal microscopy and used to calculate displacement and strain fields as a function of spatial position. The second objective was to assess the feasibility of encapsulating primary human chondrocytes in physiologically stiff agarose. The third objective was to determine if primary human chondrocytes could deform in high-stiffness agarose gels. Primary human chondrocyte viability was assessed using live–dead imaging following 24 and 72h in tissue culture. Chondrocyte shape was measured before and after application of 10% compression. These data indicate that (1) displacement and strain precision are ~1% and 6.5% respectively, (2) high-stiffness agarose gels can maintain primary human chondrocyte viability of >95%, and (3) compression of chondrocytes in 4.5% agarose can induce shape changes indicative of cellular compression. Overall, these results demonstrate the feasibility of using high-concentration agarose for applying in vitro compression to chondrocytes as a model for understanding how chondrocytes respond to in vivo loading.

The chondrotoxicity of single-dose corticosteroids

Corticosteroids are commonly injected into the joint space. However, studies have not examined the chondrotoxicity of one-time injection doses. The purpose of this study is to evaluate the effect of dexamethasone sodium phosphate (Decadron), methylprednisolone acetate (Depo-Medrol), betamethasone sodium phosphate and betamethasone acetate (Celestone Soluspan), and triamcinolone acetonide (Kenalog) on human chondrocyte viability in vitro. Single-injection doses of each of the corticosteroids were separately delivered to human chondrocytes for their respective average duration of action and compared to controls using a bioreactor containing a continuous infusion pump constructed to mimic joint fluid metabolism. A 14-day time-controlled trial was also performed. A live/dead reduced biohazard viability/cytotoxicity assay was used to quantify chondrocyte viability. Over their average duration of action, betamethasone sodium phosphate/acetate solution and triamcinolone acetonide caused significant decreases in chondrocyte viability compared to control media (19.8 ± 2.9% vs. 5.2 ± 2.1%, P = 0.0025 and 10.2 ± 1.3% vs. 4.8 ± 0.9%, P = 0.0049, respectively). In the 14-day trial, only betamethasone sodium phosphate/acetate solution caused a significant decrease in chondrocyte viability compared to control media (21.5% vs. 4.6%, P < 0.001). A single-injection dose of betamethasone sodium phosphate and betamethasone acetate solution illustrated consistent and significant chondrotoxicity using a physiologically relevant in vitro model and should be used with caution. Given the observed chondrotoxicity of triamcinolone acetonide in a single trial, there may be some evidence that this medication is chondrotoxic. However, at 14 days, betamethasone sodium phosphate and betamethasone acetate was the only condition that caused significant cell death.

The effect of local anesthetic and corticosteroid combinations on chondrocyte viability

Local anesthetic and corticosteroid combination injections are often used in clinical practice, however research investigating the chondrotoxic properties of these combinations is minimal. The goal of this study was to evaluate the effect of single injection doses of 1% lidocaine or 0.25% bupivacaine in combination with single injection doses of dexamethasone sodium phosphate (Decadron), methylprednisolone acetate (Depo-Medrol), betamethasone sodium phosphate and betamethasone acetate (Celestone Soluspan), or triamcinolone acetonide (Kenalog) on human chondrocyte viability. All treatment conditions were delivered to human chondrocytes in vitro for the medication's respective average duration of action using a bioreactor containing a continuous infusion pump constructed to mimic joint fluid metabolism. A two-color fluorescence assay was used to evaluate cell viability. A mixed-effects regression model was used to evaluate the mean differences in cell viability between treatment groups. At 14 days, a single injection dose of 1% lidocaine or 0.25% bupivacaine in combination with betamethasone sodium phosphate and betamethasone acetate solution illustrated significant chondrotoxicity when compared with the local anesthetics alone (P < 0.01). Methylprednisolone acetate and Triamcinolone acetonide both showed significant evidence of chondrotoxicity (P = 0.013; P = 0.016, respectively) when used in combination with 1% lidocaine compared with lidocaine alone, but showed no significant chondrotoxicity in combination with 0.25% bupivacaine (P's = n.s.). Clinicians should use caution when injecting 1% lidocaine or 0.25% bupivacaine in conjunction with betamethasone sodium phosphate and betamethasone acetate solution due to its pronounced chondrotoxic effect in this study. 1% lidocaine used in combination with methylprednisolone acetate or triamcinolone acetonide also led to significant chondrotoxicity.

In vitro assessment of human chondrocyte viability after treatment with local anaesthetic, magnesium sulphate or normal saline

Local anaesthetic agents are often used as an intra-articular analgesic following arthroscopic procedures. However, there is increasing evidence of a potential toxic effect to chondrocytes within the articular cartilage. The aim of this study was to compare the effect on human chondrocyte viability of treatment with bupivacaine, levobupivacaine and ropivacaine. The second aim was to compare the effect on chondrocyte viability of the local anaesthetics with magnesium, a potential alternative analgesic agent. Chondrocytes were exposed to one of the local anaesthetic agents (levobupivacaine 0.13, 0.25, 0.5%; bupivacaine 0.13, 0.25, 0.5%; ropivacaine 0.19, 0.38, 0.75%), normal saline or 10% magnesium sulphate for 15 min. Cells exposed to cell culture media served as controls. Twenty-four hours after exposure, cell viability was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. There was no significant difference in chondrocyte viability after treatment with either normal saline or magnesium sulphate. With the exception of 0.13% levobupivacine, all local anaesthetic treatment showed significantly greater toxic effects than either normal saline or magnesium sulphate. Statistically significant dose-dependent responses of decreasing cell viability were found with increasing local anaesthetic concentration. A dose-dependent reduction in chondrocyte viability after treatment with common local anaesthetic agents was confirmed. Local anaesthetic agents had a greater deleterious effect on chondrocytes than did 10% magnesium sulphate. These findings suggest the need for continuing caution with the use of intra-articular local anaesthetic. Magnesium sulphate is a potential alternative intra-articular analgesic agent.

The effect of Lidocaine on the viability of cultivated mature human cartilage cells: an in vitro study

More and more orthopedic procedures are performed in an outpatient setting. A commonly used strategy in pain management is the intra-articular injection of local anesthetics. Recent attention has been drawn to their possible toxic effect on chondrocytes. Local anesthetics, and in particular Lidocaine, are also used for diagnostic joint infiltrations. A controlled laboratory study was performed to investigate the possible toxic effect of Lidocaine on human articular chondrocytes. Mature human articular chondrocytes were harvested from the knees of human tissue donors or patients undergoing total knee replacement. The cells were exposed to Lidocaine 1 and 2% with and without epinephrine and to a saline 0.9% control group, with variable exposure times in different experiments. The activity and viability of the cells were assessed by lactate dehydrogenase activity, interleukin-6 production and a live/dead cell count. After a 1-h exposure, devastating results were seen for Lidocaine 1, 2 and 2% with epinephrine showing cell death rates of 91, 99 and 97%, respectively, compared with 26% in the saline control group (P-values of 0.004, 0.010, 0.006, respectively). Exposing the chondrocytes to a 50/50 mixture of culture medium and local anesthetics substantially decreased cytotoxicity but still showed high toxicity when compared with the saline group (90% dead cells for Lidocaine 2%, P = 0.047). Lidocaine also showed a time-dependent cytotoxicity with gradually more dead cells after exposure for 15, 30 or 60 min. In vitro, local anesthetics containing Lidocaine are significantly more toxic to mature human articular chondrocytes than a saline 0.9% control group. The effect of Lidocaine on the viability of human chondrocytes in vivo needs further investigation. However, based on our in vitro results, cautious use of intra-articular Lidocaine in clinical practice is recommended.

Toxicity of antiseptics against chondrocytes: what is best for the cartilage in septic joint surgery?

In septic joint surgery, the most frequently used antiseptics are polyhexanide, hydrogen peroxide and taurolidine. The aim of this study was to examine the effects of these antiseptics on viability of human chondrocytes. Our hypothesis was that antiseptics and supplemental irrigation with sodium chloride lavage are less toxic on human chondrocytes than treatment with antiseptics only. Primary human chondrocytes were isolated and cultured from six donated human knee joints. Polyhexanide, hydrogen peroxide or taurolidine were added to the cultures. Toxicity analysis was performed by visualisation of cell structure using light microscopy and LDH activity. The determination of vital cells and total cell numbers of chondrocytes treated with antiseptics partly followed by irrigation with sodium chloride solution was performed by using Casy Cell-Counter. Light microscopic data revealed a defect in cell structure after addition of antiseptics. We showed a significant increase of LDH enzyme activity after the treatment with polyhexanide or taurolidine. After treatment with antiseptics followed by sodium chloride solution a significant increase of vital and total cell numbers resulted in comparison with the chondrocytes that were only treated with antiseptics. The data show that treatment with polyhexanid, hydrogen peroxide or taurolidine induces cell death of human chondroctes in vitro. The application of sodium chloride solution after the treatment with polyhexanide and hydrogen peroxide possibly has a protective effect on chondrocyte viability.