The production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism .     

Effects of hypotonic shock on intracellular pH in bovine articular chondrocytes

Chondrocytes inhabit an unusual environment, in which they are repeatedly subjected to osmotic challenges as fluid is expressed from the extracellular matrix during static joint loading. In the present study, the effects of hypotonic shock on intracellular pH, pH(i), have been studied in isolated bovine articular chondrocytes using the pH-sensitive fluroprobe BCECF. Cells subjected to a 50% dilution rapidly alkalinised, by approximately 0.2 pH units, a sustained plateau being achieved within 300 s. The effect was not altered by inhibitors of pH regulators, such as amiloride, bafilomycin and SITS, but was absent when cells were subjected to hypotonic shocks in solutions in which Na(+) ions were replaced by NMDG(+). The response was found to be sensitive to Gd(3+) ions, blockers of stretch-activated cation channels. Alkalinisation was also inhibited by treatment with Zn(2+) ions, at a concentration reported to block voltage-activated H(+) channels (VAHC). Depolarisation using high K(+) solutions supplemented with valinomycin also induced intracellular alkalinisation. Measurements using a membrane potential (E(m)) fluorescent dye showed that E(m) was approximately -44 mV, but was depolarised by over 50 mV following HTS. The depolarisation was also inhibited by Na(+) substitution with NMDG(+) or treatment with Gd(3+). We conclude that in response to HTS the opening of a stretch-activated cation channel leads to Na(+) influx, which results in a membrane depolarisation. Subsequent activation of VAHC permits H(+) ion efflux along the prevailing electrochemcial gradient, leading to the alkalinisation, which we record.     

Influence of perfusion on metabolism and matrix production by bovine articular chondrocytes in hydrogel scaffolds

Articular cartilage has a limited capacity for self-repair after damage. Engineered cartilage is a promising treatment to replace or repair damaged tissue. The growth of engineered cartilage is sensitive to the extracellular culture environment. Chondrocytes were seeded into alginate beads and agarose scaffolds at 4 millions/mL, and the response to static and perfusion culture was examined over period of up to 12 days. For both types of scaffolds, the chondrocytes kept their differentiated morphology over 12 days in all culture conditions. In alginate beads, more glycosaminoglycans (GAGs) were produced in perfusion culture than in static conditions. GAG distribution in alginate constructs was more uniform in perfusion culture than in static culture. However, in agarose constructs there was no significant difference in GAG production between static culture and perfusion culture. Under perfusion culture, the retention rate of GAG in alginate was higher than in agarsoe. It is suggested that the positive effect of perfusion culture only can be achieved by an appropriate choice of other factors such as scaffold materials.     

Effects of cell swelling on intracellular calcium and membrane currents in bovine articular chondrocytes

Chondrocytes experience a dynamic extracellular osmotic environment during normal joint loading when fluid is forced from the matrix, increasing the local proteoglycan concentration and therefore the ionic strength and osmolarity. To exist in such a challenging environment, chondrocytes must possess mechanisms by which cell volume can be regulated. In this study, we investigated the ability of bovine articular chondrocytes (BAC) to regulate cell volume during a hypo-osmotic challenge. We also examined the effect of hypo-osmotic stress on early signaling events including [Ca2+](i) and membrane currents. Changes in cell volume were measured by monitoring the fluorescence of calcein-loaded cells. [Ca2+](i) was quantified using fura-2, and membrane currents were recorded using patch clamp. BAC exhibited regulated volume decrease (RVD) when exposed to hypo-osmotic saline which was inhibited by Gd3+. Swelling stimulated [Ca2+](i) transients in BAC which were dependent on swelling magnitude. Gd3+, zero [Ca2+](o), and thapsigargin all attenuated the [Ca2+](i) response, suggesting roles for Ca2+ influx through stretch activated channels, and Ca2+ release from intracellular stores. Inward and outward membrane currents significantly increased during cell swelling and were inhibited by Gd3+. These results indicate that RVD in BAC may involve [Ca2+](i) and ion channel activation, both of which play pivotal roles in RVD in other cell types. These signaling pathways are also similar to those activated in chondrocytes subjected to other biophysical signals. It is possible, then, that these signaling events may also be involved in a mechanism by which mechanical loads are transduced into appropriate cellular responses by chondrocytes.     

Characterisation of inorganic phosphate transport in bovine articular chondrocytes.

In mineralising tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV) possess membrane transporters that accumulate Ca(2+) and inorganic phosphate (P(i)), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. In the present study, P(i) transport pathways in isolated bovine articular chondrocytes have been characterised. P(i) uptake was temperature-sensitive and could be resolved into Na(+)-dependent and Na(+)-independent components. The Na(+)-dependent component saturated at high concentrations of extracellular P(i), with a K(m) for P(i) of 0.17 mM. In solutions lacking Na(+), uptake did not fully saturate, implying that under these conditions carrier-mediated uptake is supplemented by a diffusive pathway. Both Na(+)-dependent and Na(+)-independent components were sensitive to the P(i) transport inhibitors phosphonoacetate and arsenate, although a fraction of Na(+)-independent P(i) uptake was resistant to these anions. Total P(i) uptake was optimal at pH 7.4, and reduced as pH was made more acidic or more alkaline, an effect that represented reduced Na(+)-dependent influx. RT-PCR analysis confirmed that two members of the NaPi III family, Pit-1 and Pit-2, are expressed, but that NaPi II transporters are not.     

The in vitro production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E₂ and PGI₂ (assayed as 6 keto PGF₁α), rather less PGF₂α and irregular quantities of thromboxane (Tx) B₂. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE₂ and a twofold increase in 6 keto PGF₁α).Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE₂α levels remained high. Indomethacin (10-⁶M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10-⁴M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism $\text{in vivo}$.     

Comparison of cellular response in bovine intervertebral disc cells and articular chondrocytes: effects .of lipopolysaccharide on proteoglycan metabolism

Lipopolysaccharide (LPS) induces matrix degradation and markedly stimulates the production of several cytokines, i.e., interleukin-1β, −6, and −10, by disc cells and chondrocytes. We performed a series of experiments to compare cellular responses of cells from the bovine intervertebral disc (nucleus pulposus and annulus fibrosus) and from bovine articular cartilage to LPS. Alginate beads containing cells isolated from bovine intervertebral discs and articular cartilage were cultured with or without LPS in the presence of 10% fetal bovine serum. The DNA content and the rate of proteoglycan synthesis and degradation were determined. In articular chondrocytes, LPS strongly suppressed cell proliferation and proteoglycan synthesis in a dose-dependent manner and stimulated proteoglycan degradation. Compared with articular chondrocytes, nucleus pulposus cells responded in a similar, although less pronounced manner. However, treatment of annulus fibrosus cells with LPS showed no significant effects on proteoglycan synthesis or degradation. A slight, but statistically significant, inhibition of cell proliferation was observed at high concentrations of LPS in annulus fibrosus cells. Thus, LPS suppressed proteoglycan synthesis and stimulated proteoglycan degradation by articular chondrocytes and nucleus pulposus cells. The effects of LPS on annulus fibrosus cells were minor compared with those on the other two cell types. The dissimilar effects of LPS on the various cell types suggest metabolic differences between these cells and may further indicate a divergence in pathways of LPS signaling and a differential sensitivity to exogenous stimuli such as LPS.This work was supported in part by NIH grants 2-P50-AR39239 and 1-P01-AR48152.     

Antigenic profiles of human,bovine and canine articular chondrocytes

Summary Human, bovine and canine articular chondrocytes have been shown to bear cartilage matrix, chondrocyte-specific and histocompatibility antigens. These cell-surface antigens of chondrocytes were demonstrated both simultaneously and separately either by complement-mediated cytotoxicity or by immunohistochemical reactions. The chondrocyte-specific antigens involve subsets of species-common and species-specific determinants, which are also present on the surfaces of rib and laryngeal chondrocytes. In addition to these antigens, human and calf articular chondrocytes also express unique cell-surface components that are capable of producing a blastogenic stimulation of autologous T-lymphocytes in vitro. These putative autoantigens segregated from lymphocytes in vivo could be released in trauma and in inflammatory joint diseases triggering the immune system of the host.     

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3α mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.     

Cycle number and waveform of fluid flow affect bovine articular chondrocytes

Many studies have shown that a loading-induced (bio)physical signal regulates chondrocyte behavior. In a recent study our group has demonstrated the shear stress level- and frequency-dependent effect of sinusoidal oscillatory fluid flow on bovine articular chondrocyte (BAC) cytosolic calcium concentration ([Ca(2+)](i)), neglecting the fact that chondrocytes are not likely to see these ideal waveform in vivo or in vitro. Furthermore, possible overload of articular cartilage or excessive shear stress in chondrocyte cultures are more likely to be of a short nature. Therefore, in this study we choose to investigate a saw-tooth waveform oscillating fluid flow at varying exposure times in comparison to the established sinusoidal oscillatory waveform. [Ca(2+)](i), as an early signaling molecule, was quantified using the fluorescent dye fura-2. BAC were exposed to 1 Hz sinusoidal or saw-tooth waveform oscillating fluid flow at 2.2 Pa flow rates in a parallel plate flow chamber for 8 different loading times. As little as 5 cycles of oscillatory fluid flow were sufficient to increase [Ca(2+)](i) significantly over baseline. The number of responding cells could not be increased any further after a sufficient number of cycles (11), regardless of the waveform. Furthermore, a saw-tooth waveform appeared to be more stimulatory than regular sinusoidal oscillating flow at higher cycle numbers. BAC appear to be able to respond to these biophysical stimuli in a differentiated manner. This ability might give every single chondrocyte the capability to maintain its territory autonomously, since chondrocytes distributed in articular cartilage without the possibility to interact, e.g., via cell processes.     

Oscillating fluid flow regulates cytosolic calcium concentration in bovine articular chondrocytes.

Mechanical loading is a well-known regulator of cartilage metabolism. This suggests that a loading-induced physical signal regulates chondrocyte behavior. Previous studies have focused on the effects of steady fluid flow on chondrocytes. In contrast to steady flow, loading induced fluid flow occurs in an oscillatory pattern and includes a reversal of flow direction with each loading event. In this study we examined the hypothesis that oscillating fluid flow increases cytosolic Ca2+ concentration ([Ca2+]i) in bovine articular chondrocytes (BAC) in a frequency-dependent manner and that the presence of serum affects this response. The aims of our study were to examine (1) whether BAC respond to physiologic oscillating fluid flow in vitro and compare these results to steady fluid flow, (2) the effect of fetal bovine serum on fluid flow responsiveness of BAC and (3) whether the response of BAC to fluid flow is flow rate and/or frequency dependent. [Ca2+]i was quantified using the fluorescent dye fura-2. BAC were exposed to steady, 0.5, 1, or 5 Hz sinusoidal oscillating fluid flow at five different flow rates in a parallel plate flow chamber. Our findings demonstrate that BAC respond to oscillating fluid flow with an increase in [Ca2+]i (p > 0.05), and furthermore, chondrocyte responsiveness to fluid flow increases with peak flow rate (p < 0.0001) and decreases with increasing frequencies (p < 0.0001). Finally, the presence of serum in the media potentiated the responsiveness of BAC to fluid flow (p < 0.0001). Our results suggest an important role for mechanical load-induced oscillating fluid flow in chondrocyte mechanotransduction.     

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

Summary Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CM^R filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.     

Changes in intracellular calcium concentration in response to hypertonicity in bovine articular chondrocytes

Intracellular calcium concentration ([Ca2+]i) in articular chondrocytes changes during mechanical challenges associated with joint movements, because of the fluctuation of the extracellular osmotic environment during joint loading. Matrix synthesis by chondrocytes is modulated by loading patterns, possibly mediated by variations in intracellular composition, including [Ca2+]i. The present study has employed the Ca(2+)-sensitive fluoroprobe Fura-2 to determine the effects of hypertonic shock on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a hypertonic shock, [Ca2+]i rapidly increased by approximately 300%, reaching a maximal value within 50 s following the hypertonic shock with a recovery of more than 90% towards the initial [Ca2+]i within 5 min. The effect was inhibited by removal of extracellular Ca2+ ions, but not by thapsigargin, indicating that the rise in [Ca2+]i is only a result of influx from the extracellular medium. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels, TRPV channels or stretch-activated cation channels. Non-specific inhibitors of Ca2+ channels like CdCl2, NiCl2, LaCl3 and ZnCl2 significantly attenuated the response, although the extent in which CdCl2 and NiCl2 (both of them inhibitors of annexin-mediated Ca2+ fluxes) inhibited the response was significantly greater. The rise was also sensitive to KBR7943, inhibitor of NCE reverse mode and trifluoperazine, inhibitor of the activity of annexins. Hypertonic shock also produced also hyperpolarisation of chondrocytes (Em measured by means of Di-BA-C4(3), a membrane potential sensitive dye), which was inhibited by TEA-Cl and BaCl, but was not affected by changing the extracellular solution to Ca(2+)-free HBS. Inhibition of hyperpolarisation completely abolished the [Ca2+]i rise following hypertonic shock. Treatment with retinoic acid, which can increase the activity of annexins as Ca2+ transport pathways caused a significant increase in [Ca2+]i. The recovery of [Ca2+] was inhibited by benzamil and was dependent on extracellular Na+, but was unaffected by Na-orthovanadate, an inhibitor of plasma Ca(2+)-ATPase. We conclude that in response to hypertonic shock, NCE reverse mode and annexins are the pathways responsible for the [Ca2+]i increase, while forward mode operation of NCE is responsible for the subsequent extrusion of Ca2+ and recovery of [Ca2+]i towards initial values.     

Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes

Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.     

Electrophysiological demonstration of voltage- activated H+ channels in bovine articular chondrocytes.

Matrix synthesis by articular chondrocytes is sensitive to changes in intracellular pH (pH(i)), so characterising the membrane transport pathways that determine pH(i) is important for understanding how chondrocytes regulate the turnover of cartilage matrix. In the present study, the whole-cell patch-clamp technique has been employed to demonstrate the operation of voltage-activated H(+) channels (VAHC) in bovine articular chondrocytes. Using solutions designed to minimise the contribution of ions other than H(+), the application of step voltage-protocols elicited whole-cell currents. These currents were slow activating, observed only in the outward direction, dependent on both extracellular pH (pH(o)) and pH(i), and inhibited by Zn(2+). The reversal potential values, measured by tail current analysis, over a range of different pHo and pHi values, were in good agreement with predicted values for membrane channels having a high selectivity for protons. The results presented here are consistent with the operation of VAHC in articular chondrocytes.     

Effects of autophagy on apoptosis of articular chondrocytes in adjuvant arthritis rats

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease that eventually leads to joint deformities and loss of joint function. Previous studies have demonstrated a close relationship between autophagy and the development of RA. Although autophagy and apoptosis are two different forms of programmed death, the relationship between them in relation to RA remains unclear. In this study, we explored the effect of autophagy on apoptosis of articular chondrocytes in vivo and in vitro. Adjuvant arthritis (AA) and acid‐induced primary articular chondrocyte apoptosis were used as in vivo and in vitro models, respectively. Articular chondrocyte autophagy and apoptosis were both observed dynamically in AA rat articular cartilage at different stages (15 days, 25 days and 35 days). Moreover, chondrocyte apoptosis and articular cartilage injury in AA rats were increased by the autophagy inhibitor 3‐methyladenine (3‐MA) and decreased by the autophagy activator rapamycin. In addition, pre‐treatment with 3‐MA increased acid‐induced chondrocyte apoptosis, while pre‐treatment with rapamycin reduced acid‐induced chondrocyte apoptosis in vitro. These results suggest that autophagy might be a potential target for the treatment of RA.     

TRPV4 channels activity in bovine articular chondrocytes: Regulation by obesity-associated mediators

Turnover of the cartilage extracellular matrix depends exclusively on chondrocytes and varies in response to load and osmolarity fluctuations. Obesity can affect chondrocyte physiology; adipokines, insulin and proinflammatory cytokines levels are all altered in the obese and are related to matrix turnover impairments and thus to osteoarthritis. TRPV4, a mechanosensitive cation channel, is responsible for reacting to hypotonic variations. In this study, the presence and activity of TRPV4 channels in bovine chondrocytes were evaluated using the whole-cell patch-clamp technique and fluorescence measurements to perform characterisations of these channels and to determine intracellular calcium responses. The expression of TRPV4 was determined by RT-PCR. The TRPV4 regulation by hypotonic shock, insulin and adipokines were analysed. Hypoosmolarity induced a Gd³⁺-, ruthenium red-, and HC-067047-sensitive current, predominantly inward, an intracellular Ca²⁺ concentration increase and a membrane depolarisation. The current had a reversal potential of +28 ± 4 mV and exhibited preferential permeability to Ca²⁺; 4αPDD, a specific TRPV4 agonist, evoked the same response. TNFα, IL-1β, insulin, and, to a lesser degree, leptin and resistin attenuated the TRPV4-mediated effects; in contrast, adiponectin did not affect them. These results confirm the function of TRPV4 in bovine articular chondrocytes and its regulation by obesity-associated mediators.     

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E2 and F2 alpha, 6 keto PGF1 alpha and thromboxane B2 by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10(-7)M - 10(-13)M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E2 and F2 alpha, and to a lesser extent, 6 keto PGF 1 alpha, but TxB2 production was only slightly inhibited by the drug in the absence of arachidonic acid and markedly increased in its presence. Colchicine (10(-7)M-10(-3)M) had the opposite effect, causing an inhibition of TxB2 and stimulating PGE2 and 6 keto PGF1 alpha production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A2 and cyclo-oxygenases, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using 35SO4 labeling methodology. The results showed that at the concentrations tested (10(-5)M to 10(-7)M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited 35SO4 incorporation into newly synthesized proteoglycan molecules both in the presence (10(-6)M) and absence of exogenous arachidonic acid. In the same concentration range chloroquine had no effect. These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.