M. Beekhuizen

Cytokine profiles in the joint depend on pathology, but are different between synovial fluid, cartilage tissue and cultured chondrocytes

IntroductionThis study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.MethodsSamples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).ResultsIn synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.ConclusionsSeveral pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.

Interleukin-6 is elevated in synovial fluid of patients with focal cartilage defects and stimulates cartilage matrix production in an in vitro regeneration model

IntroductionThis study aimed to determine whether, as in osteoarthritis, increased levels of interleukin-6 (IL-6) are present in the synovial fluid of patients with symptomatic cartilage defects and whether this IL-6 affects cartilage regeneration as well as the cartilage in the degenerated knee.MethodsIL-6 concentrations were determined by ELISA in synovial fluid and in conditioned media of chondrocytes regenerating cartilage. Chondrocytes were obtained from donors with symptomatic cartilage defects, healthy and osteoarthritic donors. The effect of IL-6 on cartilage regeneration and on metabolism of the resident cartilage in the knee was studied by both inhibition of endogenous IL-6 and addition of IL-6, in a regeneration model and in osteoarthritic explants in the presence of synovial fluid, respectively. Readout parameters were DNA and glycosaminoglycan (GAG) content and release. Differences between controls and IL-6 blocked or supplemented samples were determined by univariate analysis of variance using a randomized block design.ResultsSynovial fluid of patients with symptomatic cartilage defects contained more IL-6 than synovial fluid of healthy donors (P = 0.001) and did not differ from osteoarthritic donors. IL-6 production of osteoarthritic chondrocytes during cartilage regeneration was higher than that of healthy and defect chondrocytes (P < 0.001). Adding IL-6 increased GAG production by healthy chondrocytes and decreased GAG release by osteoarthritic chondrocytes (P < 0.05). Inhibition of IL-6 present in osteoarthritic synovial fluid showed a trend towards decreased GAG content of the explants (P = 0.06).ConclusionsOur results support a modest anabolic role for IL-6 in cartilage matrix production. Targeting multiple cytokines, including IL-6, may be effective in improving cartilage repair in symptomatic cartilage defects and osteoarthritis.

Osteoarthritic synovial tissue inhibition of proteoglycan production in human osteoarthritic knee cartilage: Establishment and characterization of a long‐term cartilage–synovium coculture

OBJECTIVE Although both cartilage and synovium are affected in osteoarthritis (OA), no in vitro coculture models of human OA tissue have been described. The aim of this study was to develop an in vitro model that includes both the synovium and cartilage of patients with knee OA. METHODS Explants of human OA cartilage and synovium were cultured alone or in coculture for 21 days. Histologic evaluation and analyses of lactate dehydrogenase release, matrix metalloproteinase (MMP) activity, content, release, and synthesis of glycosaminoglycan (GAG), and cytokine production were used to evaluate synovial tissue functionality and its effect on cartilage metabolism. To assess the possibility of intervention in the model system, the effect of triamcinolone was studied. RESULTS Throughout the entire culture period, OA synovial tissue remained viable and produced cytokines. Monocultures of synovial and cartilage explants produced different cytokine subsets, with the subsets found in coculture being most similar to those previously described in OA synovial fluid. MMP activity was detectable only in the synovial explant monoculture and in coculture. Cocultures showed a reduction in final GAG content (P < 0.02), attributable to an inhibition of GAG production (P < 0.001) rather than an increase in GAG release. Addition of triamcinolone inhibited cytokine production and MMP activity in coculture and synovial tissue monoculture and counteracted the inhibition of GAG production induced by coculture. In cartilage monoculture, however, triamcinolone reduced GAG production. CONCLUSION OA synovium affects cartilage metabolism by reducting GAG production. Triamcinolone can relieve this effect of synovial tissue, while being inhibitory when added to cartilage monoculture. These results clearly indicate the importance of tissue coculture as a promising tool for studying OA pathophysiology and for development of possible interventions.

Pronounced biomaterial dependency in cartilage regeneration using nonexpanded compared with expanded chondrocytes

AIM We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials. MATERIALS & METHODS Chondrocytes were both directly and after expansion to passage 2, incorporated into four biomaterials: Polyactive™, Beriplast®, HyStem® and a type II collagen gel. Early cartilage matrix gene expression, cytokine production and glycosaminoglycan (GAG) and DNA content in response to these biomaterials were evaluated. RESULTS HyStem induced more GAG production, compared with all other biomaterials (p ≤ 0.001). Nonexpanded cells did not always produce more GAGs than expanded chondrocytes, as this was biomaterial-dependent. Cytokine production and early gene expression were not predictive for final regeneration. CONCLUSION For chondrocyte-based cartilage treatments, the biomaterial best supporting cartilage matrix production will depend on the chondrocyte differentiation state and cannot be predicted from early gene expression or cytokine profile.

Inflammatory Mediators in Posttraumatic Radiocarpal Osteoarthritis

PURPOSE To identify the mediator profile in healthy, pre-osteoarthritis (OA) and end-stage OA radiocarpal joints. We hypothesized that there would be an increase in soluble mediators in posttraumatic wrist OA. METHODS We obtained radiocarpal synovial fluid samples from 3 groups of patients: healthy control (n = 12) samples were collected during wrist ganglion resection; pre-osteoarthritic (n = 16) samples, during a 3-ligament tenodesis procedure for complete scapholunate dissociation; and end-stage OA (n = 20) samples in patients with proven radiological OA changes. Using a multiplex enzyme-linked immunosorbent assay, we measured 12 mediators: interleukin (IL)-1β, tumor necrosis factor-α, oncostatin-M, interferon-γ, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-1RA, and osteoprotegerin. Statistical analysis was performed using analysis of variance and Bonferroni-corrected post hoc tests. RESULTS Mediators IL-6, IL-10, and interferon-γ were increased in OA wrists compared to healthy and pre-OA samples. Tumor necrosis factor-α, oncostatin-M, osteoprotegerin, IL-8, and IL-1RA were detected but not at increased levels in OA wrists. We found no differences between healthy and pre-OA joints in all 12 mediators. Mediators IL-4, IL-7, IL-13, and IL-1β were not detected in either healthy, pre-OA or end-stage OA samples. CONCLUSIONS We identified no differences between healthy and pre-OA samples, suggesting no alteration in inflammatory status at the time of the 3-ligament tenodesis procedure. Consequently, mechanical disturbance seems to be the driving force toward OA and OA-associated inflammation in this stage of scapholunate dissociation. Increased levels of interferon-γ, IL-6, and IL-10 confirm inflammatory changes in the mechanically disturbed posttraumatic radiocarpal joint.

Early evolving joint degeneration by cartilage trauma is primarily mechanically controlled

BACKGROUND Mechanical and inflammatory processes add to osteoarthritis (OA). To what extent both processes contribute during the onset of OA after a cartilage trauma is unknown. This study evaluates whether local cartilage damage leads to focally confined or more generalized cartilage damage with synovial inflammation in the early development of joint tissue degeneration. METHODS In nine goats, cartilage damage was surgically induced on the weight bearing area of exclusively the medial femoral condyle of the right knee joint. The other tibio-femoral compartments, lateral femoral condyle and lateral medial tibial plateau, were left untouched. The contralateral left knee joint of each animal served as an intra-animal control. Twenty weeks post-surgery changes in cartilage matrix integrity in each of the four compartments, medial and lateral synovial tissue inflammation, and synovial fluid IL-1β and TNFα were evaluated. RESULTS In the experimental medial femoral plateau, significant macroscopic, histologic, and biochemical cartilage damage was observed versus the contralateral control compartments. Also the articulating cartilage of the experimental medial tibial plateau was significantly more damaged. Whereas, no differences were seen between the lateral compartments of experimental and contralateral control joints. Synovial tissue inflammation was mild and only macroscopically (not histologically) significantly increased in the experimental medial compartments. Synovial fluid IL-1β level was not different between experimental and contralateral control joints, and TNFα was overall beneath the detection limit. CONCLUSIONS Local cartilage damage is a trigger for development of OA, which in early onset seems primarily mechanically driven. Early treatment of traumatic cartilage damage should take this mechanical component into consideration.

FRI0314 Aetiology of experimentally induced osteoarthritis of the knee; biomechanical or biochemical factors?

Background At the moment, osteoarthritis (OA) is seen as a multifactorial disease, caused by a combination of i.a. genetics, overloading, obesity and trauma in the past. Disturbance of joint homeostasis, due to several risk factors, can also cause OA1. In the present study we investigated the role of biomechanical loading and biochemical joint homeostasis in the development of osteoarthritis, in an experimental caprine model of joint damage. Objectives Is the development of experimentally induced OA more due to changed biomechanics or is it due to a changed joint homeostasis? Methods In nine skeletally mature female milk goats, cartilage damage was introduced in the right stifle joint according to the Groove model2. Grooves were made with a K-wire with a bend tip, only on the medial femoral condyle and maximally 0.5mm deep; preventing the subchondral plate from damage. The left stifle joint served as a control. After 20 weeks the goats were sacrificed and the cartilage and synovial tissue was analyzed on macroscopical, histological (both OARSI goat score) and biochemical (proteoglycan (PG) turnover) OA characteristics. Results Macroscopic analysis showed a significant increase in the OARSI goat cartilage score for both the femoral and tibial experimental medial compartment compared to control (femur and tibia; +225% p<0.001 and +42.9% p<0.04 respectively). No effect on the ungrooved lateral compartment was seen. The histological analysis of the cartilage corroborated these results. A significant increase in the OARSI score for the experimental medial compartment (+225% p<0.001 and +81,7% p<0.006, respectively for femur and tibia) was seen. Biochemical analysis showed a decreased PG content in the medial experimental compartment compared to control (femur -12.1% p=0.038 and tibia -7.3% p=0.031). There was a slight increase in synovial inflammation in the experimental joint (p<0.003). For all previous mentioned analyses, no changes in the experimental lateral compartment or the control joint were found. Conclusions The present study shows clearly that the development of joint damage is much more dependent on the biomechanical component, since all detectable damage is in the medial compartment. Although joint homeostasis is disturbed, indicated by moderate synovial inflammation, this did not lead to generalised OA in this time period. References Abramson et al. Arthritis Res Ther. 2009. Mastbergen et al. Osteoarthritis and Cartilage 2006. Disclosure of Interest None Declared

222 INHIBITION OF PROSTAGLANDIN E2 BY CELECOXIB DECREASES GLYCOSAMINOGLYCAN RELEASE, HOWEVER DOES NOT STIMULATE REPAIR OF OSTEOARTHRITIC CARTILAGE TISSUE

Results: BAP level was higher in OA synovial fluid than in simple synovitis, and d-ROM level was slightness lower in OA than control synovitis, but this differences were not significant. BAP and d-ROM levels were not related with OA grade. After intra-articular injection of HA, BAP level was significantly increased and d-ROM level was reduced. On the other hand, BAP and d-ROM level were significantly decreased after injection of corticosteroid. Conclusions: Oxidative stress and anti-oxidative potential in synovial fluid were not significant difference between OA and simple synovitis. Our results showed that hyaluronan increase the anti-oxidant potential and decreased oxidative stress, but corticosteroid decreased both oxidative stress and the anti-oxidative stress potential.