Cartilage Synthesis Research Articles

Mesenchymal stem cells--a new pathway for tissue engineering in reconstructive surgery

Mesenchymal stem cells (MSC) have the capacity to differentiate into chondrocytes with the synthesis of cartilage. This report presents the use of human adult bone marrow derived mesenchymal stem cells for tissue engineering of autologous cartilage grafts. Human bone marrow aspirates were obtained from the iliac crest and fractionated on a Percoll gradient. The isolated hMSC were plated at 20 x 10 (6) cells per 100 mm (2) culture dish. After 21 days in culture at 37 degrees C with 5 % CO 2, the adherent multiplied MSC were trypsinized, counted, and tested for viability by trypan blue assay. The hMSCs were loaded into a sterile 15 ml polypropylene tube (0.5 Mio cells/ml) and centrifuged on the bottom of the tube at 500 g for 5 minutes. The MSC were cultivated for 3 weeks in vitro in a specific chondrogenetic medium composed of Dulbecco's Modified Eagles Medium-High Glucose supplemented with 10 ng/ml transforming growth factor-beta 1, 1 % ITS-Premix medium, 80 micro M ascorbic acid, and 100 nM dexamethasone. Histological and immunohistochemical studies performed after 3 weeks in three dimensional culture demonstrated the expression of cartilage specific collagen type II and X as well as proteoglycans. Human adult mesenchymal stem cells derived from bone marrow aspirates have the ability to differentiate into chondrocytes under specific culture conditions by growth factors. The use of adult mesenchymal stem cells may be a promising tool for tissue engineering of autologous cartilage grafts in reconstructive surgery in the future.

Diminished Callus Size and Cartilage Synthesis in α1β1 Integrin-Deficient Mice during Bone Fracture Healing

Integrins mediate cell adhesion to extracellular matrix components. Integrin alpha 1 beta 1 is a collagen receptor expressed on many mesenchymal cells, but mice deficient in alpha 1 integrin (alpha1-KO) have no gross structural defects. Here, the regeneration of a fractured long bone was studied in alpha1-KO mice. These mice developed significantly less callus tissue than the wild-type (WT) mice, and safranin staining revealed a defect in cartilage formation. The mRNA levels of nine extracellular matrix genes in calluses were evaluated by Northern blotting. During the first 9 days the mRNA levels of cartilage-related genes, including type II collagen, type IX collagen, and type X collagen, were lower in alpha1-KO mice than in WT mice, consistent with the reduced synthesis of cartilaginous matrix appreciated in tissue sections. Histological observations also suggested a diminished number of chondrocytes in the alpha 1-KO callus. Proliferating cell nuclear antigen staining revealed a reduction of mesenchymal progenitors at the callus site. Although, the number of mesenchymal stem cells (MSCs) obtained from WT and alpha 1-KO whole marrow was equal, in cell culture the proliferation rate of the MSCs of alpha 1-KO mice was slower, recapitulating the in vivo observation of reduced callus cell proliferation. The results demonstrate the importance of proper collagen-integrin interaction in fracture healing and suggest that alpha1 integrin plays an essential role in the regulation of MSC proliferation and cartilage production.

Development of gene-based therapies for cartilage repair.

Articular cartilage is particularly vulnerable to injury and degenerative conditions, and has a limited capacity for self-repair. Although current clinical procedures cannot restore a normal articular surface, there are a growing number of proteins that may be used to augment a repair process, or protect cartilage from degeneration. Because proteins are often difficult to administer effectively, gene therapy approaches are being developed to provide their sustained synthesis at sites of injury or disease. To promote cartilage repair, cDNAs can be targeted to synovium, or cartilage. Gene transfer to the synovium is generally considered more suitable for chondroprotective therapies that rely on expression of large amounts of anti-inflammatory mediators. The delivery of genes to cartilage defects to promote enhanced repair can be performed by either direct administration of gene delivery vectors, or by implantation of genetically modified chondrogenic cells. Variations of these methods have been used to demonstrate that exogenous cDNAs encoding growth factors can be delivered locally to sites of cartilage damage where they are expressed at physiologically relevant levels. Data is beginning to emerge that suggests that delivery and expression of these genescan influence a repair response toward the synthesis of normal articular cartilage in vivo. This article reviews the current status of gene delivery for cartilage healing and presents some of the remaining challenges.

UDP-glucose pyrophosphorylase: up-regulation in hypertrophic cartilage and role in hyaluronan synthesis.

Previously, we have performed subtractive hybridization to identify genes up-regulated in hypertrophic chondrocytes of the avian epiphyseal growth plate. In the present study, we report the identification of one of the clones as UDP-glucose pyrophosphorylase (UDPG-PPase) and propose a possible function for this enzyme in regulating hyaluronan (HA) synthesis in hypertrophic cartilage. We have cloned the 2.6 kb full-length cDNA for avian UDPG-PPase and confirmed its up-regulation in hypertrophic versus non-hypertrophic cartilage by Northern-blot analysis. The 6-fold increase in mRNA was paralleled by an equivalent increase in enzymic activity. The enzyme catalyses the conversion of glucose 1-phosphate into UDP-glucose, which is used to synthesize a number of cellular components, including HA. Overexpression of enzymically active UDPG-PPase in non-hypertrophic chondrocytes resulted in a 2-3-fold increase in total HA, as determined by a competitive binding assay and immunohistochemistry. In the developing growth plate, HA synthesis was elevated in the hypertrophic zone along with the up-regulation of the HA synthase (HAS)-2 gene. Our data suggest that an increase in both activities, UDPG-PPase and HAS-2, is required for non-hypertrophic chondrocytes to synthesize an amount of HA comparable with that in hypertrophic chondrocytes. Therefore we conclude that HA synthesis during chondrocyte differentiation is regulated at the level of the substrate-provider gene, UDPG-PPase, as well as the HAS genes.

UDP-glucose pyrophosphorylase: up-regulation in hypertrophic cartilage and role in hyaluronan synthesis

Previously, we have performed subtractive hybridization to identify genes up-regulated in hypertrophic chondrocytes of the avian epiphyseal growth plate. In the present study, we report the identification of one of the clones as UDP-glucose pyrophosphorylase (UDPG-PPase) and propose a possible function for this enzyme in regulating hyaluronan (HA) synthesis in hypertrophic cartilage. We have cloned the 2.6kb full-length cDNA for avian UDPG-PPase and confirmed its up-regulation in hypertrophic versus non-hypertrophic cartilage by Northern-blot analysis. The 6-fold increase in mRNA was paralleled by an equivalent increase in enzymic activity. The enzyme catalyses the conversion of glucose 1-phosphate into UDP-glucose, which is used to synthesize a number of cellular components, including HA. Overexpression of enzymically active UDPG-PPase in non-hypertrophic chondrocytes resulted in a 2–3-fold increase in total HA, as determined by a competitive binding assay and immunohistochemistry. In the developing growth plate, HA synthesis was elevated in the hypertrophic zone along with the up-regulation of the HA synthase (HAS)-2 gene. Our data suggest that an increase in both activities, UDPG-PPase and HAS-2, is required for non-hypertrophic chondrocytes to synthesize an amount of HA comparable with that in hypertrophic chondrocytes. Therefore we conclude that HA synthesis during chondrocyte differentiation is regulated at the level of the substrate-provider gene, UDPG-PPase, as well as the HAS genes.

A single injection of fibronectin fragments into rabbit knee joints enhances catabolism in the articular cartilage followed by reparative responses but also induces systemic effects in the non-injected knee joints

Objective To investigate effects on cartilage metabolism and degeneration of injection of fibronectin fragments (Fn-fs) into rabbit knee joints.Design The knees of adolescent New Zealand white rabbits were intraarticularly injected with rabbit Fn-fs. Cartilage sections from both injected and non-injected joints were treated with Safranin-O, with antibodies to the VDIPEN and NITEGE neoepitopes of degraded aggrecan and to matrix metalloproteinase-3 (MMP-3). Proteoglycan (PG) content of cartilage was measured by a dimethylmethylene blue assay of papain digests. PG synthesis rates were measured by35S-sodium sulfate incorporation into explanted cartilage.Results In the injected joint cartilage, the Fn-fs bound cells in the upper superficial zone maximally between 6 and 24h. By day 2, MMP-3 protein was enhanced and cartilage PG content and PG synthesis rates were reduced 40% and 70%, respectively. MMP-3 epitope and VDIPEN and NITEGE neoepitopes were also enhanced. The PG content then increased to supernormal levels from days 14 to 35 and then declined to normal levels by day 70, as did PG synthesis rates.In the non-injected joint cartilage, Fn-fs were not detected. Although MMP-3 expression was enhanced between days 2 and 21 as well as VDIPEN neoepitope, the PG content was never reduced but rather enhanced to supernormal levels from days 21 to 35. This was associated with enhanced PG synthesis by day 7, which decreased to control levels by day 70.Conclusions In this cartilage degeneration model, loss of cartilage PG is followed by supernormal anabolic responses that facilitate PG restoration. Further, the damage causes a systemic effect of enhanced PG synthesis and content in the non-injected joint cartilage.

Accumulation of advanced glycation endproducts reduces chondrocyte-mediated extracellular matrix turnover in human articular cartilage

Objective The prevalence of osteoarthritis (OAs) increases with age and coincides with the accumulation of advanced glycation endproducts (AGEs) in articular cartilage, suggesting that accumulation of glycation products may be involved in the development of OA. This study was designed to examine the effects of accumulation of AGEs on the turnover of the extracellular matrix of human articular cartilage.Design Chondrocyte mediated cartilage degradation (GAG release, colorimetric) was measured in human articular cartilage of donors aged 19–82 years (N=30, 4-day culture). In addition, to mimic the age-related increase in AGE levels in vitro, cartilage was cultured in the absence or presence of glucose, ribose or threose. Cartilage degradation and proteoglycan synthesis (35SO2−4 incorporation) were measured and related to the degree of cartilage AGE levels (fluorescence at 360/460nm).Results Chondrocyte-mediated degradation of articular cartilage (i.e. GAG release) decreased with increasing age of the cartilage donor (r=−0.43, P< 0.02). In vitro incubation of cartilage with glucose, ribose or threose resulted in a range of AGE levels that was highly correlated to the chondrocyte-mediated cartilage degradation (r=−0.77, P< 0.001, N=26). In addition, in these in vitro glycated cartilage samples, a decrease in proteoglycan synthesis was observed at increasing AGE levels (r=−0.54, P< 0.005,N =25).Conclusions This study shows that an increase in AGE levels negatively affects the proteoglycan synthesis and degradation of articular cartilage. In combination, these two effects reduce the turnover of the cartilage and thereby the maintenance and repair capacity of the tissue. By this mechanism, the age-related increase in cartilage AGE levels may contribute to the development of OA.

Diagnosis of arthrosis

Pain and loss of function are the major clinical symptoms leading to the diagnosis of osteoarthritis (OA). Further clinical findings allow the diagnosis of OA with a high level of sensitivity and specificity at the hip, knee and hand. Standard x-rays of the joints confirm the definitive diagnosis while standard serological examinations are mostly only necessary for differential diagnosis. The analysis of synovial fluid is an important diagnostic tool. Many biochemical markers in the serum have been established for anabolic and catabolic substances, which play a role in the synthesis and degradation of bone, cartilage and synovium in OA. These are of high scientific interest, but play only a minor role in daily practise. At present no single clinically important biochemical parameter exists for the early diagnosis, treatment, control or prognosis of OA.

Regulation of the nitric oxide production resulting from the glucocorticoid-insensitive expression of iNOS in human osteoarthritic cartilage

Objective Nitric oxide (NO) produced by cartilage and synovial membranes is implicated in the pathogenesis of osteoarthritis (OA) and inhibitors of NO synthesis may have indications in the treatment or prevention of joint destruction in OA. Because the signaling mechanisms as well as the NOS isoform involved in induction of NO production in human cartilage remain in many parts unclear, the present study was designed to investigate the regulation of inducible NO synthesis in human intact OA cartilage.Methods Cartilage specimens were collected from OA patients undergoing knee replacement surgery and studied for iNOS expression and NO production in organ culture to allow intact chondrocyte–matrix interactions. J774 macrophages were used for comparison as a well-documented source of iNOS.Results OA cartilage expressed iNOS and produced NO in the absence of exogenous cytokines. Addition of interleukin-1β (IL-1β), tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS) into the culture medium enhanced NO production in a dose-and timedependent manner. Various NOS inhibitors suppressed NO production in the following order of potency: 1400W (novel selective iNOS inhibitor)=L-NIO>L-NMMA>L-NAME. Cycloheximide (an inhibitor of protein synthesis), pyrrolidine dithiocarbamate (PDTC; an NF-κB inhibitor) and genistein (an inhibitor of tyrosine protein kinases) inhibited cytokine-induced NO production, while dexamethasone, diaminohydroxypyrimidine (DAHP; an inhibitor of tetrahydrobiopterin synthesis) and PD 98059 (p42/44 MAP kinase inhibitor) had no effect.Conclusions The results suggest that NO synthesis in human osteoarthritic cartilage derives from the glucocorticoid-insensitive expression of iNOS. Very similar mechanisms appear to regulate inducible NO synthesis in human osteoarthritic cartilage and J774 macrophages with the exception that dexamethasone inhibited NO production in J774 cells but not in osteoarthritic cartilage.

Use of synovial fluid markers of cartilage synthesis and turnover to study effects of repeated intra-articular administration of methylprednisolone acetate on articular cartilage in vivo

In vivo the effects of intra-articular (IA) corticosteroids on articular cartilage remain controversial. This study was designed to examine this issue using synovial fluid (SF) markers of cartilage metabolism. Paired radiocarpal joints, without clinical or radiographic signs of joint disease, were studied in 10 adult horses. Aseptic arthrocentesis was performed weekly for 13 weeks. IA injections of methylprednisolone acetate (MPA) into the treatment joint and the vehicle into the control joint were performed at weeks 3, 5 and 7. We used radioimmunoassays on SF samples which measure a keratan sulfate epitope (KS) and the 846 epitope on cartilage aggrecan (PG) and the C-propeptide (CPII) of cartilage type II procollagen which is released following synthesis of this molecule. Gel chromatography was performed on selected SF samples to evaluate the sizes of SF PG molecules. The total joint KS and the 846 epitopes were both present on a heterogeneous population of mainly molecules which, from chromotographic analysis, appeared to be mainly fragments of the articular cartilage aggrecan. They were significantly elevated in MPA joints whereas CPII was significantly reduced compared to the control during the treatment period. These results indicate that the repeated use of IA MPA leads to a potentially harmful inhibition of procollagen II synthesis and an increased release of degradation products of the PG aggrecan from articular cartilage.

Misoprostol therapeutics revisited.

Misoprostol, a prostaglandin E1 analog, is a racemate of four stereoisomers. On administration it rapidly de-esterifies to its active form, misoprostolic acid. Misoprostolic acid is 85% albumin bound and has a half-life of approximately 30 minutes. It is excreted in urine as inactive metabolites. No significant drug interactions have been reported. Besides its gastrointestinal protective and uterotonic activities, misoprostol regulates various immunologic cascades. It inhibits platelet-activating factor and leukocyte adherence, and modulates adhesion molecule expression. It protects against gut irradiation injury, experimental gastric cancer, enteropathy, and constipation. It improves nutrient absorption in cystic fibrosis. Misoprostol has utility in acetaminophen and ethanol hepatotoxicity, hepatitis, and fibrosis. It is effective in asthmatics and aspirin-sensitive asthmatic and allergic patients. It lowers cholesterol and severity of peripheral vascular diseases, prolongs survival of cardiac and kidney transplantation, synergizes cyclosporine, and protects against cyclosporine-induced renal damage. It works against drug-induced renal damage, interstitial cystitis, lupus nephritis, and hepatorenal syndrome. It is useful in periodontal disease and dental repair. Misoprostol enhances glycosoaminoglycan synthesis in cartilage after injury. It prevents ultraviolet-induced cataracts and reduces intraocular pressure in glaucoma and ocular hypertension. It synergizes antiinflammatory and analgesic effects of diclofenac or colchicine and has been administered to treat trigeminal neuralgic pain. It reduces chemotherapy-induced hair loss and recovery time from burn injury, and is effective in treating sepsis, multiple sclerosis, and pancreatitis.

Neurophysiological basis for neurogenic-mediated articular cartilage anabolism alteration.

This study was designed to investigate the pathways involved in neurogenic-mediated articular cartilage damage triggered by a nonsystemic distant subcutaneous or intra-articular inflammation. The cartilage damage was assessed 24 h after subcutaneous or intra-articular complete Freund's adjuvant (CFA) injection measuring patellar proteoglycan (PG) synthesis (ex vivo [Na(2)(35)SO(4)] incorporation) in 96 Wistar rats. Unilateral subcutaneous or intra-articular injection of CFA induced significant decrease (25-29%) in PG synthesis in both patellae. Chronic administration of capsaicin (50 mg. kg(-1). day(-1) during 4 days), which blunted the normal response of C fiber stimulation, prevented the bilateral significant decrease in cartilage synthesis. Similarly, intrathecal injection of MK-801 (10 nmol/day during 5 days), which blocked the glutamatergic synaptic transmission at the dorsal horn of signal originating in primary afferent C fibers, eliminated the CFA-induced PG synthesis decrease in both patellae. Chemical sympathectomy, induced by guanethidine (12.5 mg. kg(-1). day(-1) during 6 wk), also prevented PG synthesis alteration. Finally, compression of the spinal cord at the T3-T5 level had a similar protective effect on the reduction of [Na(2)(35)SO(4)] incorporation. It is concluded that the signal that triggers articular cartilage synthesis damage induced by a distant local inflammation 1) is transmitted through the afferent C fibers, 2) makes glutamatergic synaptic connections with the preganglionic neurons of the sympathetic system, and 3) involves spinal and supraspinal pathways.

Osteoarthritis and cartilage: the role of cytokines.

The pathogenesis of osteoarthritis involves multiple etiologies, including mechanical, biochemical, and genetic factors that contribute to the imbalance in the synthesis and destruction of articular cartilage. It is now well documented that interleukin-1 and tumor necrosis factor-alpha are the predominant proinflammatory and catabolic cytokines involved in disease initiation and progression. Other proinflammatory cytokines may amplify or modulate this process, whereas anti-inflammatory cytokines, which are often detected, paradoxically, in osteoarthritis tissues, may counteract the tissue destruction and inflammation. This review focuses on the role of cytokines in the pathogenesis of osteoarthritis with special emphasis on how findings in culture and animal models may be reflected in the human disease process.

The effects of methylprednisolone on normal and monocyte-conditioned medium-treated articular cartilage from dogs and horses.

To study in vitro (1) the dose-response relationships between proteoglycan metabolism in normal and corticosteroid-treated articular cartilage; (2) long-term proteoglycan metabolism after treatment of articular cartilage with corticosteroids; and (3) the effect of corticosteroids on proteoglycan metabolism in articular cartilage treated with monocyte-conditioned medium (MCM). Equine and canine articular cartilage explants were treated with corticosteroids and MCM. Proteoglycan synthesis and degradation were measured by radioactive labeling in short-term culture, and the long-term effect of corticosteroid treatment on proteoglycan metabolism was studied in normal explants. Two young cross-breed horses and 3 young Labrador retrievers. Equine articular cartilage explants were incubated in medium containing methylprednisolone sodium succinate (MPS) at 0, .001, .01, .1, 1, and 10 mg/mL (final concentration) for 1 day and then in fresh medium without MPS. Proteoglycan synthesis was measured by incorporation of sodium [35S]sulfate at 1, 3, 7, 10, and 13 days after initial treatment with MPS. Proteoglycan release was measured from separate explants prelabeled with sodium [35S]sulfate and treated similarly. Equine articular cartilage explants were treated with equine MCM simultaneously with, and 24 hours before MPS, at 0, 0.01, 0.1, 1, or 5 mg/mL for 72 hours. Proteoglycan synthesis and degradation in these explants was compared. Proteoglycan synthesis and degradation were measured similarly in canine articular cartilage explants treated simultaneously with canine MCM and MPS at 0, 0.001, 0.01, 0.1, 1 and 10 mg/mL for 72 hours. Equine articular cartilage explants treated with 0, 0.01, 0.1, 1, and 5 mg/mL of MPS for 72 hours were evaluated histologically. Proteoglycan synthesis in normal equine articular cartilage was severely depressed by 10 mg/mL MPS for 24 hours, and proteoglycan synthesis failed to recover after 13 days of culture in medium without MPS. Cartilage treated with 5 mg/mL MPS had pyknotic chondrocyte nuclei and empty lacunae. Concentrations of 1 and 0.1 mg/mL MPS depressed proteoglycan synthesis in normal equine cartilage explants. For these 2 concentrations, proteoglycan synthesis recovered 2 days after MPS removal and increased significantly (P < .05) 7 days after treatment with MPS compared with controls without MPS. Concentrations of 0.001 and 0.01 mg/mL MPS did not significantly affect proteoglycan synthesis in normal equine cartilage explants. Cumulative proteoglycan loss over 13 days in culture from normal equine explants treated for 24 hours with different concentrations of MPS was not significantly different between treatment groups at any time point. MCM significantly depressed proteoglycan synthesis in both canine and equine articular cartilage explants and significantly increased proteoglycan release. These effects were prevented in the canine explants by simultaneous treatment with MPS at 1 and 0.1 mg/mL, and proteoglycan release induced by MCM in equine articular cartilage was inhibited by 1 mg/mL MPS. Concentrations of 1.0 and 0.1 mg/mL MPS alleviated articular cartilage degradation in MCM-treated articular cartilage in vitro. These concentrations of MPS in contact with normal cartilage explants for 24 hours are unlikely to be detrimental in the long term to proteoglycan synthesis. The response of articular cartilage to MPS was affected by treatment with MCM so that results of experiments with normal articular cartilage explants may not reflect results obtained with abnormal cartilage. It may be possible to find an intraarticular concentration of corticosteroid that protects articular cartilage against cytokine-induced matrix degradation yet not have prolonged or permanent detrimental effects on chondrocyte matrix synthesis.

Adenovirus-mediated gene transfer of insulin-like growth factor 1 stimulates proteoglycan synthesis in rabbit joints.

To examine the effect of insulin-like growth factor 1 (IGF-1) on the regulation of cartilage synthesis and other articular events in vivo. A first-generation adenoviral vector expressing human IGF-1 (AdIGF-1) from the cytomegalovirus promoter was constructed. Particles of AdIGF-1 (5 x 10(9)) were injected through the patellar tendon into normal rabbit knee joints and rabbit knee joints with antigen-induced arthritis (AIA), with the same dose of a control adenoviral vector injected into the contralateral knees. Lavage fluids were obtained from rabbit knee joints on days 3 and 7 postinjection and used for analysis of IGF-1 expression, white blood cell infiltration, and cartilage breakdown. Cartilage chips from rabbit joints were used for assay of new proteoglycan synthesis, and tissues also were harvested from the dissected knees for histologic study. Intraarticular injection of AdIGF-1 resulted in a mean of 180.6 ng/ml of IGF-1 expression in the lavage fluid from rabbit joints. IGF-1 expression stimulated new proteoglycan synthesis in both naive and AIA rabbit knees, but had no significant chondroprotective or antiinflammatory effects. Histologic analysis showed that elevated levels of IGF-1 expression in both normal and arthritic knees had no adverse pathologic effects on synovium or adjacent muscles. Gene transfer of IGF-1 into rabbit knee joints promotes proteoglycan synthesis without significantly affecting inflammation or cartilage breakdown. In addition, no adverse effects following intraarticular IGF-1 gene delivery were observed. Thus, local gene transfer of IGF-1 to joints could serve as a therapeutic strategy to stimulate new matrix synthesis in both rheumatoid arthritis and osteoarthritis.

Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRbeta tyrosine phosphorylation.

Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso-N-acetylpenicillamine (SNAP) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of (35)SO(4). IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with N(G)-monomethyl-L-arginine (L-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.

Type II interleukin-1beta receptor: a candidate for gene therapy in human arthritis.

Interleukin-1 plays a pivotal role in the pathophysiologic process of arthritis. It is released in an autocrine fashion in joints affected with arthritis. Human cartilage affected with arthritis (but not normal cartilage) showed upregulation of interleukin-1beta messenger ribonucleic acid and protein in ex vivo conditions. Type II interleukin-1 receptor, interleukin-1 receptor antagonist, and Type I soluble interleukin-1 receptor has potent interleukin-1 neutralizing activity. In view of these observations, the role of type II interleukin-1 receptor in chondrocyte function was examined. Human interleukin-1beta (5-10 ng/mL) induced nitric oxide, prostaglandin E2 and matrix metalloprotease production in bovine or human chondrocytes, which could be inhibited by 500 pg/mL of Type II interleukin-1 receptor. Interleukin-1 inhibited proteoglycan synthesis that could be reversed by Type II soluble interleukin-1 receptor. Similarly, 1 ng/mL human interleukin-1 induced (or spontaneously produced) nitric oxide and prostaglandin E2 production in human osteoarthritis-affected cartilage could be inhibited by 50% or greater with 100 pg/mL Type II interleukin-1 receptor in ex vivo conditions. Type II interleukin-1 receptor transfected chondrocytes were immune to the insults of exogenous interleukin-1. These experiments showed that endogenous or exogenous Type II interleukin-1 receptor can attenuate the effects of interleukin-1 with respect to induction of inflammatory mediators, matrix metalloprotease activity and, proteoglycan synthesis in human chondrocytes and cartilage.

Ex vivo synthesis of articular cartilage

This review discusses modern methods used for the synthesis of articular cartilage ex vivo. The value of culturing articular chondrocytes as a monolayer and in three-dimensional lattices is discussed. Of particular interest are techniques involving seeding of chondrocytes onto synthetic, biodegradable, polymeric scaffolds, and natural materials, such as collagen and agarose. Also discussed is the use of bioreactors to modulate the fluid-flow-induced shear environment of cell-seeded scaffolds. Biodegradable scaffolds are central to the efforts to tissue engineer articular cartilage ex vivo. A review of salient efforts to design and use such scaffolds is presented, along with our thoughts on potential future improvements.

Effect of polysulfated glycosaminoglycan on DNA content and proteoglycan metabolism in normal and osteoarthritic canine articular cartilage explants.

To study the effect of polysulfated glycosaminoglycan (PSGAG) on proteoglycan metabolism and DNA content of control and osteoarthritic (OA) cartilage. An in vitro study comparing the effects of PSGAG on articular cartilage explants from canine stifle joints with and without chronic OA after transection of the left cranial cruciate ligament. Five large cross-breed dogs. Cartilage explants (6 to 13 per treatment group) from the medial side of the femoral trochlea and medial femoral condyle from both stifles of each dog were incubated in a defined medium containing 0, 0.05, 0.5, or 5 mg/mL of PSGAG. After 72 hours in culture, explants were pulsed for 6 hours with sodium [35S]sulfate. Aminophenylmercuric acetate (APMA) was used to activate endogenous neutral matrix metalloproteinases (MMPs) and induce proteoglycan degradation in the radiolabeled explants. DNA content and radioactivity were measured in papain-digested explants, and radioactivity was measured in the medium by liquid scintillation counting. Proteoglycan synthesis and degradation were calculated. Cartilage was examined histologically for signs of OA. A mixed model analysis of variance and linear contrasts were used to test for significant (P < .05) effects of OA and treatment with PSGAG. Transection of the cranial cruciate ligament produced OA in operated joints. DNA content and proteoglycan synthesis of OA cartilage were significantly lower than in cartilage from control joints. For both DNA content and proteoglycan synthesis, significant interactions occurred between the concentration of PSGAG and whether the articular cartilage was from OA or control joints. The two lower concentrations of PSGAG (0.05 and 0.5 mg/mL) predominantly increased DNA content in OA cartilage (7 and 18%, respectively, compared with 0 mg/mL PSGAG) while the highest concentration (5 mg/mL) predominantly increased DNA content in control cartilage (30% compared with 0 mg/mL PSGAG). PSGAG at .05 mg/mL predominantly decreased proteoglycan synthesis in OA cartilage (19% reduction compared with 0 mg/mL PSGAG) while PSGAG at .5 and 5 mg/mL predominantly decreased proteoglycan synthesis in control cartilage (17 and 55% reduction, respectively, compared with 0 mg/mL PSGAG). Following activation of MMPs, PSGAG caused a dose-dependent decrease in degradation of radiolabeled proteoglycan in both OA and control cartilage. OA cartilage was responsive to treatment with PSGAG at 100-fold lower concentration than control cartilage. When treated with PSGAG, articular cartilage explants maintained or increased DNA content at the expense of proteoglycan synthesis. Following MMP activation, proteoglycan degradation was inhibited in OA and control explants in a dose-dependent manner. If the results of this study extend to in vivo use, treatment with PSGAG may modify the progression of OA in articular cartilage by maintaining chondrocyte viability or stimulating chondrocyte division as well as protecting against extracellular matrix degradation.

Reduction of interleukin-17-induced inhibition of chondrocyte proteoglycan synthesis in intact murine articular cartilage by interleukin-4.

To investigate the role of interleukin-4 (IL-4) and IL-10 in basal and IL-1- and IL-17-mediated inhibition of chondrocyte metabolism. Cartilage explants of patellae from naive mice were incubated with IL-17 and/or IL-1 alone or were pretreated with IL-4 and IL-10. In addition, knee joints of naive mice were injected intraarticularly with IL-4 and IL-10 alone or were coinjected with IL-1. Chondrocyte proteoglycan (PG) synthesis was measured in intact murine articular cartilage. Levels of nitric oxide (NO) were measured using the Griess reagent. IL-17, a novel cytokine secreted by CD4+ activated memory T cells, inhibited chondrocyte PG synthesis in intact murine articular cartilage, although the suppressive effect was less potent than that of IL-1. The suppressive effect of IL-17 was completely abolished in the presence of L-NIO (L-NS-[1-iminoethyl]ornithine), an inhibitor of NO metabolism, and IL-17 only slightly induced inhibition of PG synthesis in cartilage explants of patellae from iNOS (inducible NO synthase) knockout mice. This indicates that the suppressive effect of IL-17 was mediated by NO. Pretreatment with IL-4, but not IL-10, significantly reduced the inhibition of chondrocyte PG synthesis induced by IL-1 or IL-17. The IL-4-induced reduction in the inhibitory effects of IL-1 and IL-17 on chondrocyte PG synthesis was accompanied by decreased NO formation in the culture supernatants. IL-17 plays a role in the inhibition of chondrocyte PG synthesis. IL-4 and IL-10 have no effect on basal chondrocyte metabolism. However, IL-4-pretreated cartilage is less sensitive to the suppressive effect of IL-1 as well as IL-17. This suggests that IL-4 is protective in T cell-driven cartilage disturbances, probably through reduction of iNOS.