Decrease In Chondrocyte Viability Research Articles

Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Cigarette smoke extract exacerbates progression of osteoarthritic-like changes in cartilage explant cultures.

Established risk factors for osteoarthritis (OA) include obesity, joint injury, age, race, and genetics. However, the relationship between cigarette smoking and OA has yet to be established. In the present study, we have employed the use of cigarette smoke extract (CSE), the water-soluble vapor phase of cigarette smoke, with porcine cartilage explants to investigate the effects of cigarette smoking on cartilage catabolism at the tissue level. Articular cartilage explants were first exposed to 2.5%, 5%, and 10% CSE to assess its effects on cartilage homeostasis. Following, the effects of CSE on OA-like inflammation was observed by culturing explants with a combined treatment of IL-1β and TNF-α and 10% CSE (CSE + OA). Cartilage explants were assessed for changes in viability, biochemical composition, extracellular matrix (ECM) integrity, and equilibrium mechanical properties (aggregate modulus and hydraulic permeability). CSE alone leads to both a time- and dose-dependent decrease in chondrocyte viability but does not significantly affect sGAG content, percent sGAG loss, or the ECM integrity of cartilage explants. When IL-1β and TNF-α were combined with 10% CSE, this led to a synergistic effect with more significant losses in viability, significantly more sGAG loss, and significantly higher production of ROS than OA-like inflammation only. Cartilage explant equilibrium mechanical properties were unaffected. Within the timeframe of this study, CSE alone does not cause OA but when combined with OA-like inflammation leads to worsened articular cartilage degeneration as measured by chondrocyte viability, sGAG loss, proteoglycan staining, and ROS production.

Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics

Background: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown. Purpose: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. Study Design: Controlled laboratory study. Methods: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed. Results: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020). Conclusion: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. This raises concern for the risk of mechanical failure of the cartilage tissue after CHG exposure. Clinical Relevance: Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Transient Exposure of Tissue-Engineered Cartilage Analogs to Synovial Fluid Hematoma After Ankle Fracture Promotes Chondrocyte Death and Alters Gene Expression Toward an Osteoarthritic Phenotype

Category: Basic Sciences/Biologics; Ankle Arthritis Introduction/Purpose: The first stage of fracture healing consists of hematoma formation with recruitment of proinflammatory cytokines and matrix metalloproteinases. Unfortunately, when there is an intra-articular fracture, these inflammatory mediators are not retained at the fracture site, but instead, bathe the healthy cartilage of the entire joint via the synovial fluid fracture hematoma (SFFH). These inflammatory cytokines and matrix metalloproteinases are known factors in the progression of osteoarthritis and rheumatoid arthritis. Despite the known inflammatory contents of the SFFH, little research has been done on the effects of the SFFH on healthy cartilage with regard to cell death and alteration in gene expression that could lead to post- traumatic osteoarthritis (PTOA). Methods: SFFH was collected from 12 patients with intraarticular ankle fracture at the time of surgery. Separately, human chondrocytes were three-dimensionally cultured to create scaffold -free cartilage tissue analogues (CTAs) to simulate articular cartilage. Experimental CTAs (n=12) were exposed to 100% SFFH for 3 days, washed, and transferred to complete media for 3 days. Control CTAs (n=12) were simultaneously cultured in complete medium. Subsequently, CTAs were harvested and underwent biochemical, histological, and gene expression analysis. Results: Exposure of CTAs to ankle SFFH 3 days significantly decreased chondrocyte viability by 34% (p = .027). Gene expression of both COL2A1 and SOX9 were significantly decreased after exposure to SFFH (p= .012 and p=.0013 respectively). COL1A1 expression trended upward in the SFFH exposure group while there was no difference in RUNX2 and MMP13 gene expression. Quantitative analysis of Picrosirius red staining demonstrated increased collagen I deposition with poor ultrastructural organization in SFFH exposed CTAs. Conclusion: Exposure of an organoid model of healthy cartilage tissue to SFFH after intraarticular ankle fracture resulted in decreased chondrocyte viability, decreased expression of genes regulating normal chondrocyte phenotype, and altered matrix ultrastructure indicating differentiation toward an osteoarthritis phenotype.

Lithium prevents glucocorticoid‑induced chondrocyte autophagy: An in vitro study.

Glucocorticoids can induce chondrocyte autophagy. Lithium is a classical regulator of autophagy. The present study aimed to determine whether lithium can prevent glucocorticoid‑induced chondrocyte autophagy by regulating the PI3K/AKT/mTOR signaling pathway. For this purpose, rat and human chondrocytes were treated with dexamethasone (200µM) or dexamethasone (200µM) combined with lithium chloride at various concentrations (0.01, 0.1, 1 and 10mM). CYTO‑ID® autophagy fluorescence staining and transmission electron microscopy were used to detect the levels of autophagy in the chondrocytes. Reverse transcription‑quantitative PCR and western blot analysis were used to measure the expression levels of the autophagy marker, LC3B and the autophagy regulatory signaling pathway (PI3K/AKT/mTOR signaling pathways) markers, AKT and mTOR. The viability of chondrocytes was measured using the Cell Counting Kit‑8 assay. It was found that compared with that in the control group, dexamethasone induced the autophagy of chondrocytes, decreased the expression levels of AKT and mTOR, and reduced cell viability. Compared with the treatment with dexamethasone alone, lithium chloride (10mM) + dexamethasone reduced the autophagy levels, increased the expression level of AKT and mTOR, and increased cell viability. In conclusion, the present study demonstrated that lithium can prevent glucocorticoid‑induced autophagy by activating the PI3K/AKT/mTOR signaling pathway and preventing the glucocorticoid‑induced decrease in chondrocyte viability.

Poster 152: Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics

Objectives: Chlorhexidine gluconate (CHG) is commonly used in antiseptic irrigation solutions for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG to articular cartilage has been reported, CHG irrigation is utilized by some surgeons to limit surgical site infection and to salvage grafts after accidental contamination during joint surgery. The full extent of CHG irrigation-related chondrotoxicity, as well as its effects on articular cartilage extracellular matrix and mechanical properties, are unknown. The purpose of this study was to determine the in vitro effects of a single one-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. Methods: Articular cartilage explants (n=6 per group) were harvested from the femoral condyles of the porcine stifle and sectioned at the tidemark. Explants were bathed in Irrisept® chlorhexidine solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) to stimulate surgical irrigation for 1 minute, followed by complete phosphate-buffered saline wash and culture. After seven days of culture in chondrogenic medium, the explants were analyzed for viability (live/dead assay); collagen content (hydroxyproline assay); glycosaminoglycan (GAG) content (dimethyl methylene blue assay); and compressive mechanical testing (creep indentation testing). Statistical analyses were performed using a one-way ANOVA with post hoc Tukey test. Results: Seven days after CHG exposure, a dose-dependent decrease in mean chondrocyte viability was observed in the CHG groups (p&lt;0.001), with &lt;3% viability observed in the 0.05% CHG group (Figure 1). There was no significant difference in mean collagen content per wet weight among groups. Conversely, 0.05% CHG exposure led to a decrease in mean GAG per wet weight compared to negative control (p=0.029) and 0.01% CHG (p=0.046) (Figure 2). These changes in GAG content corresponded to observed changes in the compressive mechanical properties of the explants. A dose-dependent decrease in mean aggregate modulus and shear modulus was observed after CHG exposure (p&lt;0.029), indicating weakening of the cartilage matrix and increased deformation in response to compressive loads (Figure 3). Conclusions: One-minute CHG exposure to articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. These detrimental effects were observed for both 0.05% and 0.01% CHG concentrations. There is a paucity of literature examining the effects of CHG on articular cartilage matrix and mechanics, and the findings of this study suggest a lack of matrix maintenance following cell death, raising concern for risk of mechanical failure of the cartilage tissue. Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Exposure of Tissue-Engineered Cartilage Analogs to Synovial Fluid Hematoma After Ankle Fracture Is Associated With Chondrocyte Death and Altered Cartilage Maintenance Gene Expression.

The first stage of fracture healing consists of hematoma formation with recruitment of proinflammatory cytokines and matrix metalloproteinases. Unfortunately, when there is an intra-articular fracture, these inflammatory mediators are not retained at the fracture site, but instead, envelop the healthy cartilage of the entire joint via the synovial fluid fracture hematoma (SFFH). These inflammatory cytokines and matrix metalloproteinases are known factors in the progression of osteoarthritis and rheumatoid arthritis. Despite the known inflammatory contents of the SFFH, little research has been done on the effects of the SFFH on healthy cartilage with regard to cell death and alteration in gene expression that could lead to posttraumatic osteoarthritis (PTOA). SFFH was collected from 12 patients with intraarticular ankle fracture at the time of surgery. Separately, C20A4 immortalized human chondrocytes were 3-dimensionally cultured to create scaffold-free cartilage tissue analogs (CTAs) to simulate healthy cartilage. Experimental CTAs (n = 12) were exposed to 100% SFFH for 3 days, washed, and transferred to complete media for 3 days. Control CTAs (n = 12) were simultaneously cultured in complete medium without exposure to SFFH. Subsequently, CTAs were harvested and underwent biochemical, histological, and gene expression analysis. Exposure of CTAs to ankle SFFH for 3 days significantly decreased chondrocyte viability by 34% (P = .027). Gene expression of both COL2A1 and SOX9 were significantly decreased after exposure to SFFH (P = .012 and P = .0013 respectively), while there was no difference in COL1A1, RUNX2, and MMP13 gene expression. Quantitative analysis of Picrosirius red staining demonstrated increased collagen I deposition with poor ultrastructural organization in SFFH-exposed CTAs. Exposure of an organoid model of healthy cartilage tissue to SFFH after intraarticular ankle fracture resulted in decreased chondrocyte viability, decreased expression of genes regulating normal chondrocyte phenotype, and altered matrix ultrastructure indicating differentiation toward an osteoarthritis phenotype. The majority of ankle fracture open reduction and internal fixation does not occur immediately after fracture. In fact, typically these fractures are treated several days to weeks later in order to let the swelling subside. This means that the healthy innocent bystander cartilage not involved in the fracture is exposed to SFFH during this time. In this study, the SFFH caused decreased chondrocyte viability and specific altered gene expression that might have the potential to induce osteoarthritis. These data suggest that early intervention after intraarticular ankle fracture could possibly mitigate progression toward PTOA.

Effects of glycosaminoglycan content in extracellular matrix of donor cartilage on the functional properties of osteochondral allografts evaluated by micro-CT non-destructive analysis.

Osteochondral allograft (OCA) is an important surgical procedure used to repair extensive articular cartilage damage. It is known that chondrocyte viability is crucial for maintaining the biochemical and biomechanical properties of OCA, which is directly related to the clinical success of the operation and is the only standard for preoperative evaluation of OCA. However, there is a lack of systematic research on the effect of the content of cellular matrix in OCA cartilage tissue on the efficacy of transplantation. Therefore, we evaluated the effect of different GAG contents on the success of OCA transplantation in a rabbit animal model. Each rabbit OCA was treated with chondroitinase to regulate glycosaminoglycan (GAG) content in the tissue. Due to the different action times of chondroitinase, they were divided into 4 experimental groups (including control group, 2h, 4h, and 8h groups). The treated OCAs of each group were used for transplantation. In this study, transplant surgery effects were assessed using micro-computed tomography (μCT) and histological analysis. Our results showed that tissue integration at the graft site was poorer in the 4h and 8h groups compared to the control group at 4 and 12 weeks in vivo, as were the compressive modulus, GAG content, and cell density reduced. In conclusion, we evaluated the biochemical composition of OCAs before and after surgery using μCT analysis and demonstrated that the GAG content of the graft decreased, it also decreased during implantation; this resulted in decreased chondrocyte viability after transplantation and ultimately affected the functional success of OCAs.

In vitro chondrotoxicity of bupivacaine, levobupivacaine and ropivacaine and their effects on caspase activity in cultured canine articular chondrocytes.

Bupivacaine, levobupivacaine and ropivacaine are potent, long acting, amide-type local anesthetics that have several clinical applications including intra-articular administration. The objectives of this study were to evaluate their in vitro effects on cell viability and caspase activity to elucidate whether they activate the extrinsic or intrinsic pathways of apoptosis in canine articular chondrocytes. Chondrocytes in monolayer culture were treated with culture medium as the control, or with 0.062% (0.62 mg/mL) bupivacaine, 0.062% levobupivacaine, and 0.062% ropivacaine for 24 hr. Cell viability was evaluated using the live/dead, 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), and Cell Counting Kit-8 (CCK-8) assays. Evaluation of caspase-3, caspase-8, and caspase-9 activity was performed using colorimetric assays. The MTT and CCK-8 assays were used to evaluate the effect of caspase inhibitors on local anesthetic chondrotoxicity. All three local anesthetics decreased chondrocyte viability after 24 hr (P<0.001). Apoptosis was induced through both the extrinsic and intrinsic pathways. Bupivacaine increased caspase-3, caspase-8, and caspase-9 activity (P<0.001). Levobupivacaine increased caspase-3 (P=0.03) while ropivacaine did not significantly upregulate activity for all three caspases. Caspase inhibition did not suppress bupivacaine chondrotoxicity whereas inhibition of caspase-8 and caspase-9 decreased ropivacaine chondrotoxicity and mildly attenuated levobupivacaine chondrotoxicity. In summary, the level of chondrotoxicity, the type of caspase activated, the level of caspase activation, and the response to caspase inhibitors was dependent on the type of local anesthetic. Therefore, ropivacaine may be a safer choice for intra-articular administration compared to levobupivacaine and bupivacaine.

Effect of Thermal Ablation on Growth Plates: A Study to Explore the Thermal Threshold of Rabbit Growth Plates During Microwave Ablation.

To explore the temperature threshold of thermal damage to growth plates. Nine rabbits were divided into three groups for femoral ablation, exposing the growth plate to different temperatures (T1 = 43-45°C; T2 = 46-48°C; T3 = 49-51°C). After 5weeks, the changes in the femurs were assessed by macroscopic images, micro-CT, haematoxylin and eosin staining, and immunohistochemistry of Col2a1 (type II collagen). At the cellular level, rabbit epiphyseal chondrocytes were exposed to 37°C, 44°C, 47°C and 50°C for 5min. Then, proliferation and chondrogenic differentiation were detected. The rabbits in the T2 and T3 groups developed length discrepancies and axial deviations of femurs, abnormal newly formed bone in the marrow cavity, disorganized growth plates and decreased Col2a1 expression. At the cellular level, the cells exposed to 47°C and 50°C for 5min showed decreased viability, increased apoptosis, decreased extracellular matrix synthesis and decreased matrix mineralization. However, the changes in rabbits in the T1 group and cells at 44°C did not show a significant difference. The ablation of growth plates at temperatures above 45°C for 5min results in decreased chondrocyte viability and disorganized growth plates, leading to growth disturbances. Further studies are warranted to confirm these promising initial results.

Ononin ameliorates inflammation and cartilage degradation in rat chondrocytes with IL-1\u03b2-induced osteoarthritis by downregulating the MAPK and NF-\u03baB pathways

BackgroundOsteoarthritis (OA) treatment aims to improve inflammation and delay cartilage degeneration. However, there is no effective strategy presently available. Ononin, a representative isoflavone glycoside component extracted from natural Chinese herbs, exerts anti-inflammatory and proliferative effects. However, the therapeutic effect of ononin on chondrocyte inflammation remains unclear.MethodsIn this study, we explored the therapeutic effect and potential mechanism of ononin in OA by establishing an interleukin-1 beta (IL-1β)-induced chondrocyte inflammation model.ResultsOur results verified that ononin alleviated the IL-1β-induced decrease in chondrocyte viability, attenuated the overexpression of the inflammatory factors tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6), and simultaneously inhibited the expression of cartilage extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteinase-13 (MMP-13). Furthermore, the decomposition of Collagen II protein could be alleviated in the OA model by ononin. Finally, ononin improved chondrocyte inflammation by downregulating the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signalling pathways.ConclusionOur findings suggested that ononin could inhibit the IL-1β-induced proinflammatory response and ECM degradation in chondrocytes by interfering with the abnormal activation of the MAPK and NF-κB pathways, indicating its protective effect against OA.

M6A-mediated upregulation of AC008 promotes osteoarthritis progression through the miR-328-3p\u2012AQP1/ANKH axis

Long noncoding RNAs (lncRNAs) have emerged as important regulators of osteoarthritis (OA), but the biological roles and clinical significance of most lncRNAs in OA are not fully understood. Microarray analysis was performed to identify differentially expressed lncRNAs, mRNAs, and miRNAs between normal and osteoarthritic cartilage. We found that AC008440.5 (abbreviated AC008), as well as AQP1 and ANKH, were highly expressed in osteoarthritic cartilage, whereas miR-328-3p was expressed at a low level in osteoarthritic cartilage. Functional assays showed that ectopic expression of AC008, AQP1, and ANKH significantly decreased chondrocyte viability and promoted chondrocyte apoptosis and extracellular matrix (ECM) degradation, whereas knockdown of AC008, AQP1, and ANKH resulted in the opposite effects. Moreover, miR-328-3p overexpression increased chondrocyte viability and attenuated chondrocyte apoptosis and ECM degradation, whereas inhibition of miR-328-3p resulted in the opposite effects. Bioinformatics analysis, RNA immunoprecipitation (RIP), and luciferase assays revealed that AC008 functioned as a competing endogenous RNA (ceRNA) to regulate miR-328-3p, which specifically targeted the AQP1 and ANKH genes. In addition, miR-328-3p significantly ameliorated MIA-induced OA, whereas AC008 accelerated OA progression in vivo. Furthermore, fat mass and obesity-associated (FTO)-mediated N6-methyladenosine demethylation downregulated AC008 transcription, while lower FTO expression led to upregulation of AC008 transcription in OA. In conclusion, our data reveal that AC008 plays a critical role in OA pathogenesis via the miR-328-3p‒AQP1/ANKH pathway, suggesting that AC008 may be a potential therapeutic target for OA.

Aberrant Fluid Shear Stress Contributes to Articular Cartilage Pathogenesis via Epigenetic Regulation of ZBTB20 by H3K4me3.

PurposeOsteoarthritis (OA) is a common disease for human beings, characterized by severe inflammation, cartilage degradation, and subchondral bone destruction. However, current therapies are limited to relieving pain or joint replacement and no effective treatment methods have been discovered to improve degenerative changes. Currently, a variety of evidences have indicated that aberrant mechanical stimuli is closely associated with articular joint pathogenesis, while the detailed underlying mechanism remains unelucidated. In the present study, we determined to investigate the impact of excessive high fluid shear stress (FSS) on primary chondrocytes and the underlying epigenetic mechanisms.Materials and MethodsPhalloidin staining and EdU staining were used to evaluate cell morphology and viability. The mRNA level and protein level of genes were determined by qPCR, Western blot assay, and immunofluorescence staining. Mechanistic investigation was performed through RNA-sequencing and CUT&Tag sequencing. In vivo, we adopted unilateral anterior crossbites (UAC) mice model to investigate the expression of H3K4me3 and ZBTB20 in aberrant force-related cartilage pathogenesis.ResultsThe results demonstrated that FSS greatly disrupts cell morphology and significantly decreased chondrocyte viability. Aberrant FSS induces remarkable inflammatory mediators production, leading to cartilage degeneration and degradation. In depth mechanistic study showed that FSS results in more than 10-fold upregulation of H3K4me3, and the modulatory effect of H3K4me3 on cartilage was obtained by directly targeting ZBTB20. Furthermore, Wnt signaling was strongly activated in high FSS-induced OA pathogenesis, and the negative impact of ZBTB20 on chondrocytes was also achieved through activating Wnt signaling pathway. Moreover, pharmacological inhibition of H3K4me3 activation by MM-102 or treatment with Wnt pathway inhibitor LF3 could effectively alleviate the destructive effect of FSS on chondrocytes. In vivo UAC mice model validated the dysregulation of H3K4me3 and ZBTB20 in aberrant force-induced cartilage pathogenesis.ConclusionThrough the combination of in vitro FSS model and in vivo UAC model, KMT2B-H3K4me3-ZBTB20 axis was first identified in aberrant FSS-induced cartilage pathogenesis, which may provide evidences for epigenetic-based therapy in the future.

Protective Effects of Annatto Tocotrienol and Palm Tocotrienol-Rich Fraction on Chondrocytes Exposed to Monosodium Iodoacetate

Background: This study aimed to compare the chondroprotective efficacy and mechanism of annatto tocotrienol (AnTT) and palm tocotrienol-rich fraction (PT3) using SW1353 chondrocytes treated with monosodium iodoacetate (MIA). Methods: The chondrocytes were incubated with AnTT or PT3 in advance or concurrently with MIA for 24 h. The viability of the cells was tested with an MTT assay. The 8-isoprostane F2-α, extracellular matrix proteins, metalloproteinase and sex-determining region Y box protein 9 (SOX9) levels were determined using immunoassays. Results: AnTT and PT3 reversed an MIA-induced decrease in chondrocyte viability when incubated together with MIA (p &lt; 0.05). Prior incubation with both mixtures did not produce the same effects. AnTT and PT3 cotreatment could suppress 8-isoprostane F2-α level in chondrocytes exposed to MIA (p &lt; 0.01). Co-exposure to tocotrienols and MIA increased the type II collagen/type I collagen ratio in chondrocytes (p &lt; 0.01). In addition, the co-exposure of AnTT and MIA for 24 h significantly upregulated SOX9, type II collagen and aggrecan levels (p &lt; 0.05), which was not observed with co-exposure of PT3 and MIA, AnTT or PT3 exposure alone. Conclusion: AnTT and PT3 could prevent a reduction in chondrocyte viability following MIA exposure by reducing oxidative stress. In addition, AnTT might induce self-repair and anabolic activities in chondrocytes challenged with MIA.

In Vitro Effects of Bupivacaine on the Viability and Mechanics of Native and Engineered Cartilage Grafts

Background: Although the toxic effects of bupivacaine on chondrocyte monolayer culture have been well described, its cellular and mechanical effects on native and engineered articular cartilage remain unclear. For the repair of articular cartilage defects, fresh autologous and allogenic cartilage grafts are commonly used, and engineered cell-based therapies are emerging. The outcome of grafting therapies aimed at repairing damaged cartilage relies largely on maintaining proper viability and mechanical suitability of the donor tissues. Purpose: To investigate the in vitro effects of single bupivacaine exposure on the viability and mechanics of 2 cartilage graft types: native articular cartilage and engineered neocartilage. Study Design: Controlled laboratory study. Methods: Articular cartilage explants were harvested from the bovine stifle femoral condyles, and neocartilage constructs were engineered from bovine stifle chondrocytes using the self-assembling process, a scaffold-free approach to engineer cartilage tissue. Both explants and neocartilage were exposed to chondrogenic medium containing a clinically applicable bolus of 0.5%, 0.25%, or 0% (control) bupivacaine for 1 hour, followed by fresh medium wash and exchange. Cell viability and matrix content (collagen and glycosaminoglycan) were assessed at t = 24 hours after treatment, and compressive mechanical properties were assessed with creep indentation testing at t = 5 to 6 days after treatment. Results: Single bupivacaine exposure was chondrotoxic in both explants and neocartilage, with 0.5% bupivacaine causing a significant decrease in chondrocyte viability compared with the control condition (55.0% ± 13.4% vs 71.9% ± 13.5%; P < .001). Bupivacaine had no significant effect on matrix content for either tissue type. There was significant weakening of the mechanical properties in the neocartilage when treated with 0.5% bupivacaine compared with control, with decreased aggregate modulus (415.8 ± 155.1 vs 660.3 ± 145.8 kPa; P = .003), decreased shear modulus (143.2 ± 14.0 vs 266.5 ± 89.2 kPa; P = .002), and increased permeability (14.7 ± 8.1 vs 6.6 ± 1.7 × 10−15 m4/Ns; P = .009). Bupivacaine exposure did not have a significant effect on the mechanical properties of native cartilage explants. Conclusion: Single bupivacaine exposure resulted in significant chondrotoxicity in native explants and neocartilage and significant weakening of mechanical properties of neocartilage. The presence of abundant extracellular matrix does not appear to confer any additional resistance to the toxic effects of bupivacaine. Clinical Relevance: Clinicians should be judicious regarding the use of intra-articular bupivacaine in the setting of articular cartilage repair.

Exploring Penicillin G as an Intrawound Antibiotic Powder for Prevention of Postoperative Shoulder Infections: Does It Exhibit In Vitro Chondrotoxicity?

Cutibacterium acnes (C. acnes) is a significant insidious pathogen for postoperative infections in shoulder surgery. Studies have demonstrated that certain topical antibiotic powders used have the potential for chondrotoxicity. Benzylpenicillin, commonly referred to as Penicillin G (Pen G) has the lowest minimum inhibitory concentration (MIC) for C. acnes. There is no research regarding the topical application of Pen G during shoulder surgery, nor has its chondrocyte toxicity been previously investigated. This study sought to characterize the in vitro chondrocyte toxicity of Pen G. Culture-derived bovine chondrocytes were exposed to serial Pen G concentrations and compared with a positive and negative control. A negative control of growth medium and positive control of 1% Triton solution. The chondrocyte viability was assessed via spectrophotometer absorbance. The treatment groups were analyzed using one-way repeated measures analysis of variance and Pearson's correlation analysis. The chondrocyte viability was significantly higher for all Pen G concentrations as compared with the positive control (p < 0.001). All concentrations of Pen G exhibited continued chondrocyte metabolic activity over time. Analysis of variance, independent of time, demonstrated no significant decrease in chondrocyte viability for Pen G concentrations ≤6.25 mg/ml, as compared with the negative control (p > 0.05). Pen G demonstrated a significant negative correlation with its concentration and absorbance (r = 0.371, p < 0.001), however, concentrations ≤6.25 mg/ml did not demonstrate a significant decrease in chondrocyte viability (p = 0.063). Pen G in concentrations appropriate for C. acnes is not significantly chondrotoxic and may be safe for intrawound application. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:726-730, 2020.

Long noncoding RNA MALAT-1 inhibits apoptosis and matrix metabolism disorder in interleukin-1β-induced inflammation in articular chondrocytes via the JNK signaling pathway.

Proinflammatory cytokine such as interleukin (IL)-1β causes inflammation of articular cartilage. In this current study, we explored the chondroprotective effects of long noncoding RNA (lncRNA) MALAT-1 on cell proliferation, apoptosis, and matrix metabolism in IL-1β-induced inflammation in articular chondrocytes. Articular chondrocytes from knee joints of normal rats were isolated and cultured, followed by identification through observation of toluidine blue and COL II immunocytochemical stainings. The proliferation of chondrocytes at passage 2 was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The inflammatory chondrocytes induced by 10 ng/mL IL-1β were observed and identified by toluidine blue and COL II immunocytochemical stainings. pcDNA 3.1 and pcDNA-MALAT-1 were transfected in the chondrocytes. Ultrastructure of chondrocytes was observed by using a transmission electron microscope. The MTT assay was carried out to evaluate chondrocyte viability. Hoechst 33258 staining and flow cytometry were adopted to assess chondrocyte apoptosis. The chondrocytes at passage 2 with the biological characteristics of chondrocytes were used for subsequent experiments. In IL-1β-treated chondrocytes, the growth rate of chondrocytes slowed down, the cells became narrow and long, the vacuoles were seen in the cells, and the morphology of the chondrocytes was irregular. The toluidine blue staining and the immunohistochemical staining of COL II became weaker. In response to IL-1β induction, articular chondrocytes showed reduced MALAT-1 expression; moreover, obvious cartilage injury was observed with decreased chondrocyte viability and Col II expression and elevated chondrocyte apoptosis, MMP-13 expression, and p-JNK expression. With the treatment of pcDNA-MALAT-1, the cartilage injury was alleviated with increased chondrocyte viability and type II collagen (Col II) expression and reduced chondrocyte apoptosis, MMP-13 expression and p-JNK expression. Taken together these results, lncRNA MALAT-1 blocked the activation of the JNK signaling pathway; thereby, IL-1β-induced inflammation in articular chondrocytes was reduced with enhanced chondrocyte proliferation and suppressed chondrocyte apoptosis and extracellular matrix degradation.

The ghrelin paradox in the control of equine chondrocyte function: The good and the bad

Increasing evidence suggests a role for ghrelin in the control of articular inflammatory diseases like osteoarthritis (OA). In the present study we examined the ability of ghrelin to counteract LPS-induced necrosis and apoptosis of chondrocytes and the involvement of GH secretagogue receptor (GHS-R)1a in the protective action of ghrelin.The effects of ghrelin (10−7–10−11 mol/L) on equine primary cultured chondrocytes viability and necrosis in basal conditions and under LPS treatment (100 ng/ml) were detected by using both acridine orange/propidium iodide staining and annexin-5/propidium iodide staining. The presence of GHS-R1a on chondrocytes was detected by Western Blot. The involvement of the GHS-R1a in the ghrelin effect against LPS-induced cytotoxicity was examined by pretreating chondrocytes with D-Lys3-GHRP-6, a specific GHS-R1a antagonist, and by using des-acyl ghrelin (DAG, 10−7 and 10−9 mol/L) which did not recognize the GHS-R 1a.Low ghrelin concentrations reduced chondrocyte viability whereas 10−7 mol/L ghrelin protects against LPS-induced cellular damage. The protective effect of ghrelin depends on the interaction with the GHS-R1a since it is significantly reduced by D-Lys3-GHRP-6. The negative action of ghrelin involves caspase activation and could be due to an interaction with a GHS-R type different from the GHS-R1a recognized by both low ghrelin concentrations and DAG. DAG, in fact, induces a dose-dependent decrease in chondrocyte viability and exacerbates LPS-induced damage.These data indicate that ghrelin protects chondrocytes against LPS-induced damage via interaction with GHS-R1a and suggest the potential utility of local GHS-R1a agonist administration to treat articular inflammatory diseases such as OA.

Osteochondral Graft Size Is Significantly Associated With Increased Force and Decreased Chondrocyte Viability

Background: Insertion force has been shown to significantly reduce chondrocyte viability during osteochondral allograft transplantation. How graft size influences the required insertion force and chondrocyte viability has yet to be determined. Hypothesis/Purpose: The purpose was to characterize how graft size influences insertion force requirements and chondrocyte viability during osteochondral transplantation. The hypothesis was that larger grafts would require greater force and reduce chondrocyte viability. Study Design: Controlled laboratory study. Methods: Four graft sizes—15 × 5 mm, 15 × 10 mm, 25 × 5 mm, and 25 × 10 mm (diameter × depth)—were harvested from 13 thawed fresh-frozen human cadaveric distal femurs. Average, maximum, and cumulative force and number of impacts were recorded for 44 grafts by a surgical mallet embedded with a calibrated force sensor. In a separate experiment, fresh osteochondral tissues were subjected to mechanical loading. To capture a range of clinically important forces, categories were selected to correspond to impaction force data. Chondrocyte viability was assessed with confocal laser microscopy and live/dead staining. Results: Total force for all grafts averaged 4576 N. Median number of impacts for all grafts was 20 (range, 7-116). The mean number of impacts for 5-mm-deep grafts was 14.2 (95% CI, 10.8-18.6), as compared with 26.3 (95% CI, 19.9-34.4) for 10-mm-deep grafts (P < .001). The mean cumulative force for 5-mm-deep grafts was 2128 N (95% CI, 1467-3087), as opposed to 4689 N (95% CI, 3232-6803) for 10-mm-deep grafts (P = .001). For every 1 mm in graft depth, an average of 13.1% (95% CI, 6.2%-20.3%) more impacts are required when controlling for diameter and density (P < .001). For every 1 mm in graft depth, the force required increases on average by 17.1% (95% CI, 7.7%-27.4%) when controlling for diameter and density (P = .001). There was a significant reduction in chondrocyte viability for the forces required for graft thickness values >10 mm. Only forces associated with graft thickness <10 mm had chondrocyte viabilities consistently >70%. Conclusion: Insertion force increases significantly with increasing graft depth. Controlling for diameter and bone density, a 1-mm increase in graft depth is associated with 13.1% more impacts and 17.1% more force. Chondrocyte viability was significantly reduced to <70% at average forces associated with grafts thicker than 10 mm. Clinical Relevance: Based on the current data, graft depth is an important consideration for surgeons when sizing osteochondral allograft transplant for chondral lesions of the knee.

Improved Chondrotoxic Profile of Liposomal Bupivacaine Compared With Standard Bupivacaine After Intra-articular Infiltration in a Porcine Model

Background: Increasingly, liposomal bupivacaine is being used with multimodal pain management strategies. In vitro investigations have shown decreased chondrotoxicity profiles for liposomal bupivacaine; however, there is no evidence regarding its in vivo effects. Hypothesis/Purpose: This study sought to investigate the in vivo chondrotoxicity of liposomal bupivacaine, hypothesizing that there would be increased chondrocyte viability after exposure to liposomal bupivacaine when compared with standard bupivacaine. Study Design: Controlled laboratory study. Methods: Eight juvenile, female Yorkshire cross piglets underwent a lateral stifle joint injection with either 1.3% liposomal bupivacaine or 0.5% bupivacaine. Injections were performed on one joint per animal with no injection to the contralateral knee, which served as the control. Chondrocyte viability was assessed 1 week after injection with a live-dead staining protocol and histologic examination. Results: Significant chondrocyte death was seen with the live-dead staining in the bupivacaine group (33% nonviable cells) in comparison with liposomal bupivacaine (6.2%) and control (5.8%) groups (P < .01). However, histologic examination showed no differences in chondral surface integrity, fibrillation, and chondrocyte viability. Conclusion: Liposomal bupivacaine was found to be safe for intra-articular injection in this animal model. Although bupivacaine demonstrated decreased chondrocyte viability on a cellular level, histologically there were no changes. This study highlights the dichotomy between fluorescent staining and histologic appearance of articular chondrocytes in short-term analyses of viability. Clinical Relevance: This study supports the peri-articular application of liposomal bupivacaine in the setting of preserved articular cartilage. A single injection of standard bupivacaine did not produce histologic changes in the articular cartilage.