Chondrocyte Cell Cultures Research Articles

Hyaluronic acid and multiwalled carbon nanotubes as bioink additives for cartilage tissue engineering

Articular cartilage and meniscus injuries are prevalent disorders with insufficient regeneration responses offered by available treatment methods. In this regard, 3D bioprinting has emerged as one of the most promising new technologies, offering novel treatment options. Additionally, the latest achievements from the fields of biomaterials and tissue engineering research identified constituents facilitating the creation of biocompatible scaffolds. In this study, we looked closer at hyaluronic acid and multi-walled carbon nanotubes as bioink additives. Firstly, we assessed the minimal concentrations that stimulate cell viability, and decrease reactive oxygen species and apoptosis levels in 2D cell cultures of normal human knee articular chondrocytes (NHAC) and human adipose-derived mesenchymal stem cells (hMSC-AT). In this regard, 0.25 mg/ml of hyaluronic acid and 0.0625 mg/ml of carbon nanotubes were selected as the most optimal concentrations. In addition, we investigated the protective influence of 2-phospho-L-ascorbic acid in samples with carbon nanotubes. Tests conducted on 3D bioprinted constructs revealed that only a combination of components positively impacted cell viability throughout the whole experiment. Gene expression analysis of COL1A1, COL6A1, HIF1A, COMP, RUNX2, and POU5F1 showed significant changes in the expression of all analyzed genes with a progressive overall loss of transcriptional activity in most of them.

Mesenchymal stromal cells donate mitochondria to articular chondrocytes exposed to mitochondrial, environmental, and mechanical stress

Articular cartilage has limited healing capacity and no drugs are available that can prevent or slow the development of osteoarthritis (OA) after joint injury. Mesenchymal stromal cell (MSC)-based regenerative therapies for OA are increasingly common, but questions regarding their mechanisms of action remain. Our group recently reported that although cartilage is avascular and relatively metabolically quiescent, injury induces chondrocyte mitochondrial dysfunction, driving cartilage degradation and OA. MSCs are known to rescue injured cells and improve healing by donating healthy mitochondria in highly metabolic tissues, but mitochondrial transfer has not been investigated in cartilage. Here, we demonstrate that MSCs transfer mitochondria to stressed chondrocytes in cell culture and in injured cartilage tissue. Conditions known to induce chondrocyte mitochondrial dysfunction, including stimulation with rotenone/antimycin and hyperoxia, increased transfer. MSC-chondrocyte mitochondrial transfer was blocked by non-specific and specific (connexin-43) gap-junction inhibition. When exposed to mechanically injured cartilage, MSCs localized to areas of matrix damage and extended cellular processes deep into microcracks, delivering mitochondria to chondrocytes. This work provides insights into the chemical, environmental, and mechanical conditions that can elicit MSC-chondrocyte mitochondrial transfer in vitro and in situ, and our findings suggest a new potential role for MSC-based therapeutics after cartilage injury.

Selected physico-chemical properties of composite scaffolds of sintered submicrocrystalline corundum and bioglass

Presented paper contains description and interpretation of the results of selected physicochemical and structural properties of two types of composite sinters. They were constituted of a mixture of sintered microcrystalline corundum and bioglass CaO-P2O5-SiO2-Na2O system intended for scaffolds to cell culture of human chondrocytes. The composites contained a mixture of both above-mentioned components in the volumetric proportion of 50:50 (W5) and 30:70 (W7). They were obtained using powder metallurgy by free sintering in air atmosphere. Phase analysis of composites and verification of theoretical identification using X-ray diffraction were performed. The same phases were found in both cases (Al2O3 SiO2 CaAl2Si2O8, Ca3 (PO4)2, Ca2Al4O7 and NaAlSiO4). Microscopic tests of composite surfaces were performed and some differences were found. W5 sample was not completely covered with bioglass, whilst W7 sample was completely covered with bioglass with few fine pores. Tests of surface topography confirmed the presence of large and small pores. Composite surfaces immersed for 30 days in artificial blood plasma were tested and then electron microscopy analysis was performed. It was found that no significant changes occurred on the surface of the W5 composite, probably partial corrosion of the glass happened. Spherical forms characteristic of HA-hydroxyapatites were observed on the surface of sample W7. Human articular chondrocyte cells were seeded on both types of sinters and proliferation assay was performed. Results indicate that tested scaffolds support cellular attachment and proliferation of chondrocytes.

Effect of intermittent hydrostatic pressure on aging human chondrocyte cells.

In this study, a hydrostatic pressure chamber (HPC) is designed and developed to host chondrocyte cell culture. External stimuli such as forces, pressure, vibration etc. are found to be significant factors on upregulating the relevant proteins for constructing the extracellular matrix (ECM) during culturing. The aim of this paper is to design a system which provides external stimuli during chondrocyte cell culture as well as to discover the relevant gene which can generate the repair and regeneration of aging cells. The system consists of a controllable HPC that provides intermittent hydrostatic pressure (HP) on the cultured cells. The chamber is capable of applying intermittent HP in the range: 0 to1 MPa, at a frequency of 0.5-1Hz. An investigation was undertaken to determine the improvement of human chondrocyte cells viz. of 3 sub-jects whose ages are 60 and above. The effect of HP on the aging cells is observed through the extracted ribonucleic acid (RNA) after the cell is treated with HP for two hours, each day, over four days. The experiments were conducted to observe the effect of HP on the level of collagen type I, collagen type II, and aggrecan. Results show that HP did little to help in upregulating the aggrecan and collagen type II in aged-chondrocyte cells. Further, it was found that the application of HP depended on the number of days applied. The results presented the possibility of ap-plying HP in regeneration of damaged cartilage in elderly.

Effects of nanopatterned-surface dishes on chondrocyte growth and cell cycle progression.

Discovering and developing ideal cell culture methods is important for cell biology, drug development, and cell therapy. Recent studies have explored and demonstrated the use of nanoscale structures and patterns that influence cell behavior, such as 3D scaffolds. In this study, we analyzed the effects of nanopatterned-surface dishes using chondrocytes as model cells. Chondrocytes grown on nanopatterned dishes exhibited rounded shapes. Interestingly, chondrocytes have a lower COL10 mRNA level when cultured using nanopatterned dishes. The nanopatterned dishes induced G0-/G1-phase cell cycle arrest and reduced the rate of proliferation. Our results suggest that nanoscale structures can directly control cellular behaviors and can be used for chondrocyte cell culture without causing chondrocytes to lose their functions. These results help to elucidate cellular responses and behaviors in native-like environments, and this information can be used to improve human health.

New “Tribo-bioreactor” for In-situ Monitoring of the Mechanical Stress Transmission at the Cellular Level: Application to Cartilage Tissue Engineering

The mechanotransduction is an important aspect to provide suitable conditions for the cartilage engineering process. Therefore, a new bioreactor was developed to apply different mechanical stresses to cell/organ culture. It allows performing cell culture in situ with appropriate mechanical constraints according to the real-time evolution of the physical parameters with the possibility of microscopic observation. In addition to the two-dimensional mechanical stimulation of a scaffold, the originality of this device is in situ monitoring of the cells and rheological measurements. This study presents the design of the new prototype with the validation of its different functions. First, the mechanical characterization of different non-biological samples is performed to calibrate the rheological measurement. Then, the monitoring of fluorescent beads in different scaffolds is carried out to estimate the mechanical stress transmitted to the cells. Finally, the ability of the device to handle a cell culture of human chondrocytes seeded in a scaffold is evaluated. The combined functions of the new bioreactor open the possibility to develop new scaffolds with optimal mechanical stress transmission, which is the basis of tissue engineering of cartilage.

Investigation of the effects of tigecycline, a semi-synthetic derivative of minocycline, on chondrocyte cultures

Aim: Although antibiotics are generally well-tolerated, they may have cytotoxic effects. The present randomized, double-blind, in vitro study aimed to investigate the effects of tigecycline on cartilage tissue cells and the extracellular matrix. Material and Methods: Cartilage tissues of patients (n = 8) were used for the preparation of primary cell cultures. Tigecycline-treated cell cultures served as the study group. Non-treated cell cultures served as the control group. Analyses were performed at 0, 24, 48, and 72 h in both groups. The results obtained were statistically evaluated. The alpha significance value was determined to be 0.05. Results: Proliferation remained unchanged in the tigecycline-treated cell cultures. The gene expressions of the markers involved in anabolic pathways increased in the tigecycline-treated cell cultures. The results obtained were statistically significant (P < 0.05). Conclusion: Although tigecycline had no toxic effect on the chondrocyte cell cultures and caused no damage to the extracellular matrix, the present study was performed in an in vitro environment.

Hyaluronic Acid (HA), Platelet-Rich Plasm and Extracorporeal Shock Wave Therapy (ESWT) promote human chondrocyte regeneration in vitro and ESWT-mediated increase of CD44 expression enhances their susceptibility to HA treatment.

Novel strategies have been proposed for articular cartilage damage occurring during osteoarthritis (OA) and -among these- Extracorporeal Shock Wave Therapy (ESWT), intra-articular injections of Platelet-Rich Plasma (PRP) or Hyaluronic Acid (HA) revealed encouraging results. To investigate the possible mechanisms responsible for those clinical benefits, we established primary cultures of human chondrocytes derived from cartilage explants and measured the in vitro effects of ESW, PRP and HA therapies. After molecular/morphological cell characterization, we assessed those effects on the functional activities of the chondrocyte cell cultures, at the protein and molecular levels. ESWT significantly prevented the progressive dedifferentiation that spontaneously occurs during prolonged chondrocyte culture. We then attested the efficiency of all such treatments to stimulate the expression of markers of chondrogenic potential such as SOX9 and COL2A, to increase the Ki67 proliferation index as well as to antagonize the traditional marker of chondrosenescence p16INK4a (known as Cdkn2a). Furthermore, all our samples showed an ESW- and HA-mediated enhancement of migratory and anti-inflammatory activity onto the cytokine-rich environment characterizing OA. Taken together, those results suggest a regenerative effect of such therapies on primary human chondrocytes in vitro. Moreover, we also show for the first time that ESW treatment induces the surface expression of major hyaluronan cell receptor CD44 allowing the increase of COL2A/COL1A ratio upon HA administration. Therefore, this work suggests that ESW-induced CD44 overexpression enhances the in vitro cell susceptibility of human chondrocytes to HA, presumably favouring the repair of degenerated cartilage.

A Novel Regulatory Role of TRAPPC9 in Osteoarthritis

INTRODUCTIONTrafficking Protein Particle Complex 9 (TRAPPC9) is a major subunit of TRAPPII Complex. TRAPPC9 is implicated in endoplasmic to Golgi trafficking pathway and intra Golgi trafficking in yeast cells. TRAPPC9 has been reported to bind IKKb and NIK where it plays a role in NFkB signaling. Hence, plays a role in different cell types differentiation and function. In osteoarthritis, inflammation mediated by the NFkB signaling plays a role in the pathogenesis on the disease. There are two distinct pathways, which can activate the NF‐kB signaling cascades. The first one termed the canonical or the classical pathway, activation of IKKα/IKKβ/IKKβ‐NEMO complex will activate this pathway. On the other hand, the non‐canonical or the alternative pathway, activation relies on NF‐kB‐inducing kinase (NIK) signaling. Our data showed that TRAPPC9 binds IKKβ and NIK in chondrocytes. We believe such interaction could lead to a potential regulatory role of TRAPPC9 during NF‐kB signaling cascades given that the role of TRAPPC9 in OA has not been studied yet.METHODSIn this study, we used human chondrocytes and primary mouse chondrocyte cell culture, immunofluorescence, and retroviral transfection techniques.RESULTSTo explore the role of TRAPPC9 in OA, first, we assessed the expression of TRAPPC9 (mRNA and protein) levels in primary human chondrocytes treated with ILb to resemble chondrocytes within OA knee and found that TRAPPC9 expression is decreased in IL‐1β‐treated chondrocytes compared to controls. Next, we determined whether modulation of TRAPPC9 expression alters the inflammatory markers induced by IL1b that are regulated by NF‐kB signaling pathways. Human chondrocytes were transfected with either TRAPPC9 overexpression plasmid or control plasmid. Gene expression for metalloproteinases (what else a part of MMP) was measured and our data showed that TRAPPC9 overexpression induces inflammation by increasing the gene expression of MMPs‐1,3,9,13, and Interlukin‐6 (IL‐6).CONCLUSIONWe showed that TRAPPC9 binds/interacts with NIK and IKKb in chondrocytes. Also, we were able to show that TRAPPC9 expression is decreased in inflammatory conditions. In addition, our data showed that modulating TRAPPC9 expression prior to IL‐1β induction leads to elevate inflammatory conditions in chondrocytes. Studies are underway to determine the effects of modulating TRAPPC9 using genetic and pharmacologic approaches on osteoarthritis development and identifying the mechanism(s) by which inflammation in chondrocytes mediated by TRAPPC9, NIK, and IKKb interactions. Collectively, these studies are the first to report a novel role for TRAPPC9 in osteoarthritis.SIGNIFICANCE/CLINICAL RELEVANCEUnderstanding the role of TRAPPC9 in osteoarthritis are not reported previously. It is important to investigate whether TRAPPC9, NIK and IKKb interaction mediates inflammation in chondrocytes by modulating the NF‐kB signaling pathways thereby, suggesting new therapeutics for osteoarthritis disease.Support or Funding InformationKent State University, School of Biomedical Sciences. Ohio Physiological Society (Peter K Lauf Travel Award).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of Mechanical Properties and Cell Viability of Poly (3-Hydroxybutyrate)-Chitosan/Al2O3 Nanocomposite Scaffold for Cartilage Tissue Engineering.

Background:The aim of this study was to evaluate the effects of alumina nanowires as reinforcement phases in polyhydroxybutyrate-chitosan (PHB-CTS) scaffolds to apply in cartilage tissue engineering.Methods:A certain proportion of polymers and alumina was chosen. After optimization of electrospun parameters, PHB, PHB-CTS, and PHB-CTS/3% Al2O3 nanocomposite scaffolds were randomly electrospun. Scanning electron microscopy, Fourier transform infrared spectroscopy, water contact angle measurement, tensile strength, and chondrocyte cell culture studies were used to evaluate the physical, mechanical, and biological properties of the scaffolds.Results:The average fiber diameter of scaffolds was 300–550 nm and the porosity percentages for the first layer of all types of scaffolds were more than 81%. Scaffolds' hydrophilicity was increased by adding alumina and CTS. The tensile strength of scaffolds decreased by adding CTS and increased up to more than 10 folds after adding alumina. Chondrocyte viability and proliferation on scaffolds were better after adding CTS and alumina to PHB.Conclusion:With regard to the results, electrospun PHB-CTS/3% Al2O3 scaffold has the appropriate potential to apply in cartilage tissue engineering.

Concentrated adipose tissue infusion for the treatment of knee osteoarthritis: clinical and histological observations.

Osteoarthritis (OA) is characterized by articular cartilage degeneration and subchondral bone sclerosis. OA can benefit of non-surgical treatments with collagenase-isolated stromal vascular fraction (SVF) or cultured-expanded mesenchymal stem cells (ASCs). To avoid high manipulation of the lipoaspirate needed to obtain ASCs and SVF, we investigated whether articular infusions of autologous concentrated adipose tissue are an effective treatment for knee OA patients. The knee of 20 OA patients was intra-articularly injected with autologous concentrated adipose tissue, obtained after centrifugation of lipoaspirate. Patients' articular functionality and pain were evaluated by VAS and WOMAC scores at three, six and 18months from infusion. The osteogenic and chondrogenic ability of ASCs contained in the injected adipose tissue was studied in in vitro primary osteoblast and chondrocyte cell cultures, also plated on 3D-bone scaffold. Knee articular biopsies of patients previously treated with adipose tissue were analyzed. Immunohistochemistry (IHC) and scanning electron microscopy (SEM) were performed to detect cell differentiation and tissue regeneration. The treatment resulted safe, and all patients reported an improvement in terms of pain reduction and increase of function. According to the osteogenic or chondrogenic stimulation, ASCs expressed alkaline phosphatase or aggrecan, respectively. The presence of a layer of newly formed tissue was visualized by IHC staining and SEM. The biopsy of previously treated knee joints showed new tissue formation, starting from the bone side of the osteochondral lesion. Overall our data indicate that adipose tissue infusion stimulates tissue regeneration and might be considered a safe treatment for knee OA.

Comparison of botulinum toxin type A and Rho-inhibitor on ATDC5 chondrocyte

Knee osteoarthritis (OA) has an incidence rate of 25% per year and its responses to treatment are variable. The analgesic effect of intra-articular injection of botulinum toxin type A (BTX-A) has been observed in patients with knee OA. BTX-A might inhibit Rho GTPase by ADP-ribosylation of amino acid asparagine-41. Rho activity has a role in mediation of chondrocyte catabolic signaling pathways, and Rho/ROCK (Rho-associated coiled-coil containing kinase) activation induces articular cartilage degradation. In our unpublished data, chondroprotection of BTX-A was observed based on pathological findings in rat knee OA. ROCK I & II were found to be altered in chondrocytes treated with BTXA, with dose-dependent effect. This study aimed to compare the effect on chondrocyte of BTX-A and Rho-inhibitor. ATDC5 chondrocytes were compared based on cell phenotype and markers for chondrocyte differentiation and growth after administration of BTX-A, Rho-inhibitor (Y27632), and normal saline (control) to clarify the downstream signaling pathway. Reverse transcription-PCR (RT-PCR) showed that in ATDC5 chondrocyte cell culture, Y27632 and BTX-A elevated aggrecan on culture day 4 and day 7. On day 21, the level of collagen type X and MMP13 were lowered in Y27632 and BTX-A. ROCK I and II level were decreased on day 4 and 7 in all three groups, but were remarkably higher on day 14 and 21 in Y27632. On day 8, in ATDC5 cells not pretreated with insulin, decreased cell density, small and flat chondrocyte morphology were observed after addition of Y27632 and BTX-A, in comparison to control. BTX-A and Rho-inhibitor decreased ROCK I & II and increased aggrecan level during early stage, and decreased collagen type X and MMP13 during late stage. Potential dedifferentiation effect in BTX-A and Rho-inhibitor were observed as well. Rho-signaling pathway might play different roles during early and late stage in chondrocyte growth.

Iopromide- and gadopentetic acid-derived preparates used in MR arthrography may be harmful to chondrocytes

BackgroundMagnetic resonance arthrography, a procedure through which contrast agents containing gadolinium and/or iopromide are administered intra-articularly, has become a useful tool in musculoskeletal diagnosis. Nevertheless, despite being considered safe for systemic use, certain tissue toxicities have been identified for both drugs. In this study, the effects of short-term exposure of human primary chondrocyte cell cultures to gadolinium and/or iopromide contrast agents were examined by assaying for stage-specific embryonic antigen-1 (SSEA-1) protein expression (a chondrogenic differentiation marker), cell viability, toxicity, and proliferation.MethodsHuman articular chondrocytes were grown in monolayer culture and were exposed to iopromide and/or gadolinium diethylenetriamine-pentaacetate (Gd-DPT) for 2 and 6 h. Cell cultures with no drug exposure were used as the control group. Cell differentiation status was assessed according to SSEA-1 protein expression. Contrast agent effects on cell viability and proliferation were analyzed using MTT analysis. Further, changes in cell morphology in relation to the control group were evaluated using inverted light microscopy, environmental scanning electron microscopy (ESEM), and 3-tesla magnetic resonance imaging. The obtained data were statistically compared.ResultsWhen compared with the control group, both SSEA-1 protein expression and cell proliferation were lowest in the Gd-DPT group (P = 0.000). There was a statistically significant correlation between SSEA-1 expression and MTT results (rho = 0.351; P = 0.003).ConclusionsNevertheless, the data obtained from in vitro experiments may not directly correspond to clinical applications. However, the mere fact that a drug used solely for diagnostic purposes may repress chondrocyte cell proliferation should be carefully considered by clinicians.

Physiological concentrations of soluble uric acid are chondroprotective and anti-inflammatory

High uric acid levels are a risk factor for cardiovascular disorders and gout; however, the role of physiological concentrations of soluble uric acid (sUA) is poorly understood. This study aimed to clarify the effects of sUA in joint inflammation. Both cell cultures of primary porcine chondrocytes and mice with collagen-induced arthritis (CIA) were examined. We showed that sUA inhibited TNF-α- and interleukin (IL)-1β–induced inducible nitric oxide synthase, cyclooxygenase-2 and matrix metalloproteinase (MMP)-13 expression. Examination of the mRNA expression of several MMPs and aggrecanases confirmed that sUA exerts chondroprotective effects by inhibiting the activity of many chondro-destructive enzymes. These effects attenuated collagen II loss in chondrocytes and reduced proteoglycan degradation in cartilage explants. These results were reproduced in chondrocytes cultured in three-dimensional (3-D) alginate beads. Molecular studies revealed that sUA inhibited the ERK/AP-1 signalling pathway, but not the IκBα-NF-κB signalling pathway. Increases in plasma uric acid levels facilitated by the provision of oxonic acid, a uricase inhibitor, to CIA mice exerted both anti-inflammatory and arthroprotective effects in these animals, as demonstrated by their arthritis severity scores and immunohistochemical analysis results. Our study demonstrated that physiological concentrations of sUA displayed anti-inflammatory and chondroprotective effects both in vitro and in vivo.

Regulation of type II collagen, matrix metalloproteinase-13 and cell proliferation by interleukin-1β is mediated by curcumin via inhibition of NF-κB signaling in rat chondrocytes.

Curcumin possesses strong anti-inflammatory, anti-rheumatoid and anti-oxidative activities, and has the potential to inhibit nuclear factor-κB (NF-κB) signaling. Cartilage damage in osteoarthritis (OA) is largely mediated by interleukin-1β (IL-1β) via activation of various transcription factors, including NF-κB and activator protein-1. The aim of the present study was to determine whether IL-1β induces matrix metalloproteinase-13 (MMP-13) expression and inhibits type II collagen expression, as well as to examine whether cell proliferation may be inhibited by curcumin through the inhibition of NF-κB signaling. The effects of curcumin were investigated in rat articular chondrocyte cell cultures treated with IL-1β in the presence or absence of curcumin or the NF-κB inhibitor pyrrolidine dithiocarbamate. Western blotting and reverse transcription-quantitative polymerase chain reaction were conducted to evaluate protein and mRNA expression levels of type II collagen, MMP-13, NF-κB inhibitor α (IκBα), phosphorylated-IκBα and NF-κB subunit p65/RelA. Western blotting and immunofluorescence were performed to examine the effects of curcumin on the expression, phosphorylation and nuclear translocation of NF-κB-associated proteins. The effects of curcumin on cell proliferation were evaluated by Cell Counting Kit-8 (CCK-8). Curcumin was demonstrated to inhibit the IL-1β-induced activation of NF-κB by suppressing IκBα phosphorylation and p65/RelA nuclear translocation. These events were associated with the downregulation of MMP-13 expression and the upregulation of type II collagen expression, both of which are considered to be NF-κB targets. CCK-8 assays revealed that co-treatment with curcumin resulted in increased proliferation in IL-1β-treated chondrocytes. These findings implicated curcumin as a naturally occurring anti-inflammatory agent for the treatment of OA via inhibition of NF-κB signaling.

Development of tracheal reconstruction methods from scaffold engineering to injectable matrix

Backgrand: For patients with long segment of tracheal stenosis, resection and replacement is necessary. Tracheal reconstruction associated with complications such as stenosis, insufficiency of blood supply and surgical complication. Methods: In this study, we prepared collagen-chitosan scaffold and sheep's decellularized trachea plus culture medium with nanocup contain growth factors for chondrocyte and fibroblast-epithelial cell culture in rotary bioreactor. After attachment of cells, engineered trachea put into the omentum. Peritoneal stem cell interact with the epithelial and chondrocytes with attendance of growth factors released from nanocup. The bioengineered trachea with omental pedicle transposed from behind the sternum and transplanted in the position of resected trachea. In third experiment, we introduce the in situ tracheal repair technique with injectable matrix for reconstruction of long-segmental stenosis of trachea in a 29-year-old woman. Results: Severe tortuosity in the first experiment and mild stenosis was seen in the second experiment. In third experiment, normal shape in tracheal diameter was seen at injection sites. The interior portion of the trachea at virtual computed tomography (CT) scan and bronchoscopy were similarity to normal after four month fallowing up of injection. Conclusions: Severe tortuosity, mild stenosis in whole length of trachea in this study and disadvantage of trachea transplantation include open surgery, fatal consequences of anastomosis leakage, and rupture besides large mediastinal vessels; hence, we decided to introduce the novel in situ tracheal repair technique. Injectable bioresorbable scaffolds may be used as a temporary scaffolding for transplanted cells and thereby allow the cells to secrete extracellular matrix of their own to enable, in the long term, a complete and natural tissue replacement.

In vitro chondrocyte toxicity following long-term, high-dose exposure to Gd-DTPA and a novel cartilage-targeted MR contrast agent.

To determine the concentrations exhibiting toxicity of a cartilage-targeted magnetic resonance imaging contrast agent compared with gadopentetate dimeglumine (Gd-DT-PA) in chondrocyte cultures. A long-term Swarm rat chondrosarcoma chondrocyte-like cell line was exposed for 48h to 1.0-20mM concentrations of diaminobutyl-linked nitroxide (DAB4-DLN) citrate, 1.0-20mM Gd-DTPA, 1.0μM staurosporine (positive control), or left untreated. Cell appearance, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays of metabolic activity, quantitative PicoGreen assays of DNA content, and calcein-AM viability assays were compared. At 1.0-7.5mM, minimal decrease in cell proliferation was found for both agents. At all doses of both agents, cell culture appearances were similar after 24h of treatment. At the higher doses, differences in cell culture appearance were found after 48h of treatment, with dose-dependent declines in chondrocyte populations for both agents. Concentration-dependent declines in DNA content and calcein fluorescence were found after 48h of treatment, but beginning at a lower dose of DAB4-DLN citrate than Gd-DTPA. Dose-dependent decreases in MTT staining (cell metabolism) were apparent for both agents, but larger effects were evident at a lower dose for DAB-DLN citrate. Poor MTT staining of cells exposed for 48h to 20mM DAB4-DLN citrate probably indicates dead or dying cells. The minimal effect of the long-term exposure of model chondrocyte cell cultures to DAB4-DLN citrate and Gd-DTPA concentrations up to 7.5mM (3x typical arthrographic administration) is supporting evidence that these doses are acceptable for MR arthrography. The findings are reassuring given that the experimental exposure to the contrast agents at sustained concentrations was much longer than when used clinically.

Effects of platelet-derived growth factor on chondrocyte proliferation, migration and apoptosis via regulation of GIT1 expression.

The formation of fibrocartilage, cartilaginous and bony calluses is vital for bone healing following a fracture. Fibroblasts, chondrocytes and osteoblasts are critical functional cells that are involved in these three processes, respectively. Platelet‑derived growth factor (PDGF), a growth factor that is released from platelet particles and appears during the early stages at the site of fractures, is essential in bone healing via regulation of cell proliferation and differentiation. However, the effects of PDGF on the chondrocytes remain unclear. In the present study, PDGF promoted phosphorylation of Src and upregulated the expression level of G‑protein‑coupled receptor kinase interacting protein‑1 (GIT1) according to the results of the cell culture of chondrocytes invitro and western blotting. However, the effect of PDGF on the upregulation of GIT1 expression was mostly inhibited by the Src inhibitor, PP2. After knocking down GIT1 expression using siRNA, the phosphorylation of Src continued to be induced by PDGF, although the expression of GIT1 was inhibited. Furthermore, the results indicated that PDGF promoted chondrocyte proliferation and migration, however, the effect on cell apoptosis induction was suppressed after adding the Src inhibitor, PP2. Additionally, when knocking down GIT1 using siRNA, the expression level of GIT1 decreased, which is similar to the effect of the Src inhibitor, PP2. The current study demonstrates that PDGF may initially activate the phosphorylation of Src, and subsequently induce GIT1 expression to promote chondrocyte proliferation and migration, but suppress cell apoptosis.

Expression of the semicarbazide-sensitive amine oxidase in articular cartilage: its role in terminal differentiation of chondrocytes in rat and human

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the oxidation of primary amines into ammonia and reactive species (hydrogen peroxide, aldehydes). It is highly expressed in mammalian tissues, especially in vascular smooth muscle cells and adipocytes, where it plays a role in cell differentiation and glucose transport. The study aims at characterizing the expression and the activity of SSAO in rat and human articular cartilage of the knee, and to investigate its potential role in chondrocyte terminal differentiation. SSAO expression was examined by immunohistochemistry and western blot. Enzyme activity was measured using radiolabeled benzylamine as a substrate. Primary cell cultures of rat chondrocytes were treated for 21 days by a specific SSAO inhibitor, LJP 1586. Terminal chondrocyte differentiation markers were quantified by RT-qPCR. The basal and IL1β-stimulated glucose transport was monitored by the entrance of (3)[H]2-deoxyglucose in chondrocytes. SSAO was expressed in chondrocytes of rat and human articular cartilage. SSAO expression was significantly enhanced during the hypertrophic differentiation of chondrocytes characterized by an increase in MMP13 and in alkaline phosphatase (ALP) expressions. SSAO inhibition delayed the late stage of chondrocyte differentiation without cell survival alteration and diminished the basal and IL1β-stimulated glucose transport. Interestingly, SSAO activity was strongly increased in human osteoarthritic cartilage. SSAO was expressed as an active form in rat and human cartilage. The results suggest the involvement of SSAO in rat chondrocyte terminal differentiation via a modulation of the glucose transport. In man, the increased SSAO activity detected in osteoarthritic patients may trigger hypertrophy and cartilage degeneration.

Enhanced chondrocyte culture and growth on biologically inspired nanofibrous cell culture dishes.

Chondral and osteochondral defects affect a large number of people in which treatment options are currently limited. Due to its ability to mimic the natural nanofibrous structure of cartilage, this current in vitro study aimed at introducing a new scaffold, called XanoMatrix™, for cartilage regeneration. In addition, this same scaffold is introduced here as a new substrate onto which to study chondrocyte functions. Current studies on chondrocyte functions are limited due to nonbiologically inspired cell culture substrates. With its polyethylene terephthalate and cellulose acetate composition, good mechanical properties and nanofibrous structure resembling an extracellular matrix, XanoMatrix offers an ideal surface for chondrocyte growth and proliferation. This current study demonstrated that the XanoMatrix scaffolds promote chondrocyte growth and proliferation as compared with the Corning and Falcon surfaces normally used for chondrocyte cell culture. The XanoMatrix scaffolds also have greater hydrophobicity, three-dimensional surface area, and greater tensile strength, making them ideal candidates for alternative treatment options for chondral and osteochondral defects as well as cell culture substrates to study chondrocyte functions.