Differential responses of chondrocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation

Mechanical stimulation is critically important for the maintenance of normal articular cartilage integrity. Molecular events regulating responses of chondrocytes to mechanical forces are beginning to be defined. Chondrocytes from normal human knee joint articular cartilage show increased levels of aggrecan mRNA following 0.33 Hz mechanical stimulation whilst at the same time relative levels of MMP3 mRNA are decreased. This anabolic response, associated with membrane hyperpolarisation, is activated via an integrin-dependent interleukin (IL)-4 autocrine/paracrine loop. Work in our laboratory suggests that this chondroprotective response may be aberrant in osteoarthritis (OA). Chondrocytes from OA cartilage show no changes in aggrecan or MMP3 mRNA following 0.33 Hz mechanical stimulation. alpha5beta1 integrin is the mechanoreceptor in both normal and OA chondrocytes but downstream signalling pathways differ. OA chondrocytes show membrane depolarisation following 0.33 Hz mechanical stimulation consequent to activation of an IL1beta autocrine/paracrine loop. IL4 signalling in OA chondrocytes is preferentially through the type I (IL4alpha/cgamma) receptor rather than via the type II (IL4alpha/IL13R) receptor. Altered mechanotransduction and signalling in OA may contribute to changes in chondrocyte behaviour leading to increased cartilage breakdown and disease progression.     

Mechanical regulation of proteoglycan synthesis in normal and osteoarthritic human articular chondrocytes--roles for alpha5 and alphaVbeta5 integrins

The importance of biomechanical forces in regulating normal chondrocyte metabolism is well established and the mechanisms whereby mechanical forces are transduced into biochemical responses by chondrocytes are beginning to be understood. Previous studies have indicated that cyclical mechanical stimulation induces increased aggrecan gene expression in normal but not osteoarthritic chondrocytes in monolayer. It remains unclear, however, whether these effects on gene expression are associated with changes in proteoglycan production and whether any changes in proteoglycan expression is dependent on integrins or integrin associated proteins. Normal and osteoarthritic articular chondrocytes in monolayer were exposed to 0.33 Hz mechanical stimulation for 20 min in the absence or presence of function modifying anti-integrin antibodies. Following stimulation GAG and proteoglycan (PG) synthesis was assessed by DMMB assay and western blotting. Mechanical stimulation of normal chondrocytes resulted in increased GAG synthesis that was blocked by the presence of antibodies to alpha5 and alphaVbeta5 integrins and CD47. Electrophoretic patterns of PGs released from normal chondrocytes following mechanical stimulation showed an increase in newly-synthesized aggrecan that was not fragmented or degraded. Chondrocytes from osteoarthritic cartilage showed lower levels of GAG production when compared to normal chondrocytes and synthesis was not influenced by mechanical stimulation. These studies show that chondrocytes derived from normal and OA cartilage have different matrix production responses to mechanical stimulation and suggest previously unrecognised roles for alphaVbeta5 integrin in regulation of chondrocyte responses to biomechanical stimulation.     

NMDA receptor expression and roles in human articular chondrocyte mechanotransduction

Mechanical forces influence articular cartilage structure by regulating chondrocyte activity. Mechanical stimulation results in activation of an alpha5beta1 integrin dependent intracellular signal cascade involving focal adhesion kinase and protein kinase C, triggering the release of interleukin-4 from the cell. In normal HAC the response to physiological mechanical stimulation is characterised by increased levels of aggrecan mRNA and a decrease in levels of mRNA for matrix metalloproteinase 3 (MMP-3), the net result of which would be to maintain and optimise cartilage structure and function. This protective/anabolic response is not seen when chondrocytes from osteoarthritic cartilage are subjected to an identical mechanical stimulation regime. Following the observation that the neurotransmitter substance P is involved in chondrocyte mechanotransduction the present study was undertaken to establish potential roles for glutamate receptors in the control of chondrocyte mechanical responses. Using immunohistochemistry and RTPCR normal and OA chondrocytes are shown to express NR1 and NR2a subunits of the NMDA receptor. Addition of NMDA receptor agonists to chondrocytes in primary culture resulted in changes in membrane potential consistent with expression of functional receptors. NMDA receptor antagonists inhibited the hyperpolarisation response of normal chondrocytes to mechanical stimulation but had no effect on the depolarisation response of osteoarthritic chondrocytes to mechanical stimulation. These studies indicate that at least one subset of the NMDA receptor family of molecules is expressed in cartilage and may have important modulatory effects on mechanotransduction and cellular responses following mechanical stimulation. Indeed the results suggest that there is an alteration of NMDA receptor signalling in OA chondrocytes, which may be critical in the abnormal response of OA chondrocytes to mechanical stimulation. Thus NMDA receptors appear to be involved in the regulation of human articular chondrocyte responses to mechanical stimulation, and in OA, mechanotransduction pathways may be modified as a result of altered activation and function of these receptors.     

ATP in the mechanotransduction pathway of normal human chondrocytes

Extracellular nucleotides have been shown to have diverse effects on chondrocyte function, generally acting via P2 purinoceptors. We have previously shown that mechanical stimulation at 0.33 Hz of normal human chondrocyte cultures causes cellular hyperpolarisation, while chondrocytes derived from osteoarthritic (OA) cartilage depolarise. Experiments have been undertaken to establish whether ATP is involved in the response of the chondrocyte to mechanical stimulation. Chondrocytes, isolated from normal and OA cartilage obtained, with consent, from human knee joints following surgery, were cultured in non-confluent monolayer. Cells were mechanically stimulated at 0.33 Hz for 20 minutes at 37 degrees C in the presence or absence of inhibitors of ATP signalling, or were stimulated by the addition of exogenous ATP or derivatives, and electrophysiological measurements recorded. Samples of medium bathing the cells were collected before and after mechanical stimulation, and the concentration of ATP in the cell medium was measured. Total RNA was extracted from cultured chondrocytes, reverse-transcribed and used for RT-PCR with primers specific for P2Y2 purinoceptors. ATP, UTP 2-methylthioadenosine and alphabeta-methylene adenosine 5'-triphosphate all induced a hyperpolarisation response in normal human articular chondrocytes. No significant change was observed in the membrane potentials of chondrocytes isolated from OA cartilage following the addition of these nucleotides to the medium. In normal chondrocytes, the hyperpolarisation induced by ATP was blocked by the presence of apamin, indicating the involvement of small-conductance calcium-activated potassium channels. Following mechanical stimulation of normal chondrocytes, an increase was observed in ATP concentration in the cell culture medium bathing the cells. The presence within the culture medium of suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) prior to and during the period of mechanical stimulation abolished the hyperpolarisation response in normal chondrocytes. The presence of mRNA for P2Y2 purinoceptors was demonstrated in both normal and OA chondrocytes by RT-PCR. These results suggest that ATP has a role in the response of normal chondrocytes to mechanical stimulation, via P2Y2 purinoceptors. This response appears to be different in chondrocytes derived from OA cartilage, and may be important in the progression of this disease.     

Cyclic strain stimulates proliferative capacity, alpha2 and alpha5 integrin, gene marker expression by human articular chondrocytes propagated on flexible silicone membranes

Chondrocytes comprise less than 10% of cartilage tissue but are responsible for sensing and responding to mechanical stimuli imposed on the joint. However, the effect of mechanical signals at the cellular level is not yet fully defined. The purpose of this study was to test the hypothesis that mechanical stimulation in the form of cyclic strain modulates proliferative capacity and integrin expression of chondrocytes from osteoarthritic knee joints. Chondrocytes isolated from articular cartilage during total knee arthroplasty were propagated on flexible silicone membranes. The cells were subjected to cyclic strain for 24 h using a computer-controlled vacuum device, with replicate samples maintained under static conditions. Our results demonstrated increase in proliferative capacity of the cells subjected to cyclic strain compared with cells maintained under static conditions. The flexed cells also exhibited upregulation of the chondrocytic gene markers type II collagen and aggrecan. In addition, cyclic strain resulted in increased expression of the alpha2 and alpha5 integrin subunits, as well as an increased expression of vimentin. There was also intracellular reconfiguration of the enzyme protein kinase C. Our findings suggest that these molecules may play a role in the signal transduction pathway, eliciting cellular response to mechanical stimulation.     

Effects of shear stress on articular chondrocyte metabolism

The articular cartilage of diarthrodial joints experiences a variety of stresses, strains and pressures that result from normal activities of daily living. In normal cartilage, the extracellular matrix exists as a highly organized composite of specialized macromolecules that distributes loads at the bony ends. The chondrocyte response to mechanical loading is recognized as an integral component in the maintenance of articular cartilage matrix homeostasis. With inappropriate mechanical loading of the joint, as occurs with traumatic injury, ligament instability, bony malalignment or excessive weight bearing, the cartilage exhibits manifestations characteristic of osteoarthritis. Breakdown of cartilage in osteoarthritis involves degradation of the extracellular matrix macromolecules and decreased expression of chondrocyte proteins necessary for normal joint function. Osteoarthritic cartilage often exhibits increased amounts of type I collagen and synthesis of proteoglycans characteristic of immature cartilage. The shift in cartilage phenotype in response to altered load yields a matrix that fails to support normal joint function. Mathematical modeling and experimental studies in animal models confirm an association between altered loading of diarthrotic joints and arthritic changes. Both types of studies implicate shear forces as a critical component in the destructive profile. The severity of cartilage destruction in response to altered loads appears linked to expression of biological factors influencing matrix integrity and cellular metabolism. Determining how shear stress alters chondrocyte metabolism is fundamental to understanding how to limit matrix destruction and stimulate cartilage repair and regeneration. At present, the precise biochemical and molecular mechanisms by which shear forces alter chondrocyte metabolism from a normal to a degenerative phenotype remain unclear. The results presented here address the hypothesis that articular chondrocyte metabolism is modulated by direct effects of shear forces that act on the cell through mechanotransduction processes. The purpose of this work is to develop critical knowledge regarding the basic mechanisms by which mechanical loading modulates cartilage metabolism in health and disease. This presentation will describe the effects of using fluid induced shear stress as a model system for stimulation of articular chondrocytes in vitro. The fluid induced shear stress was applied using a cone viscometer system to stimulate all the cells uniformly under conditions of minimal turbulence. The experiments were carried using high-density primary monolayer cultures of normal and osteoarthritic human and normal bovine articular chondrocytes. The analysis of the cellular response included quantification of cytokine release, matrix metalloproteinase expression and activation of intracellular signaling pathways. The data presented here show that articular chondrocytes exhibit a dose- and time-dependent response to shear stress that results in the release of soluble mediators and extracellular matrix macromolecules. The data suggest that the chondrocyte response to mechanical stimulation contributes to the maintenance of articular cartilage homeostasis in vivo.     

Calcium regulates cyclic compression-induced early changes in chondrocytes during in vitro cartilage tissue formation

A single application of cyclic compression (1kPa, 1Hz, 30min) to bioengineered cartilage results in improved tissue formation through sequential catabolic and anabolic changes mediated via cell shape changes that are regulated by α5β1 integrin and membrane-type metalloprotease (MT1-MMP). To determine if calcium was involved in this process, the role of calcium in regulating cell shape changes, MT1-MMP expression and integrin activity in response to mechanical stimulation was examined. Stimulation-induced changes in cell shape and MT1-MMP expression were abolished by chelation of extracellular calcium, and this effect was reversed by re-introduction of calcium. Spreading was inhibited by blocking stretch-activated channels (with gadolinium), while retraction was prevented by blocking the L-Type voltage-gated channel (with nifedipine); both compounds inhibited MT1-MMP upregulation. Calcium A23187 ionophore restored cellular response further supporting a role for these channels. Calcium regulated the integrin-mediated signalling pathway, which was facilitated through Src kinase. Both calcium- and integrin-mediated pathways converged on ERK-MAPK in response to stimulation. While both integrins and calcium signalling mediate chondrocyte mechanotransduction, calcium appears to play the major regulatory role. Understanding the underlying molecular mechanisms involved in chondrocyte mechanotransduction may lead to the development of improved bioengineered cartilage.     

Coordination of chondrocyte differentiation and joint formation by alpha5beta1 integrin in the developing appendicular skeleton

The control point by which chondrocytes take the decision between the cartilage differentiation program or the joint formation program is unknown. Here, we have investigated the effect of alpha5beta1 integrin inhibitors and bone morphogenetic protein (BMP) on joint formation. Blocking of alpha5beta1 integrin by specific antibodies or RGD peptide (arginine-glycine-aspartic acid) induced inhibition of pre-hypertrophic chondrocyte differentiation and ectopic joint formation between proliferating chondrocytes and hypertrophic chondrocytes. Ectopic joint expressed Wnt14, Gdf5, chordin, autotaxin, type I collagen and CD44, while expression of Indian hedgehog and type II collagen was downregulated in cartilage. Expression of these interzone markers confirmed that the new structure is a new joint being formed. In the presence of BMP7, inhibition of alpha5beta1 integrin function still induced the formation of the ectopic joint between proliferating chondrocytes and hypertrophic chondrocytes. By contrast, misexpression of alpha5beta1 integrin resulted in fusion of joints and formation of pre-hypertrophic chondrocytes. These facts indicate that the decision of which cell fate to make pre-joint or pre-hypertrophic is made on the basis of the presence or absence of alpha5beta1 integrin on chondrocytes.     

Mechanical regulation of mitogen-activated protein kinase signaling in articular cartilage

Articular chondrocytes respond to mechanical forces by alterations in gene expression, proliferative status, and metabolic functions. Little is known concerning the cell signaling systems that receive, transduce, and convey mechanical information to the chondrocyte interior. Here, we show that ex vivo cartilage compression stimulates the phosphorylation of ERK1/2, p38 MAPK, and SAPK/ERK kinase-1 (SEK1) of the JNK pathway. Mechanical compression induced a phased phosphorylation of ERK consisting of a rapid induction of ERK1/2 phosphorylation at 10 min, a rapid decay, and a sustained level of ERK2 phosphorylation that persisted for at least 24 h. Mechanical compression also induced the phosphorylation of p38 MAPK in strictly a transient fashion, with maximal phosphorylation occurring at 10 min. Mechanical compression stimulated SEK1 phosphorylation, with a maximum at the relatively delayed time point of 1 h and with a higher amplitude than ERK1/2 and p38 MAPK phosphorylation. These data demonstrate that mechanical compression alone activates MAPK signaling in intact cartilage. In addition, these data demonstrate distinct temporal patterns of MAPK signaling in response to mechanical loading and to the anabolic insulin-like growth factor-I. Finally, the data indicate that compression coactivates distinct signaling pathways that may help define the nature of mechanotransduction in cartilage.     

The effect of mechanical load on integrin subunits α5 and β1 in chondrocytes from mature and immature cartilage explants

Articular cartilage is subjected to cyclic compressive stresses during joint loading. There is increasing experimental evidence that this loading is essential for the chondrocytes to maintain the functionality of the cartilage extracellular matrix (ECM) and that members of the integrin family of transmembrane receptors may play an important role in signal mechanotransduction between the ECM and chondrocytes. Of particular interest are the integrin subunits 5 and 1, which are known to form the receptor for fibronectin, an important ECM protein, and to be involved in mechanotransduction as well as in the regulation of cytokine production. In this study, we measured the amounts of the integrin subunits 5 and 1 in chondrocytes from young (immature) and adult (mature) bovine articular cartilage explants which were subjected to a continuously applied cyclic compressive stress of 1 MPa for 6 and 24 h. The integrin content per chondrocyte was measured immediately after load cessation by flow cytometry following matrix digestion to release the cells. We found that a mechanical stress induced an increase in the number of integrin subunit 5 in immature and mature cartilage but not in the integrin subunit 1 content. The integrin contents were greatest after 6 h of loading and returned to control levels after 24 h of unloading. The results of this study supply further experimental evidence that chondrocytes respond to changes in their mechanical environment and that the integrin 51 may act as a mechanical signal transducer between the chondrocyte and the ECM for the modulation of cellular physiology.This work was supported by NIH grant AR45748 to PAT and the HSS MacArthur Cartilage Fund.     

Metabolic responses induced by compression of chondrocytes in variable-stiffness microenvironments

Cells sense and respond to mechanical loads in a process called mechanotransduction. These processes are disrupted in the chondrocytes of cartilage during joint disease. A key driver of cellular mechanotransduction is the stiffness of the surrounding matrix. Many cells are surrounded by extracellular matrix that allows for tissue mechanical function. Although prior studies demonstrate that extracellular stiffness is important in cell differentiation, morphology and phenotype, it remains largely unknown how a cell’s biological response to cyclical loading varies with changes in surrounding substrate stiffness. Understanding these processes is important for understanding cells that are cyclically loaded during daily in vivo activities (e.g. chondrocytes and walking). This study uses high-performance liquid chromatography – mass spectrometry to identify metabolomic changes in primary chondrocytes under cyclical compression for 0–30 minutes in low- and high-stiffness environments. Metabolomic analysis reveals metabolites and pathways that are sensitive to substrate stiffness, duration of cyclical compression, and a combination of both suggesting changes in extracellular stiffness in vivo alter mechanosensitive signaling. Our results further suggest that cyclical loading minimizes matrix deterioration and increases matrix production in chondrocytes. This study shows the importance of modeling in vivo stiffness with in vitro models to understand cellular mechanotransduction.     

Integrin-regulated secretion of interleukin 4: A novel pathway of mechanotransduction in human articular chondrocytes

Chondrocyte function is regulated partly by mechanical stimulation. Optimal mechanical stimulation maintains articular cartilage integrity, whereas abnormal mechanical stimulation results in development and progression of osteoarthritis (OA). The responses of signal transduction pathways in human articular chondrocytes (HAC) to mechanical stimuli remain unclear. Previous work has shown the involvement of integrins and integrin-associated signaling pathways in activation of plasma membrane apamin-sensitive Ca2+-activated K+ channels that results in membrane hyperpolarization of HAC after 0. 33 Hz cyclical mechanical stimulation. To further investigate mechanotransduction pathways in HAC and show that the hyperpolarization response to mechanical stimulation is a result of an integrin-dependent release of a transferable secreted factor, we used this response. Neutralizing antibodies to interleukin 4 (IL-4) and IL-4 receptor alpha inhibit mechanically induced membrane hyperpolarization and anti-IL-4 antibodies neutralize the hyperpolarizing activity of medium from mechanically stimulated cells. Antibodies to interleukin 1beta (IL-1beta) and cytokine receptors, interleukin 1 receptor type I and the common gamma chain/CD132 (gamma) have no effect on me- chanically induced membrane hyperpolarization. Chondrocytes from IL-4 knockout mice fail to show a membrane hyperpolarization response to cyclical mechanical stimulation. Mechanically induced release of the chondroprotective cytokine IL-4 from HAC with subsequent autocrine/paracrine activity is likely to be an important regulatory pathway in the maintenance of articular cartilage structure and function. Finally, dysfunction of this pathway may be implicated in OA.     

Membrane channel gene expression in human costal and articular chondrocytes

Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca²⁺ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought.     

Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation

Indian hedgehog (Ihh), a member of the vertebrate hedgehog morphogen family, is a key signaling molecule that controls chondrocyte proliferation and differentiation. In this study, we show a novel function of Ihh. Namely, it acts as an essential mediator of mechanotransduction in cartilage. Cyclic mechanical stress greatly induces the expression of Ihh by chondrocytes. This induction is abolished by gadolinium, an inhibitor of stretch-activated channels. This suggests that the IHH gene is mechanoresponsive. The mechano-induction of Ihh is essential for stimulating chondrocyte proliferation by mechanical loading. The presence of an Ihh functional blocking antibody during loading completely abolishes the stimulatory effect of mechanical load on proliferation. Furthermore, Ihh mediates the mechanotransduction process in a bone morphogenic protein (BMP)-dependent and parathyroid hormone-related peptide-independent manner. BMP 2/4 are up-regulated by mechanical stress through the induction of Ihh, and BMP antagonist noggin inhibits mechanical stimulation of chondrocyte proliferation. This suggests BMP lies downstream of Ihh in mechanotransduction pathway. Our data suggest that Ihh may transduce mechanical signals during cartilage growth and repair processes.     

Ankle flexor muscles in the cat: Length-active tension and muscle unit properties as related to locomotion

The mechanical properties of the whole muscle and fast-twitch muscle units of the cat hindlimb pretibial flexors have been explored and related to normal locomotion. Tibialis anterior (TA) is parallel-fibered and functionally crosses a single joint, the ankle, whereas extensor digitorum longus (EDL) is pinnate and spans the ankle, knee, metatarsophalangeal and interphalangeal joints. The active tetanic tension of TA remains near its peak value over a range of muscle lengths associated with normal ankle movement. In contrast, the length-tension curve of EDL is sharply peaked. However, normal corollary action of the knee, ankle and metatarsophalangeal joints during stepping minimizes EDL's excursion and maintains it at or near a length optimal for peak tension development. EDL is capable of producing synchronous but sterotyped digit and ankle movements while TA provides for independent ankle flexion at all relevant joint angles. The mechanical properties of 84 TA and 98 EDL fast-twitch muscle units were studied by measuring twitch contraction time (≤45 msec), peak tetanic tension, response to repetitive stimulation, and contractile fatigue resistance during electrical stimulation of single alpha axons, functionally isolated from ventral root filaments. These mechanical properties were essentially similar for both muscles with the exception of mean peak tetanic tension which was 30% lower for TA units (14 gm-wt) than for EDL units (20 gm-wt). A high proportion of units in both muscles demonstrated fatigue resistance which is reflective of the repetitive, phasic demand upon these muscles during locomotion.     

Differing in vitro biology of equine,ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.     

The Identification of CD163 Expressing Phagocytic Chondrocytes in Joint Cartilage and Its Novel Scavenger Role in Cartilage Degradation

Background

Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation.

Methods

Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163⁺ chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated.

Results

In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P<0.05). However, the levels of ACP-1, NO and ROS, which relate to cellular digestion capability were unchanged (P>0.05). CD163⁺ chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163⁺ chondrocytes with enhanced phagocytic activity were present in Col-II⁺ chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163⁺ chondrocytes were also found in isolated Col-II⁺ chondrocytes stimulated with TNF-α (P<0.05). Mid-zone distribution of CD163⁺ cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II⁺ chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05).

Conclusions

An increased number of CD163⁺ chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a role in eliminating degraded tissues. Targeting these cells provides a new strategy for the treatment of arthritis.     

Chondrocyte intracellular calcium, cytoskeletal organization, and gene expression responses to dynamic osmotic loading

While chondrocytes in articular cartilage experience dynamic stimuli from joint loading activities, few studies have examined the effects of dynamic osmotic loading on their signaling and biosynthetic activities. We hypothesize that dynamic osmotic loading modulates chondrocyte signaling and gene expression differently than static osmotic loading. With the use of a novel microfluidic device developed in our laboratory, dynamic hypotonic loading (–200 mosM) was applied up to 0.1 Hz and chondrocyte calcium signaling, cytoskeleton organization, and gene expression responses were examined. Chondrocytes exhibited decreasing volume and calcium responses with increasing loading frequency. Phalloidin staining showed osmotic loading-induced changes to the actin cytoskeleton in chondrocytes. Real-time PCR analysis revealed a stimulatory effect of dynamic osmotic loading compared with static osmotic loading. These studies illustrate the utility of the microfluidic device in cell signaling investigations, and their potential role in helping to elucidate mechanisms that mediate chondrocyte mechanotransduction to dynamic stimuli. cartilage; calcium signaling; actin cytoskeleton; aggrecan