Pathophysiological mechanisms in osteoarthritis lead to novel therapeutic strategies

Osteoarthritis (OA) is a debilitating, progressive disease of diarthrodial joints associated with aging. At the molecular level, OA is characterized by an imbalance between anabolic (i.e. extracellular matrix biosynthesis) and catabolic (i.e. extracellular matrix degradation) pathways in which articular cartilage is the principal site of tissue injury responses. The pathophysiology of OA also involves the synovium in that 'nonclassical' inflammatory synovial processes contribute to OA progression. Chondrocytes are critical to the OA process in that the progression of OA can be judged by the vitality of chondrocytes and their ability to resist apoptosis. Growth factors exemplified by insulin-like growth factor-1, its binding proteins and transforming growth factor-beta contribute to anabolic pathways including compensatory biosynthesis of extracellular matrix proteins. Catabolic pathways are altered by cytokine genes such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) which are upregulated in OA. In addition, IL-1 and TNF-alpha downregulate extracellular matrix protein biosynthesis while concomitantly upregulating matrix metalloproteinase (MMP) gene expression. When MMPs are activated, cartilage extracellular matrix degradation ensues apparently because levels of endogenous cartilage MMP inhibitors cannot regulate MMP activity. Therapeutic strategies designed to modulate the imbalance between anabolic and catabolic pathways in OA may include neutralizing cytokine activity or MMP gene expression or inhibiting signaling pathways which result in apoptosis dependent on mature caspase activity or mitogen-activated protein kinase (MAPK) activity. MAPK activity appears critical for regulating chondrocyte and synoviocyte apoptosis and MMP genes.     

The chrondoprotective actions of a natural product are associated with the activation of IGF-1 production by human chondrocytes despite the presence of IL-1β

Background

Cartilage loss is a hallmark of arthritis and follows activation of catabolic processes concomitant with a disruption of anabolic pathways like insulin-like growth factor 1 (IGF-1). We hypothesized that two natural products of South American origin, would limit cartilage degradation by respectively suppressing catabolism and activating local IGF-1 anabolic pathways. One extract, derived from cat's claw (Uncaria guianensis, vincaria^®), is a well-described inhibitor of NF-κB. The other extract, derived from the vegetable Lepidium meyenii (RNI 249), possessed an uncertain mechanism of action but with defined ethnomedical applications for fertility and vitality.

Methods

Human cartilage samples were procured from surgical specimens with consent, and were evaluated either as explants or as primary chondrocytes prepared after enzymatic digestion of cartilage matrix. Assessments included IGF-1 gene expression, IGF-1 production (ELISA), cartilage matrix degradation and nitric oxide (NO) production, under basal conditions and in the presence of IL-1β.

Results

RNI 249 enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7 fold, an effect that was further enhanced to 3.8 fold by co-administration with vincaria. Enhanced basal IGF-1 production by RNI 249 alone and together with vincaria, was confirmed in both explants and in primary chondrocytes (P <0.05). As expected, IL-1β exposure completely silenced IGF-1 production by chondrocytes. However, in the presence of IL-1β both RNI 249 and vincaria protected IGF-1 production in an additive manner (P <0.01) with the combination restoring chondrocyte IGF-1 production to normal levels. Cartilage NO production was dramatically enhanced by IL-1β. Both vincaria and RNI 249 partially attenuated NO production in an additive manner (p < 0.05). IL-1β – induced degradation of cartilage matrix was quantified as glycosaminoglycan release. Individually RNI 249 or vincaria, prevented this catabolic action of IL-1β.

Conclusion

The identification of agents that activate the autocrine production of IGF-1 in cartilage, even in the face of suppressive pro-inflammatory, catabolic cytokines like IL-1β, represents a novel therapeutic approach to cartilage biology. Chondroprotection associated with prevention of the catabolic events and the potential for sustained anabolic activity with this natural product suggests that it holds significant promise in the treatment of debilitating joint diseases.     

Interleukin-4 downregulates the cyclic tensile stress-induced matrix metalloproteinases-13 and cathepsin B expression by rat normal chondrocytes

Mechanical stress plays a key role in the pathogenesis of cartilage destruction seen in osteoarthritis (OA). We investigated the effect of cyclic tensile stress (CTS) on the anabolic and catabolic gene expression of rat cultured normal chondrocytes using the Flexercell strain unit. The effects of interleukin (IL)-4, a chondroprotective cytokine, on the changes in gene expression induced by CTS were also investigated. CTS (7% elongation at 0.5 Hz) for 24 h did not affect the expression of aggrecan and type II collagen, whereas CTS significantly upregulated matrix metalloproteinase (MMP)-13 and cathepsin B mRNA expression by chondrocytes. IL-1beta expression was also signifi cantly upregulated by CTS up to 12 h. The upregulation of MMP-13 was observed at 3 h, which was earlier than that of IL-1beta. Furthermore, pre-treatment with IL-4 (10 ng/ml) suppressed both MMP-13 and cathepsin B induction by mechanical stress, as well as CTS-induced IL-1beta expression. Our results suggest that IL-4 might have a therapeutic value in the treatment of OA by downregulation of mechanical stress-induced MMP-13 and cathepsin B expression by chondrocytes.     

Cytokines as Therapeutic Targets for Osteoarthritis

Osteoarthritis (OA) is a debilitating, progressive disease of diarthrodial joints associated with the aging process. With the exception of anti-inflammatory corticosteroids and nonsteroidal anti-inflammatory drugs which inhibit cyclo-oxygenase-2, the enzyme responsible for prostaglandin biosynthesis in inflammation, no specific therapy based on fundamental intracellular pathways of chondrocytes and synoviocytes exists for the medical management of OA. At the molecular level, OA is characterized by an imbalance between chondrocyte anabolism and catabolism. Disruption of chondrocyte homeostasis primarily affects the cartilage extracellular matrix (ECM), which is responsible for the biomechanical properties of the tissue. Recent evidence has implicated cytokines, among which interleukin (IL)-1, tumor necrosis factor-alpha, IL-6, and IL-17 seem most involved in the OA process of cartilage destruction. The primary role of these cytokines is to modulate the expression of matrix metalloproteinases and cartilage ECM proteins. Cartilage repair that could restore the functional integrity of the joint is also impaired because chondrocytes in OA cartilage appear unable to respond to insulin-like growth factor-1 or respond abnormally to transforming growth factor-beta. As these growth factors also modulate cytokine expression, they may prove useful in designing strategies for suppressing 'chondrocyte activation'. Although cytokines and growth factors provide a potential therapeutic target for OA, it will be necessary to elucidate the fundamental mechanisms that cytokines employ to cause chondrocyte and synoviocyte dysfunction before 'anti-cytokine' therapy can be employed in the medical management of the disease.     

Standardized butanol fraction of WIN-34B suppresses cartilage destruction via inhibited production of matrix metalloproteinase and inflammatory mediator in osteoarthritis human cartilage explants culture and chondrocytes

Background

WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes.

Methods

The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1β-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed.

Results

WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1β-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1β. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic acid or mangiferin in IL-1β-stimulated chondrocytes.

Conclusions

WIN-34B is potentially valuable as a treatment for OA by virtue of its suppression of MMPs, ADAMTSs, and inflammatory mediators, and it’s up-regulation of TIMP-1 and TIMP-3 involved in the MAPK pathway.

     

Effects of high mobility group box protein-1, interleukin-1β, and interleukin-6 on cartilage matrix metabolism in three-dimensional equine chondrocyte cultures

The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.     

Regulation of Gelatinases Expression by Cytokines,Endotoxin, and Pharmacological Agents in the Human Osteoarthritic Knee

We examined the amount of gelatinases (matrix metalloproteinase-2 and -9 [MMP-2 and MMP-9] in a series of chondral, meniscal, and synovial cultures of early osteoarthritis (OA) after treatment with or without catabolic cytokines. These included interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), and pharmacological agents, including plasmin/serine proteinase antagonist aprotinin, protein synthesis inhibitor cycloheximide, and protein kinase C (PKC) inhibitors staurosporine, H7, and Gö6976 for investigation of their effects on MMP-2 and -9 production in OA. Gelatin zymography revealed that IL-α, TNF-α, and LPS could elevate MMP-2 secretion in all tissue cultures and also increase MMP-9 production in all synovial and some meniscal cultures. In contrast, aprotinin, cycloheximide, staurosporine, H7, and Gö6976 could suppress MMP-2 secretion in all tissue cultures and also decrease MMP-9 production in all synovial and some meniscal cultures. Our data indicate that catabolic cytokines and LPS may promote tissue destruction and disintegration of extracellular matrix in early OA. Agents that target on the PKC pathway, plasmin/serine proteinase or protein synthesis for MMP-2 and -9 in early OA may inhibit the production of MMPs. These findings might contribute to the design of more efficacious therapies.     

Jumonji domain containing-3 (JMJD3) inhibition attenuates IL-1β-induced chondrocytes damage in vitro and protects osteoarthritis cartilage in vivo

Objectives

This study aimed to explore the effects and relative mechanism of JMJD3 on knee osteoarthritis (OA).

Methods

In this study, we first analyzed the expression of JMJD3 in OA cartilage using western blot and immunohistochemistry. In an in vitro study, the effects of GSK-J4, JMJD3 inhibitor, on ATDC-5 chondrocytes were evaluated by CCK-8 assay. Real-time PCR and western blot were used to examine the inhibitory effect of GSK-J4 on the inflammation and ECM degradation of chondrocytes. NF-κB p65 phosphorylation and nuclear translocation were measured by western blot and immunofluorescence. In the animal study, twenty mice were randomized into four experimental groups: sham group, DMM-induced OA + DMSO group, OA + low-dose GSK-J4 group, and OA + high-dose GSK-J4 group. After the treatment, hematoxylin–eosin and safranin O/fast green staining were used to evaluate cartilage degradation of knee joint, with OARSI scores for quantitative assessment of cartilage damage.

Results

Our results revealed that JMJD3 was overexpressed in OA cartilage and GSK-J4 could suppress the IL-1β-induced production of pro-inflammatory cytokines and catabolic enzymes, including IL-6, IL-8, MMP-9 and ADAMTS-5. Consistent with these findings, GSK-J4 could inhibit IL-1β-induced degradation of collagen II and aggrecan. Mechanistically, GSK-J4 dramatically suppressed IL-1β-stimulated NF-κB signal pathway activation. In vivo, GSK-J4 prevented cartilage damage in mouse DMM-induced OA model.

Conclusions

This study elucidates the important role of JMJD3 in cartilage degeneration in OA, and our results indicate that JDJM3 may become a novel therapeutic target in OA therapy.

     

Coordinate synthesis of stromelysin, interleukin-1, and oncogene proteins in experimental osteoarthritis. An immunohistochemical study.

Metalloproteases appear to play an important role in the pathophysiology of osteoarthritis (OA) and their expression is believed to be regulated by cytokines such as interleukin-1 (IL-1). Nuclear oncogene products are suggested as mediators through which IL-1 induces metalloprotease gene expression. Little data are available on the in vivo involvement of these agents in the pathophysiology of OA. This study examined by immunohistochemistry, using specific antibodies, the distribution of stromelysin, IL-1 alpha, IL-1 beta, and oncogene products (c-FOS, c-JUN, and c-MYC) in synovium and cartilage from normal and experimental canine models of OA. In the OA synovium, stromelysin and IL-1 were localized in the cytoplasm of superficial synovial lining cells, infiltrating mononuclear cells, and endothelial and smooth muscle cells of the blood vessels, whereas oncoproteins were detected predominantly in the synovial lining cells. Normal synovial membranes demonstrated low levels of specific staining in synovial lining cells with occasional staining of blood vessel cells for IL-1 alpha, IL-1 beta, and stromelysin. In OA cartilage, chondrocytes at the superficial and middle layers as well as in fibrillated areas were found to be involved in the synthesis of stromelysin, IL-1, and oncoproteins. Diffuse staining of stromelysin and IL-1 beta in OA cartilage matrix was also identified. In normal cartilage, only a few chondrocytes at the superficial layer showed a low level of antigens. These results demonstrate the in vivo concomitant cellular and/or matrical presence of stromelysin, IL-1, and oncogene proteins in tissues from experimentally induced OA with the most intense staining at the sites of cartilage erosion and synovial proliferation. These findings suggest that they may be involved in the pathophysiology of OA, and that the regulatory mechanisms involved in the expression of these proteins may be associated.     

Regulation of gelatinases expression by cytokines, endotoxin, and pharmacological agents in the human osteoarthritic knee

We examined the amount of gelatinases (matrix metalloproteinase-2 and -9 [MMP-2 and MMP-9] in a series of chondral, meniscal, and synovial cultures of early osteoarthritis (OA) after treatment with or without catabolic cytokines. These included interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and pharmacological agents, including plasmin/serine proteinase antagonist aprotinin, protein synthesis inhibitor cycloheximide, and protein kinase C (PKC) inhibitors staurosporine, H7, and G?6976 for investigation of their effects on MMP-2 and -9 production in OA. Gelatin zymography revealed that IL-alpha, TNF-alpha, and LPS could elevate MMP-2 secretion in all tissue cultures and also increase MMP-9 production in all synovial and some meniscal cultures. In contrast, aprotinin, cycloheximide, staurosporine, H7, and G?6976 could suppress MMP-2 secretion in all tissue cultures and also decrease MMP-9 production in all synovial and some meniscal cultures. Our data indicate that catabolic cytokines and LPS may promote tissue destruction and disintegration of extracellular matrix in early OA. Agents that target on the PKC pathway, plasmin/serine proteinase or protein synthesis for MMP-2 and -9 in early OA may inhibit the production of MMPs. These findings might contribute to the design of more efficacious therapies.     

Matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro

Interleukin (IL)-1β plays an important role in promoting osteoarthritis (OA) lesions by inducing chondrocytes to secrete matrix metalloproteinases (MMPs), which degrade the extracellular matrix and facilitate chondrocyte apoptosis. Matrine was shown to exert anti-inflammatory effects. However, the role of matrine in OA is still unclear. Therefore, in this study, we investigated the effects of matrine on the expression of MMPs in IL-1β-treated human chondrocytes and the underlying mechanism. The cell viability of chondrocytes was detected by MTT assay. The cell apoptosis of chondrocytes was measured by flow cytometric analysis. The protein production of MMPs was determined by ELISA. The protein expression of phosphorylation of mitogen-activated protein kinases (MAPKs) and the inhibitor of kappaB alpha (IκBα) was determined by Western blot. Matrine significantly inhibited the IL-1β-induced apoptosis in chondrocytes. It also significantly inhibited the IL-1β-induced release of MMP-3 and MMP-13, and increased the production of TIMP-1. Furthermore, matrine inhibits the phosphorylation of p-38, extracellular regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and IκBα degradation induced by IL-1β in chondrocytes. Taken together, our results show that matrine inhibits IL-1β-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-κB in human chondrocytes in vitro. Therefore,-matrine may be beneficial in the treatment of OA.     

The effects of cytokines on metalloproteinase inhibitors (TIMP) and collagenase production by human chondrocytes and TIMP production by synovial cells and endothelial cells.

It has been suggested that IL-1 produces cartilage matrix degradation by metalloproteinases such as collagenase, and that such degradation is regulated by metalloproteinase inhibitors (TIMP). Therefore, the balance between collagenase and TIMP is an important factor for tissue destruction in inflammatory joints. In the present study the effects of cytokines on collagenase and TIMP production in chondrocytes as well as the effects of cytokines on TIMP production in connective tissue cells were studied. IL-1 beta inhibited TIMP production in endothelial cells while enhancing TIMP production in synovial cells and chondrocytes. In addition, tumour necrosis factor-alpha (TNF-alpha) significantly inhibited and IL-6 significantly enhanced TIMP production in endothelial cells, synovial cells and chondrocytes. In the chondrocyte supernatant, collagenase activity/TIMP ratio was significantly elevated by the addition of either IL-1 beta or TNF-alpha to the cells, whereas the ratio was significantly decreased by IL-6. These results suggest that the cytokine effects on TIMP production are different among the different cell types, and that either IL-1 beta or TNF-alpha induce cartilage matrix degradation by disrupting the collagenase/TIMP balance, while, on the other hand, IL-6 protects the tissue through an opposite effect.     

Role of oxygen radicals and IL-6 in IL-1-dependent cartilage matrix degradation

It has been suggested that IL-1 produces cartilage matrix degradation by metalloproteinases such as collagenase and that such degradation is regulated by metalloproteinase inhibitors. In the present study, the effects of IL-6 and oxygen radical scavengers on cartilage matrix degradation were studied. Superoxide dismutase, catalase, or methionine all significantly inhibited cartilage matrix degradation both in IL-1-stimulated and unstimulated experimental conditions. Both 10 mM EDTA and 100 nM tissue inhibitor of metalloproteinase (TIMP) significantly inhibited cartilage matrix degradation. The addition of methionine significantly inhibited collagenase activity produced in the culture supernatants of chondrocytes stimulated with IL-1. IL-6 significantly suppressed cartilage matrix degradation produced spontaneously or by IL-1 stimulation in chondrocytes. IL-6 inhibited superoxide production by chondrocytes both in IL-1-stimulated or unstimulated conditions. These results suggest that oxygen radicals are involved in cartilage matrix degradation mediated by both paracrine and autocrine IL-1 mechanisms and that oxygen radicalmediated activation of collagenase in chondrocytes may explain the mechanisms of how oxygen radicals are involved in cartilage matrix degradation. IL-6 inhibited superoxide production in chondrocytes and thus inhibited cartilage matrix degradation.     

Effects of high mobility group box protein-1, interleukin-1β, and interleukin-6 on cartilage matrix metabolism in three-dimensional equine chondrocyte cultures

The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.     

An inflammatory equine model demonstrates dynamic changes of immune response and cartilage matrix molecule degradation in vitro

The molecular aspects of inflammation were investigated in equine articular cartilage explants using quantitative proteomics. Articular cartilage explants were stimulated with interleukin (IL)-1β in vitro for 25 days, and proteins released into cell culture media were chemically labeled with isobaric mass tags and analyzed by liquid chromatography-tandem mass spectrometry. A total of 127 proteins were identified and quantified in media from explants. IL-1β-stimulation resulted in an abundance of proteins related to inflammation, including matrix metalloproteinases, acute phase proteins, complement components and IL-6. Extracellular matrix (ECM) molecules were released at different time points, and fragmentation of aggrecan and cartilage oligomeric matrix protein was observed at days 3 and 6, similar to early-stage OA in vivo. Degradation products of the collagenous network were observed at days 18 and 22, similar to late-stage OA. This model displays a longitudinal quantification of released molecules from the ECM of articular cartilage. Identification of dynamic changes of extracellular matrix molecules in the secretome of equine explants stimulated with IL-1β over time may be useful for identifying components released at different time points during the spontaneous OA process.     

Cue-Signal-Response Analysis in 3D Chondrocyte Scaffolds with Anabolic Stimuli

Articular cartilage is an avascular connective tissue responsible for bearing loads. Cell signaling plays a central role in cartilage homeostasis and tissue engineering by directing chondrocytes to synthesize/degrade the extracellular matrix or promote inflammatory responses. The aim of this paper was to investigate anabolic, catabolic and inflammatory pathways of well-known and underreported anabolic stimuli in 3D chondrocyte cultures and connect them to diverse cartilage responses including matrix regeneration and cell communication. A cue-signal-response experiment was performed in chondrocytes embedded in alginate scaffolds subjected to a 9-day treatment with 7 anabolic cues. At the signaling level diverse pathways were measured whereas at the response level glycosaminoglycan (GAG) synthesis and cytokine releases were monitored. A significant increase of GAG was observed for each stimulus and well known anabolic phosphoproteins were activated. In addition, WNK1, an underreported protein of chondrocyte signaling, was uncovered. At the extracellular level, inflammatory and regulating cytokines were measured and DEFB1 and CXCL10 were identified as novel contributors to chondrocyte responses, both closely linked to TLR signaling and inflammation. Finally, two new pro-growth factors with an inflammatory potential, Cadherin-11 and MGP were observed. Interestingly, well-known anabolic stimuli yielded inflammatory responses which pinpoints to the pleiotropic roles of individual stimuli.