Cultured Bovine Articular Chondrocytes Research Articles

3D culture of bovine articular chondrocytes in viscous medium encapsulated in agarose hydrogels for investigation of viscosity influence on cell functions.

The mechanical properties of an extracellular microenvironment can affect cell functions. The effects of elasticity and viscoelasticity on cell functions have been extensively studied with hydrogels of tunable mechanical properties. However, investigation of the viscosity effect on cell functions is still very limited and it can be tricky to explore how viscosity affects cells in three-dimensional (3D) culture due to the lack of appropriate tools. In this study, agarose hydrogel containers were prepared and used to encapsulate viscous media for 3D cell culture to investigate the viscosity effect on the functions of bovine articular chondrocytes (BACs). Polyethylene glycol of different molecular weights was used to adjust culture medium viscosity in a large range (72.8-679.2 mPa s). The viscosity affected gene expression and secretion of cartilagenious matrices, while it did not affect BAC proliferation. The BACs cultured in the lower viscosity medium (72.8 mPa s) showed a higher level of cartilaginous gene expression and matrix secretion.

Cyclic compressive loading activates angiotensin II type 1 receptor in articular chondrocytes and stimulates hypertrophic differentiation through a G-protein-dependent pathway.

Angiotensin II type 1 receptor (AT1R) appears to have a mechanosensing function in a number of cell types. The purpose of this study was to examine whether AT1R expressed in articular chondrocytes is involved in osteoarthritis (OA) progression in vivo and whether cyclic compressive loading activates the AT1R and stimulates hypertrophic differentiation of chondrocytes in vitro. The relationships between the modified Mankin score for cartilage degeneration and the expression of AT1R and type X collagen (Col X) were studied in mouse knees with OA induced using the destabilization‐of‐medial‐meniscus model. Cyclic compressive loads were applied to cultured bovine articular chondrocytes in three‐dimensional agarose scaffolds. Expression of Col X and runt‐related transcription factor 2 (Runx2) was analyzed using RT‐PCR and western blotting. We dissected the downstream pathway for intracellular signal transductions of AT1R including G‐protein‐dependent and G‐protein‐independent pathways. Positive significant correlations between the Mankin score and the rate of AT1R‐immunopositive cells and between the rates of AT1R and Col X expression were noted. The expression of Col X and Runx2 was increased by compressive loading but suppressed by addition of olmesartan, an Ang II receptor blocker, to the agarose scaffolds. Compressive loading upregulated the phosphorylation of c‐Jun N‐terminal kinase (JNK), Src, and STAT1, but olmesartan significantly suppressed only JNK phosphorylation. We conclude that AT1R expressed by articular chondrocytes may be involved in OA progression in vivo. Mechanical stress can activate AT1R and stimulate hypertrophic differentiation of chondrocytes through the G‐protein‐dependent pathway. AT1R has a mechanosensing function in chondrocytes and may be a new therapeutic target in OA.

Fabrication of Highly Crosslinked Gelatin Hydrogel and Its Influence on Chondrocyte Proliferation and Phenotype.

Gelatin methacrylate (GelMA) hydrogels have been widely studied for biomedical applications, such as tissue engineering and drug delivery, because of their good biocompatibility and injectability. However, the quick degradation and low mechanical property of GelMA hydrogels need to be improved for further applications, especially for long-term implantation. In this study, a sequential double modification of gelatin was used to achieve high density of photocrosslinkable double bonds in gelatin derivatives. The amino groups in gelatin were first reacted with methacrylic anhydride. After this, the hydroxyl and carboxyl groups in gelatin were reacted with glycidyl methacrylate to obtain the double modified gelatin macromer. The double modified gelatin macromer was used to prepare gelatin hydrogels with high crosslinking density. The hydrogels exhibited high storage modulus and low degradation. Culture of bovine articular chondrocytes in the gelatin hydrogels showed that chondrocytes had round morphology and maintained a cartilaginous phenotype while cell proliferation was hampered. This method for increasing crosslinking density should be useful for preparation of stable hydrogels for cartilage tissue engineering.

Photocrosslinkable polyaspartamide/polylactide copolymer and its porous scaffolds for chondrocytes

With the aim to produce, by a simple and reproducible technique, porous scaffolds potentially employable for tissue engineering purposes, in this work, we have synthesized a methacrylate (MA) copolymer of α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) and polylactic acid (PLA). PHEA-PLA-MA has been dissolved in organic solvent at different concentrations in the presence of NaCl particles with different granulometry, and through UV irradiation and further salt leaching technique, various porous scaffolds have been prepared. Obtained samples have been characterized by scanning electron microscopy and their porosity has been evaluated as well as their degradation profile in aqueous medium in the absence or in the presence of esterase from porcine liver. PHEA-PLA-MA scaffold that has shown homogeneous porosity and the best degradation profile has been further characterized to study its mechanical properties along with its capacity to incorporate and to control the release of dexamethasone. Finally, the ability to allow a three-dimensional culture of bovine articular chondrocytes have been also investigated.

Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of Fetal Calf Serum.

Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood, however, pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology, and clustering of cultured bovine articular chondrocytes within stiff (2% w/v) and soft (0.2% w/v) three-dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; fetal calf serum (FCS) 1-10%;380 mOsm) for up to 7 days. Chondrocytes were fluorescently labeled after 1, 3, and 7 days with 5-chloromethylfluorescein-diacetate (CMFDA) and propidium iodide (PI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology, and clustering were quantified using Volocity™ software. In stiff gels after 7 d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However, in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P < 0.0001) and enhanced clustering (P < 0.05), compared to stiff gels. FCS exacerbated changes to density (P < 0.01), abnormal morphology (P < 0.001) and clustering (P < 0.01) compared to lower concentrations at the same gel strength. Reduced gel stiffness and/or increased FCS concentrations promoted chondrocyte proliferation and clustering, increased cell volume, and stimulated abnormal morphology, producing similar changes to those occurring in OA. The increased penetration of factors in FCS into soft gels may be important in the development of these abnormal chondrocyte properties. J. Cell. Physiol. 232: 1041-1052, 2017. © 2016 Wiley Periodicals, Inc.

Cell response to single-walled carbon nanotubes in hybrid porous collagen sponges

Three-dimensional (3D) porous collagen sponges incorporated with single-walled carbon nanotubes (SWCNTs) were prepared and used for 3D culture of bovine articular chondrocytes (BACs). The pore structures of the sponges were controlled by using ice particulates as a porogen material. The responses of cells to SWCNTs were investigated in this 3D cell culture system by evaluation of cell functions and cellular uptake of SWCNTs. The results showed that cells adhered and spatially distributed in the porous sponges. The incorporation of SWCNTs in the porous sponges promoted cell proliferation and production of sulfated glycosaminoglycans (sGAG). Confocal Raman imaging revealed that SWCNTs could be internalized by cells. The hybrid porous sponges not only provided nanostructured pore surfaces to facilitate cell proliferation and extracellular matrix (ECM) secretion but also supplied nanomaterials for cellular uptake which may be useful for biomedical applications.

Clodronate exerts an anabolic effect on articular chondrocytes mediated through the purinergic receptor pathway

Bisphosphonates are commonly used anti-osteoporotic drugs which have controversial effects on joint diseases including osteoarthritis. Certain bisphosphonates have been shown to have anabolic effects on cartilage which could have important ramifications for their proposed effects in vivo; however, the underlying mechanisms are poorly understood. Thus, the purpose of this study was to characterize the effects of clodronate on primary articular chondrocyte metabolism and to determine the underlying signaling pathways responsible. The effects of clodronate and pamidronate on extracellular matrix (ECM) biosynthesis, accumulation and MMP-13 activity were observed in high density, 3D cultures of bovine articular chondrocytes for up to 4 weeks were evaluated. Mechanisms were delineated by measuring intracellular Ca(2+) signaling and the effects of pharmacologic inhibition of the purinergic receptor pathway. Clodronate (100 μM) induced an anabolic effect (increased biosynthesis by 13-14%) which resulted in an 89-90% increase in ECM accumulation after 4 weeks of culture and without an associated effect on matrix turn-over. Stimulation by clodronate resulted in a 3.3-fold increase in Ca(2+) signaling and pharmacological inhibitor experiments suggested that the anabolic effects exerted by clodronate are transduced through the purinergic receptor pathway. These findings support the previous notion that certain bisphosphonates may be useful as adjunctive therapies to potentially ameliorate progression of cartilage degeneration and improve arthritis management.

Collagen Scaffolds with Controlled Insulin Release and Controlled Pore Structure for Cartilage Tissue Engineering

Controlled and local release of growth factors and nutrients from porous scaffolds is important for maintenance of cell survival, proliferation, and promotion of tissue regeneration. The purpose of the present research was to design a controlled release porous collagen-microbead hybrid scaffold with controlled pore structure capable of releasing insulin for application to cartilage tissue regeneration. Collagen-microbead hybrid scaffold was prepared by hybridization of insulin loaded PLGA microbeads with collagen using a freeze-drying technique. The pore structure of the hybrid scaffold was controlled by using preprepared ice particulates having a diameter range of 150–250 μm. Hybrid scaffold had a controlled pore structure with pore size equivalent to ice particulates and good interconnection. The microbeads showed an even spatial distribution throughout the pore walls. In vitro insulin release profile from the hybrid scaffold exhibited a zero order release kinetics up to a period of 4 weeks without initial burst release. Culture of bovine articular chondrocytes in the hybrid scaffold demonstrated high bioactivity of the released insulin. The hybrid scaffold facilitated cell seeding and spatial cell distribution and promoted cell proliferation.

Preparation of collagen porous scaffolds with a gradient pore size structure using ice particulates

Direct comparison using scaffolds with a gradient pore size structure is important to investigate the effect of pore size on tissue regeneration. Collagen porous scaffolds with a pore size gradient were prepared by using pre-prepared ice particulates as a porogen material. The ice particulates had diameters of 150–250, 250–355, 355–425 and 425–500μm. The gradient collagen scaffolds had well-interconnected pore structures with compactly packed spherical pores. The gradient pore scaffolds were used for culture of bovine articular chondrocytes, directly examining the effect of pore size on cartilage regeneration. Chondrocytes adhered and showed a homogenous distribution throughout the scaffolds. In vivo implantation results indicated that the micropores in the scaffolds prepared with ice particulates in the range of 150–250μm showed the best effect on cartilage regeneration.

Uptake and intracellular distribution of collagen-functionalized single-walled carbon nanotubes

Carbon nanotubes (CNTs) have shown great potential for biological and medical applications because of their intrinsic unique properties. However, applications of CNTs have been severely restricted by their super-hydrophobicity and easy aggregation in aqueous medium, which are related to cytotoxicity and other negative cellular effects. In this study, single-walled carbon nanotubes (SWCNTs) were functionalized with collagen (collagen-SWCNTs). The collagen-SWCNTs retained the inherent properties of SWCNTs and the suspension solution was stable for months. The cellular effects, uptake and intracellular distribution of the collagen-SWCNTs were investigated by using them for culture of bovine articular chondrocytes (BACs). The collagen-SWCNTs showed no obvious negative cellular effects and high amount of SWCNTs were internalized by cells. The internalized collagen-SWCNTs were distributed in the perinuclear region and retained in the cells for more than one week. Adsorption of SWCNTs by extracellular matrix (ECM) was shown to be an important step for cellular uptake of SWCNTs. The high stability, easy cellular uptake and long retention in cells of the collagen-SWCNTs will facilitate the biomedical and biotechnological applications of SWCNTs.

Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges**This work was presented at the International Union of Materials Research Societies (International Conference in Asia) (Qingdao, China, Sep. 25–28 2010).

Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering.

Induction of hypertrophic chondrocyte-like phenotypes by oxidized LDL in cultured bovine articular chondrocytes through increase in oxidative stress

It has been reported that the lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor 1 (LOX-1) is expressed by chondrocytes in osteoarthritis (OA) cartilage and that Ox-LDL binding to LOX-1 increases intracellular oxidative stress in cultured bovine articular chondrocytes (BACs). It was recently demonstrated that reactive oxygen species (ROS) induce hypertrophic differentiation of chondrocytes in the growth plate. It has also been shown that activated chondrocytes in OA have hypertrophic chondrocyte-like phenotypes. The purpose of this study was to determine whether Ox-LDL induces hypertrophic chondrocyte-like phenotypes in BACs. Changes in type X collagen (COL10) and runt-related transcription factor 2 (Runx2) mRNA expression in BACs after Ox-LDL stimulation were investigated using real-time polymerase chain reaction (PCR). Western blotting and immunofluorescent cell staining were used to investigate changes in protein level. The antioxidant N-acetyl cysteine (NAC) was used to ascertain whether oxidative stress is involved in COL10 and Runx2 expression. We induced LOX-1 knockdown cells using small interfering RNA (siRNA) to examine the receptor specificity of Ox-LDL. COL10 expression was upregulated by Ox-LDL in a time- and dose-dependent manner. Immunofluorescent staining showed that Ox-LDL increased COL10 production in the extracellular matrix. Ox-LDL-induced upregulation of COL10 was suppressed by pretreatment with NAC and siRNA. Expression of Runx2 was upregulated by Ox-LDL and H(2)O(2), and these effects were suppressed by NAC pretreatment. Ox-LDL binding to LOX-1 induces a hypertrophic chondrocyte-like phenotype through oxidative stress, indicating that Ox-LDL plays a role in the degeneration of cartilage.

Induction of bovine articular chondrocyte senescence with oxidized low‐density lipoprotein through lectin‐like oxidized low‐density lipoprotein receptor 1

Findings of recent in vivo and in vitro studies suggest that oxidized low-density lipoprotein (ox-LDL) plays a role in the degeneration of cartilage. The purpose of this study was to determine whether ox-LDL induces chondrocyte senescence through binding to lectin-like ox-LDL receptor 1 (LOX-1). The effects of ox-LDL on senescence of cultured bovine articular chondrocytes (BACs) were investigated by observing senescence-associated (SA) beta-galactosidase (beta-gal) activity, cell proliferation activity, and telomerase activity. Telomerase activity was measured after adding LY294002 (a specific inhibitor of phosphatidylinositol 3-kinase [PI3K]) or after adding insulin-like growth factor 1 (IGF-1; an activator of PI3K) plus ox-LDL to the culture medium to elucidate the involvement of the PI3K/Akt pathway. Immunoblot analysis was used to investigate whether ox-LDL affects the phosphorylation of Akt. To ascertain whether these effects were attributable to ox-LDL binding to LOX-1, BACs were preincubated with TS-20, an anti-bovine LOX-1 blocking antibody. The activity of SA beta-gal was increased and the incorporation of bromodeoxyuridine into BACs was decreased by ox-LDL in a dose-dependent manner. The telomerase activity of BACs was suppressed by the addition of ox-LDL in a time- and dose-dependent manner. LY294002 suppressed the telomerase activity of BACs, and IGF-1 reversed the ox-LDL-induced suppression of telomerase activity. In addition, ox-LDL rapidly decreased the amount of phosphorylated Akt in BACs. Pretreatment of cultured BACs with TS-20 recovered these effects. These data show that ox-LDL binding to LOX-1 induces stress-induced premature senescence of chondrocytes and results in suppression of telomerase activity by inactivating the PI3K/Akt pathway. Oxidized LDL may play an important role in the pathogenesis of osteoarthritis by inducing chondrocyte senescence.

Monitoring of mechanical properties of serially passaged bovine articular chondrocytes by atomic force microscopy

Atomic force microscopy (AFM) is a powerful tool in imaging cells and tissues and probing their mechanical properties. Articular chondrocytes, the cells responsible for the production and maintenance of cartilaginous extracellular matrix in the knee joint, change their morphology and dedifferentiate during in vitro expansion culture. It was unclear if the mechanical properties of chondrocytes change accompanying phenotype variation. The elasticity of in vitro serially cultured bovine articular chondrocytes was investigated using AFM. The chondrocytes changed their morphology from round to spindle-like. The freeze-dried P0 chondrocytes showed significantly higher modulus than did the serially passaged (P1–P4) chondrocytes. The change of chondrocyte morphology was accompanied with a decrease of elastic modulus.

Control of chondrocyte regulatory volume decrease (RVD) by [Ca 2+] i and cell shape

Optimal matrix metabolism by articular chondrocytes is controlled by the 'set-point' volume which is determined mainly by membrane transporters. The signal transduction pathway(s) for the key membrane transporter which responds to cell swelling ('osmolyte channel') and mediates regulatory volume decrease (RVD) is poorly understood, so here the role of Ca2+ and the effects of 2D culture have been clarified. Changes to the volume and intracellular calcium levels ([Ca2+]i) of freshly isolated and 2D cultured bovine articular chondrocytes subjected to hypotonic challenge using a 43% reduction in medium osmolarity were studied by single-cell fluorescence microscopy. The effects of ethylene glycol tetraacetic acid (EGTA), REV5901 and Gd(3+) were studied and the role of Ca2+ influx determined by Mn2+ quench. In freshly isolated cells, approximately 50% of chondrocytes exhibited 'robust RVD' (6[120]). RVD was inhibited by REV 5901 (4+/-2% responding) (3[23]) and 2 mM EGTA (18+/-5% responding) (4[166]) whereas Gd3+ had no effect (3[89]). The hypotonic challenge resulted in a Gd3+-insensitive rise in [Ca2+]i that did not correlate with RVD in all cells. Following 2D culture, chondrocytes also demonstrated Gd3+-insensitive RVD, but in contrast, the [Ca2+]i rise was blocked by this agent. The data suggested that in freshly isolated and 2D cultured chondrocytes, the rise in [Ca2+]i occurring during hypotonic challenge could be related to RVD, but only in some cells. However, with 2D culture, the Ca2+ response switched to being Gd3+-sensitive, suggesting that as a result of changes to chondrocyte shape, stretch-activated cation channels although present, do not appear to play a role in volume regulation.

The action of TNFα and TGFβ include specific alterations of the glycosylation of bovine and human chondrocytes

Joint destruction in arthritis is often associated with high levels of inflammatory cytokines. Previous work has shown that inflammatory conditions can alter the activities of glycosyltransferases that synthesize the glycan chains of glycoproteins, and that these changes in turn can influence the functions of glycoproteins. We therefore examined glycosyltransferases involved in glycoprotein biosynthesis in primary cultures of bovine articular chondrocytes and human chondrocytes isolated from knee cartilage of osteoarthritis patients. Bovine chondrocytes exhibited enzyme activities involved in the synthesis of bi-antennary complex Asn-linked N-glycans, as well as the enzymes involved in the synthesis of GalNAc-Ser/Thr-linked O-glycans with the core 1 structure. Human chondrocytes, in addition, were able to synthesize more complex O-glycans with core 2 structures. TNFα was found to induce apoptosis in chondrocytes, and this process was associated with significant changes in lectin binding to chondrocyte cell surface glycans. TGFβ increased cell proliferation, and had significant effects on cell surface glycosylation in bovine but not in human cells. These cytokine-specific effects were partially correlated with changes in glycosyltransferase activities. Thus, chondrocytes have many of the enzymes necessary for the synthesis of N- and O-glycan chains of glycoproteins. The O-glycosylation pathways and the effects of TNFα and TGFβ on glycosylation differed between bovine and human chondrocytes. These alterations are of potential importance for the regulation of the functions of cell surface receptors on chondrocytes, and for an understanding of the pathophysiology of arthritis.

Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-γ

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-γ was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-γ inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-γ and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.

Cyclic tensile stretch load and oxidized low density lipoprotein synergistically induce lectin‐like oxidized ldl receptor‐1 in cultured bovine chondrocytes, resulting in decreased cell viability and proteoglycan synthesis

Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 microg/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 microg/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 microg/mL ox-LDL significantly suppressed chondrocytes viability (84.6% +/- 3.4% and 80.9% +/- 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% +/- 7.1% and 85.7% +/- 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% +/- 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 microg/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% +/- 4.9% and 48.7% +/- 6.7% of control at 24 h, respectively (n = 3; p < 0.01 when compared with individual application of cyclic loading or 10 microg/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis.

Arthritis and cannabinoids: HU-210 and Win-55,212–2 prevent IL-1 α-induced matrix degradation in bovine articular chondrocytes in-vitro

Cannabinoids have analgesic, immunomodulatory and anti-inflammatory properties and attenuate joint damage in animal models of arthritis. In this study the mechanisms of action of the synthetic cannabinoid agonists, HU-210 and Win-55,212-2, were studied to determine if they affected interleukin-1 alpha (IL-1alpha)-induced proteoglycan and collagen degradation in bovine nasal cartilage explant cultures and prostaglandin E2 (PGE2) production in primary cultures of bovine articular chondrocytes. The effects of the inactive enantiomer, Win-55,212-3, were compared with those of the active enantiomer, Win-55,212-2, to determine if the effects were cannabinoid (CB)-receptor mediated. The chondrocytes and explants were stimulated by IL-1alpha (100 U mL(-1) identical with 0.06 nM and 500 U mL(-1) identical with 0.3 nM, respectively). Proteoglycan breakdown was determined as sulfated glycosaminoglycan (sGAG) release using the dimethylmethylene blue assay. Collagen degradation was determined as hydroxyproline in the conditioned culture media and cartilage digests. PGE2 was determined by ELISA. Expression of cannabinoid receptors, CB1 and CB2; cyclooxygenase-1 and -2 (COX-1 and COX-2); inducible nitric oxide synthase (iNOS); as well as activation of nuclear factor-kappa B (NF-kappaB) in chondrocytes were studied using immunoblotting techniques and immunofluorescence. The results showed that HU-210 and Win-55,212-2 (5-15 microM) significantly inhibited IL-1-alpha stimulated proteoglycan (P < 0.001) and collagen degradation (P < 0.001). Win-55,212-2 (5-10 microM) also significantly inhibited PGE2 production (P < 0.01). At 5 microM, Win-55,212-2 inhibited the expression of iNOS and COX-2 and activation of NF-kappaB. Chondrocytes appeared to constitutively express cannabinoid receptors CB1 and CB2. It is concluded that biologically stable synthetic cannabinoids protect cartilage matrix from degradation induced by cytokines and this effect is possibly CB-receptor mediated and involves effects on prostaglandin and nitric oxide metabolism. Cannabinoids could also be producing these effects via inhibition of NF-kappaB activation.

A novel method for assessing effects of hydrostatic fluid pressure on intracellular calcium: a study with bovine articular chondrocytes

Chondrocytes in articular cartilage are exposed to hydrostatic pressure and distortional stress during weight bearing and joint loading. Because these stresses occur simultaneously in articular cartilage, the mechanism of mechanosignal transduction due to hydrostatic pressure alone in chondrocytes is not clear. In this study, we attempted to characterize the change in intracellular calcium concentration ([Ca2+]i) in response to the application of hydrostatic fluid pressure (HFP) to cultured bovine articular chondrocytes isolated from defined surface (SZ) and middle zones (MZ) by using a fluorescent indicator (X-rhod-1 AM), a novel custom-made pressure-proof optical chamber, and laser confocal microscopy. Critical methodology implemented in this experiment involved application of high levels of HFP to the cells and the use of a novel imaging apparatus to measure the peak [Ca2+]i in individual cells. The peak [Ca2+]i in MZ cells cultured for 5 days showed a significant twofold increase after the application of HFP at constant 0.5 MPa for 5 min. The peak [Ca2+]i in SZ cells was lower (43%) than that of MZ cells. The peak was suppressed with an inhibitor of dantrolene, gadolinium, or a calcium ion-free buffer, but not with verapamil. This study indicated that the increase in [Ca2+]i in chondrocytes to HFP is dependent on the zonal origin. HFP stimulates calcium mobilization and stretch-activated channels.